EXPLORING GROWTH-INHIBITORY AND IMMUNO-STIMULATORY
ACTIVITY IN BRITISH COLUMBIA WILD MUSHROOMS AND LICHENS
by
Vicky Myhre
B. Sc., University of Northern British Columbia, 2010

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

UNIVERSITY OF NORTHERN BRITISH COLUMBIA
July 2018
© Vicky Myhre, 2018

Abstract
Four mushroom and two lichen species were collected from selected forests in north-central
British Columbia and screened for anti-proliferative and immuno-stimulatory activities. All
species investigated were immuno-stimulatory with the strongest activity found in the hot
water extracts of Laetiporus sp. and Phaeolepiota aurea. Anti-proliferative activity was
primarily seen in the ethanol extracts of Laetiporus sp., P. aurea, Letharia vulpina, Vulpicida
canadensis, and Hydnellum diabolus. Using Sevag extraction method, DEAE Sephadex
anion-exchange and Sephacryl S-500 size-exclusion chromatography, an immunostimulatory compound was purified from the hot water extract of P. aurea. The purified
compound, made up of carbohydrate and protein, has an average molecular weight of 390
kDa. Its carbohydrate component is rich in β-glucan. The compound can stimulate the innate
immune system, causing a pro-inflammatory response. Additional studies are required to
further characterize its carbohydrate and protein components in an effort to determine
whether it is a novel immuno-stimulatory compound.

ii

Table of Contents
Abstract .................................................................................................................................... ii
Table of Contents ................................................................................................................... iii
List of Tables ........................................................................................................................... v
List of Figures ......................................................................................................................... vi
Acknowledgements ................................................................................................................ xi
Chapter 1: Introduction ......................................................................................................... 1
1.1

A History of Mushrooms in Medicine ....................................................................... 1

1.2

Anticancer Activity of Medicinal Mushrooms........................................................... 4

1.2.1 Phellinus linteus ....................................................................................................... 7
1.2.2 Ganoderma lucidum................................................................................................. 8
1.2.3 Other Anti-cancerous Mushrooms ........................................................................... 8
1.3

Project Goals ............................................................................................................ 10

Chapter 2: Collection, Identification, and Screening of British Columbian Wild
Mushrooms and Lichens for Anti-Proliferative and Immuno-Stimulatory Activities ... 11
2.1 Introduction ................................................................................................................... 11
2.2 Methods......................................................................................................................... 12
2.2.1 Sample Collection and Identification..................................................................... 12
2.2.2 Chemical Extraction............................................................................................... 14
2.2.3 Biological Assays for Primary Screening .............................................................. 17
2.3 Results ........................................................................................................................... 22
2.3.1 Species Identification ............................................................................................. 22
2.3.2 Chemical Extraction............................................................................................... 32
2.3.3 Cell Viability MTT Assay ..................................................................................... 42
2.3.4 Immuno-stimulatory activity ................................................................................. 60
2.4 Discussion ..................................................................................................................... 73
Chapter 3: Purification and Characterization of an Immuno-Stimulatory Compound
from Phaeolepiota aurea ....................................................................................................... 80
3.1 Introduction ................................................................................................................... 80
3.2 Methods......................................................................................................................... 80
3.2.1 Collection, Identification, and Re-extraction of Phaeolepiota aurea .................... 80
iii

3.2.2 Column Chromatography....................................................................................... 81
3.2.3 Carbohydrate Quantification .................................................................................. 87
3.2.4 Protein Quantification ............................................................................................ 88
3.2.5 Precipitation Methods ............................................................................................ 88
3.2.6 Enzyme Digestion .................................................................................................. 89
3.3 Results ........................................................................................................................... 90
3.3.1 Sephadex LH-20 .................................................................................................... 90
3.3.2 Carbohydrate and Protein Quantification .............................................................. 94
3.3.3 Precipitation Methods ............................................................................................ 97
3.3.4 DEAE Sephadex A50 ............................................................................................ 99
3.3.5 Sephacryl S500 HR .............................................................................................. 102
3.3.6 Enzyme Digestion ................................................................................................ 109
3.3.7 Mouse Cytokine Array/Chemokine Array 32-Plex ............................................. 115
3.4 Discussion ................................................................................................................... 118
Chapter 4: General Discussion .......................................................................................... 122
4.1 Introduction ................................................................................................................. 122
4.2 Sample Identification and Screening for Growth-Inhibitory and Immuno-stimulatory
Activities ........................................................................................................................... 123
4.3 Purification and Characterization of an Immuno-Stimulatory Compound from
Phaeolepiota aurea. .......................................................................................................... 125
4.4 Final Conclusions and Future Directions .................................................................... 126
References ............................................................................................................................ 129
Appendix .............................................................................................................................. 141
A1. DNA Consensus Sequences ....................................................................................... 141
Hericium coralloides .................................................................................................... 141
Hydnellum diabolus ...................................................................................................... 141
Phaeolepiota aurea ....................................................................................................... 141
Laetiporus sp................................................................................................................. 142
Letharia vulpina ............................................................................................................ 142
Vulpicida canadensis .................................................................................................... 142

iv

List of Tables
Table 1. Traditional uses of medicinal mushrooms. ................................................................ 2
Table 2. Some medicinal mushrooms showing anticancer activity. ........................................ 9
Table 3. Thermocycler program for PCR of isolated DNA samples. .................................... 14
Table 4. A summary of fungi collected in British Columbia. ................................................ 32
Table 5. Summary of chemical extraction results. ................................................................. 42
Table 6. Cytokine/Chemokine concentrations (pg/mL) obtained from Eve Technologies of
post-column fractions treated onto Raw 264.7 cells. ............................................................ 117

v

List of Figures
Figure 1. Flowchart outlining the manual chemical extraction procedure for obtaining crude
extracts from mushrooms. ....................................................................................................... 16
Figure 2. Flowchart outlining the speed extractor procedure for obtaining crude extracts
from mushrooms. .................................................................................................................... 17
Figure 3. DNA analysis of PCR products. ............................................................................. 23
Figure 4. Dose-dependent MTT-assay of H. coralloides crude extracts. .............................. 44
Figure 5. Time-dependent MTT-assay of H. coralloides crude extracts. .............................. 45
Figure 6. Time-dependent MTT-assay of H. coralloides crude extracts. .............................. 45
Figure 7. Dose-dependent MTT-assay of H. diabolus crude extracts. .................................. 46
Figure 8. Time-dependent MTT assay of growth-inhibitory extracts from H. diabolus. ...... 47
Figure 9. Time-dependent MTT-assay of crude extracts isolated from H. diabolus. ............ 48
Figure 10. Dose-dependent MTT-assay of P. aurea crude extracts. ..................................... 49
Figure 11. Time-dependent MTT-assay of P. aurea crude fractions..................................... 50
Figure 12. Time-dependent MTT-assay of P. aurea crude fractions..................................... 50
Figure 13. Time-dependent MTT-assay of P. aurea crude fraction. ..................................... 51
Figure 14. Multiple cell line dose-dependent MTT-assay of P. aurea crude ethanol extract.
................................................................................................................................................. 52
Figure 15. Dose-dependent MTT-assay of Laetiporus sp. crude extracts. ............................ 53
Figure 16. Time-dependent MTT-assay of crude extracts isolated from Laetiporus sp. ....... 54
Figure 17. Time-dependent MTT-assay of E1 from Laetiporus sp... .................................... 54
Figure 18. Dose-dependent MTT-assay of crude extracts isolated from L. vulpina. ............. 55
Figure 19. Dose-dependent MTT-assay of crude extract E1 from L. vulpina. ...................... 56
vi

Figure 20. Time-dependent MTT-assay of crude extract E1 from L. vulpina. ...................... 57
Figure 21. Time-dependent MTT-assay of E1 from L. vulpina. ............................................ 57
Figure 22. Dose-dependent MTT-assay of crude extracts isolated from V. canadensis. ....... 58
Figure 23. Time-dependent MTT-assay of E1 from V. canadensis. ...................................... 59
Figure 24. Time-dependent MTT-assay of E1 from V. canadensis. ...................................... 59
Figure 25. ELISA 1 results for crude extracts of H. coralloides. .......................................... 61
Figure 26. ELISA 2 results for crude extracts of H. coralloides. .......................................... 61
Figure 27. ELISA 3 results for crude extracts of H. coralloides. .......................................... 62
Figure 28. ELISA 1 results for crude extracts of H. diabolus. .............................................. 63
Figure 29. ELISA 2 results for crude extracts of H. diabolus. .............................................. 64
Figure 30. ELISA 3 results for crude extracts of H. diabolus. .............................................. 64
Figure 31. ELISA 1 results for crude extracts of P. aurea. ................................................... 65
Figure 32. ELISA 2 results for crude extracts of P. aurea. ................................................... 66
Figure 33. ELISA 3 results for crude extracts of P. aurea. ................................................... 66
Figure 34. ELISA 1 results for crude extracts of Laetiporus sp.. .......................................... 68
Figure 35. ELISA 2 results for crude extracts of Laetiporus sp.. .......................................... 68
Figure 36. ELISA 3 results for crude extracts of Laetiporus sp. ........................................... 69
Figure 37. ELISA 1 results for crude extracts of L. vulpina. ................................................. 70
Figure 38. ELISA 2 results for crude extracts of L. vulpina. ................................................. 70
Figure 39. ELISA 3 results for crude extracts of L. vulpina. ................................................. 71
Figure 40. ELISA 1 results for crude extracts of V. canadensis. ........................................... 72
Figure 41. ELISA 2 results for crude extracts of V. canadensis. ........................................... 72

vii

Figure 42. ELISA 3 results for crude extracts of V. canadensis. Raw 264.7 cells were plated
at a density of 100,000 cells/well. ........................................................................................... 73
Figure 43. ELISA of post-Sephadex LH20 fractions from the first small column run.......... 91
Figure 44. ELISA of post-Sephadex LH20 fractions, comparing small column runs 1 and 2.
................................................................................................................................................. 92
Figure 45. MTT assay of post-Sephadex LH-20 small column run #3.................................. 92
Figure 46. MTT assay of post-Sephadex LH-20 large column runs 1 and 2. ........................ 93
Figure 47. ELISA of post-Sephadex LH20 fractions, comparing large column runs 1 and 2.
................................................................................................................................................. 94
Figure 48. Total Carbohydrate Assay Kit results for large column Sephadex LH-20 runs 1
and 2. ....................................................................................................................................... 95
Figure 49. Pierce BCA Protein Assay Kit results for Sephadex LH-20 large column runs 1
and 2. ....................................................................................................................................... 96
Figure 50. Effects of heat on immuno-stimulatory activities seen in post-Sephadex LH-20
fractions................................................................................................................................... 96
Figure 51. ELISA of precipitates from E3 purification: comparing Sevag method with
ethanol precipitation................................................................................................................ 98
Figure 52. Concentration-dependent ELISA of precipitates from E3 purification: comparing
Sevag method with ethanol precipitation. ............................................................................... 98
Figure 53. ELISA of post-DEAE Sephadex A50 collections, comparing the effectiveness of
different buffer systems. ....................................................................................................... 100
Figure 54. ELISA of post-DEAE Sephadex A50 using water as a buffer system. .............. 101
Figure 55. ELISA of post-DEAE Sephadex A50 using water as a buffer system. .............. 102

viii

Figure 56. ELISA of fractions collected from Sephacryl S500 HR column chromatography.
............................................................................................................................................... 103
Figure 57. TNF-α production of the first Sephacryl S500 HR column run in relation to
carbohydrate and protein content. ......................................................................................... 104
Figure 58. ELISA of post-Sephacryl S500 HR column chromatography fractions, with NaCl
wash data, for the second column run. .................................................................................. 105
Figure 59. TNF-α production of the second Sephacryl S500 HR column run in relation to
carbohydrate and protein content. ......................................................................................... 105
Figure 60. TNF-α production of the second Sephacryl S500 HR column NaCl wash in
relation to carbohydrate and protein content. ....................................................................... 106
Figure 61. TNF-α production of the third Sephacryl S500 HR column run in relation to
carbohydrate and protein content. ......................................................................................... 107
Figure 62. TNF-α production of the third Sephacryl s500 HR column NaCl wash in relation
to carbohydrate and protein content. ..................................................................................... 107
Figure 63. TNF-α production of the fourth Sephacryl S500 HR column run. ..................... 108
Figure 64. TNF-α production of the fourth Sephacryl S500 HR column NaCl wash. ........ 109
Figure 65. Effect of proteinase K, cellulose and amyloglucosidase on the immunostimulatory activity of PSK. .................................................................................................. 110
Figure 66. Effect of cellulase, amyloglucosidase and proteinase-K on the immunostimulatory activity of bioactive compound(s) from P. aurea. ............................................. 111
Figure 67. Effect of cellulase, amyloglucosidase and proteinase-K, on the immunostimulatory activity of PSK.. ................................................................................................. 112

ix

Figure 68. Effect of cellulase or amyloglucosidase on the immuno-stimulatory activity of
PSK. ...................................................................................................................................... 113
Figure 69. Effect of cellulase, amyloglucosidase and proteinase-K on the immunostimulatory compound(s) from P. aurea. .............................................................................. 114
Figure 70. Effect of cellulase or amyloglucosidase on the immuno-stimulatory compound(s)
from P. aurea. ........................................................................................................................ 115

x

Acknowledgements
I would like to thank my co-supervisors, Dr. Chow Lee and Dr. Kerry Reimer, for
giving me the opportunity to participate in the Mushroom Project and pursue a MSc degree.
To my committee members, Dr. Keith Egger and Dr. Hugues Massicotte, your “tips” on
where to locate specimens and assistance in identifying them were greatly appreciated.
Thank you to Dr. Maggie Li for performing additional DNA analysis on my collected
samples and for your recommendations that helped to progress my research. I would also like
to thank Aaron Smith for his pioneering of this project and support in keeping me on track.
Thank you as well to Dr. Andrea Gorrell, whose advice was greatly appreciated in my pursuit
of column chromatography. I would also like to thank Sebastian Mackedenski for his
assistance in running the AKTA Pure system, and the much-needed coffee breaks,
discussion, and support during this study. Lastly, I am very grateful to my friends and family
for their unwavering belief and support throughout my degree. This project would not have
been possible without you.

xi

Chapter 1: Introduction
1.1 A History of Mushrooms in Medicine
Fungi comprise the second largest kingdom of eukaryotic life on this planet1. Only
10% of the estimated 140,000 species are actually known2–4. Being rich sources of proteins,
carbohydrates, amino acids, fatty acids, sterols, terpenoids, flavonoid compounds, vitamins
and minerals, mushrooms have been used as food and medicine since the earliest
civilizations2,5,6. Some of the compounds that mushrooms produce to defend themselves from
predation and infection are what make them useful as medicine7. As well, structural
compounds and other secondary metabolites have shown beneficial activities. The high
diversity of fungi and the wide range of potential defensive interactions allow for a great
array of biological responses8. As seen in Table 1, mushrooms have been used by traditional
healers to treat a variety of ailments.
The use of mushrooms medicinally can be tracked throughout history, and through a
variety of cultures2. Earliest records of mushroom use come from ancient Egypt, where
poems devoted to fungi were found in temples2. It was determined that mushrooms were
reserved only for royalty, and no one of the lower classes could consume them without harsh
penalty2. Greeks and Romans continued the use of fungi medicinally, importing many of
them from Libya2. The Greek physician Hippocrates (450 BC) was said to have used Fomes
fomentarius as an anti-inflammatory agent, and to aid in cauterizing wounds7. Even the Ice
Man, a naturally preserved mummy from the Alps, was found with a type of birch polypore
bound to his clothing, indicating that mushrooms were valued as early as 5300 years ago7,9.
While some cultures, such as those from China and Japan, have maintained documented
records of the mushrooms they use, other cultures rely on oral communications to pass on
1

knowledge2. Due to this, much knowledge of traditional mushroom medicine is not directly
available to researchers, having been lost with the passing of cultural elders2.

Table 1. Traditional uses of medicinal mushrooms.
Scientific Name

Medicinal Use

Calvatia
cyathiformis

Common
Name
Purple-Spored
Puffball

Leucorrhea, infertility, and hiccups

2,10

Calvatia species

Puff Balls

Wound Healing

2,7

Cordyceps
militaris

Dong Chong
Xia Cao

Chronic cough, asthma, hemoptysis in phthisis,
impotence, and seminal emission

11,12

Daldinia
concentrica

Coal Fungus

Upset stomach, ulcers, skin disease, whooping
cough, and prevention of excessive growth of fetus

2,10

Fomes
fomentarius

Tinder Fungus

Anti-inflammatory, and cauterizing wounds

Ganoderma
applanatum

Artist’s Conk

Antioxidant, reducing blood sugar levels, and
antihypertensive

Ganoderma
lucidum

Reshi

Chronic bronchitis, hepatitis, hypertension,
hypercholesterolemia, immunological disorders,
arthritis, neoplasia, and cancers

Ganoderma
resinaceum

Laquered
Bracket

Reduce blood sugar, and protect liver

2,10

Grifola frondosa

Maitake

Spleen and stomach ailments, hemorrhoids, and to
calm nerves and mind

15,16

Inonotus obliquus

Chaga

Worms, tuberculosis, diabetes, gastrointestinal
cancer, cardiovascular disease, liver disease,
stomach ailments, and as an internal cleansing agent

17,18

Lasiosphaera
fenzlii

Ma Bo

Sore throat, cough and hoarseness, epistaxis and
traumatic bleeding

11,12

Lentinus edodes

Shiitake

Tumors, influenza, heart diseases, high blood
pressure, obesity, diabetes, liver ailments,
respiratory diseases, exhaustion, weakness, and
problems related to sexual dysfunction and ageing

19

Lentinus
squarrosulus

-

Mumps and heart disease

2

References

7

2,10

2,10,13,14

2,10

Lignosus
rhinocerus

Tiger Milk
Mushroom

Breast cancer, fever, cough, asthma, food
poisoning, liver cancer, chronic hepatitis, and
gastric ulcers

20

Phellinus linteus

Black Hoof
Mushroom

Detoxification, hepatoprotection, allergies, diabetes,
oral ulcers, gastric disorders, lymphatic disease, and
to improve blood circulation

21

Pleurotus tuberrigium

King Tuber
Mushroom

Headache, cold, fever, stomach ache, and
constipation

Termitomyces
microcarpus

-

Gonorrhea, health promotor and induction of
lactation

2,10,22

Termitomyces
titanicus

-

Abdominal pain, stomach ache, ulcers, and
constipation

2,22

2,10

Advances in biochemical techniques have allowed for the isolation and purification of
chemical compounds. This has led to the discovery of over 150 novel enzymes, as well as
various other compounds from mushroom sources7,23. Isolated compounds can elicit an array
of bioactivities, including antioxidant, antimicrobial, antitumor, immunomodulatory,
antiviral, antihyperglycemic, anti-inflammatory, cardiovascular-protective, antidiabetic, and
hepatoprotective activities2,6,13. Due to these activities, and their low toxicity, mushroom
products have attracted the attention of researchers in the field of drug discovery and
development13,23,24. Since Alexander Fleming discovered penicillin, the first β-lactam
antibiotic, from Penicillium notatum in 1928, fungi have garnered much attention from
pharmaceutical companies as a source of novel bioactive compounds24. Due to this, many
beneficial drugs have been discovered, allowing for advances in medical practices. For
example, cyclosporine, an immunosuppressive drug discovered in the soil fungus
Tolypocladium inflatum, has significantly increased survival rates in organ transplant patients
by preventing rejection24,25. The isolation of statin compounds mevastatin (1976) and
lovastatin (1986) from Penicillium citrinum and Monascus ruber have led to the production
3

of synthetic analogue drugs such as Lipitor®, Crestor®, and Livalo®, some of the most potent
cholesterol-lowering agents on the market today24,26,27. Even pets have seen a benefit from
fungus-derived drugs with the discovery of insecticidal nodulisporic acid from
Nodulisporium sp., used as an effective flea treatment in cats and dogs24,28,29. In addition to
being sources of novel compounds, fungi have also been utilized as bio-factories for the
synthesis of a variety of other compounds. They are effectively used for the
biotransformation of steroidal molecules commonly used in pharmaceutical practices24.
Many basidiomycetes have been shown to contain biologically active polysaccharides30.
1.2 Anticancer Activity of Medicinal Mushrooms
Anticancer activity was first documented in mushrooms by Lucas et al. in 1957 when
treating sarcoma-180 in mice using an extract from the fruiting body of Boletus edulis3,30.
From this time forward, anticancer activity has been investigated in a variety of medicinal
mushrooms (Table 2). Anticancer activity of mushroom-derived compounds has been linked
to immunomodulatory and/or direct cytotoxic effects30–32. Isolated compounds with these
activities include polysaccharides, polysaccharide-protein complexes, proteins, terpenes,
lipids, and phenols32. Studies have shown that the immunomodulatory effects arise from the
stimulation and activation of various immune cells, leading to cytokine release and
downstream effects32. The most commonly studied, and marketed, immuno-stimulatory
compounds found in mushrooms are β-glucans33. As they are a major component of fungal
cell walls, it is believed that glucans are recognition molecules for fungal infections utilized
by the immune system and, as such, are the targets of immune cell receptors such as
complement receptor 3, dectin-1, and toll like receptors TLR-2, TLR-4, and TLR-6 32–35.
However, the type and effectiveness of immune response is linked to the complexity of the β4

glucan molecule, with some of the more complex polysaccharides showing more potent
activity33. Some of the best characterized anticancer β-glucans derived from mushrooms are
polysaccharide K (PSK), lentinan, and schizophyllan23,30,33.
Polysaccharide-K
Polysaccharide-K, also known as Krestin, is a protein-bound β-glucan derived from
Trametes versicolor. The polysaccharide component consists of a (1→3)-β-glycan backbone
with (1→6)-β-glycosidic side chains36,37. Anticancer activity of T. versicolor was first
identified in 1965 when the remission of cancer was observed following regular consumption
of this mushroom38. In-vitro trials showed the ability of PSK to inhibit tumor cell
proliferation, induce apoptosis, and regulate the immune system36,38–40. Clinical trials began
around 1970 and were found to significantly increase survival in patients with stomach,
breast, colorectal, and esophageal cancers when used as an adjuvant to radiotherapy or
chemotherapy41. PSK is now commercially produced in Japan for use as an adjuvant therapy
in the treatment of breast, liver, prostate, lung, stomach, esophagus, nasopharynx, rectum,
and colon cancer30,38. It has also been used in veterinary medicine to treat a wide array of
animal conditions, including fibrosarcoma, adenosarcoma, mastocytoma, plasmacytoma,
melanoma, sarcoma, and carcinoma30. This compound is orally available, spreading to the
salivary glands, brain, liver, bone marrow, pancreas, and tumors within 24 hours of
consumption38,40,42. Research has also identified the ability of PSK to activate dendritic cells
and suggests that it may be useful as a vaccine adjuvant43. The immunomodulatory activity
of PSK is suggested to involve TLR-4, where it is believed to act as a ligand to induce TNF-α
and IL-6 inflammatory cytokines34. However, due to its broad range of activities, it is
believed to activate multiple immunological pathways to achieve its anticancer effects44,45.
5

Lentinan
Lentinan is obtained from the hot water extract of Lentinus edodes (Shiitake)31. It is a
β-glucan, consisting of a (1→3)-β-glycan main chain with two (1→6)-β-D- glucopyranoside
branches for every 5 glucose units of the backbone37,40,46. Anticancer activity was first
documented in 1970, when researchers were able to inhibit the growth of sarcoma-180 tumor
cells in mice31. Additional animal studies have identified its antitumor, metastasispreventative, and oncogenesis-preventative activities in cancers induced by chemical and
viral agents23. Approved for pharmaceutical use in the 1980’s, lentinan was shown to
clinically increase the survival of patients suffering from advanced gastric and colorectal
cancers, and recurrent ovarian cancer when used as an adjuvant to conventional
therapies23,31,47. Patients were observed to have an overall prolonged life, regression of
tumors, and improved immune response31. By reducing debilitating symptoms such as
nausea, pain and hair loss, lentinan used as an adjuvant therapy has been shown to improve
the quality of life of patients undergoing chemotherapy. It is currently used in Japan and
China for that purpose23. It is believed that lentinan binds to scavenger receptors on myeloid
cells33,48.
Schizophyllan
Schizophyllan (SPG), also known as Sizofiran, is a water-soluble exopolysaccharide
derived from the fruiting body of Schizophyllum commune. It consists of a β-(1→3)-glucose
backbone with single β-(1→6)-glucose side chains attached at approximately every third
glucose backbone unit49, and is known to form a triple helical structure in water50. Its first
documented anticancer activity was noted in 1969, when it was found to inhibit sarcoma-180
in mice. Studies show that SPG’s antitumor activity is largely due to stimulation of a host6

mediated immune response49,51, and is believed to be T-cell mediated52. A stimulatory effect
on the production of IL-1, IL-2, IL-3 and IFN-γ was seen following treatment with SPG53,54.
SPG started clinical trials in 1983, where it was found to increase the survival of patients
with gastric, head and neck cancers55,56. As an adjuvant to radio- and chemotherapy, SPG
was shown to increase the survival and time to recurrence of stage 2, but not stage 3, cervical
cancer23,49. SPG is currently produced and marketed as an immunopotentiator in Japan, and
current research is investigating its potential use as a delivery vehicle in vaccines49,57.
1.2.1 Phellinus linteus
Phellinus linteus has been studied for its anticancer activity via in-vitro and animal
models. This fungus is a well-known oriental medicine, found to contain polysaccharides,
protein complexes, and low molecular weight complexes58. Crude extracts can elicit
immunomodulatory, as well as direct antitumor activity through apoptosis58. Phellinus linteus
shows promise in the treatment of lung cancer59, invasive breast cancer60, benign prostatic
hyperplasia61, and colon adenocarcinoma62; it is also able to inhibit tumor growth in mice
inoculated with sarcoma-180 tumor cells63. Interestingly, a primarily polysaccharide extract
of P. linteus has been seen to have a synergistic effect when used in conjunction with
doxorubicin (a chemotherapeutic), sensitizing prostate cancer cells to the cytotoxic effects of
this drug, activating caspase cascade leading to apoptosis64. A similar synergistic effect was
seen when used with camptothecin (a topoisomerase inhibitor-based drug) in colon cancer
cells65. An isolated phenol compound, Hispolon, has been shown to inhibit the growth of
nasopharyngeal carcinoma cells, primarily through caspase activation leading to apoptosis
through the ERK1/2, JNK1/2 and p38 MAPK patway66. As many studies still involve

7

mixtures of compounds from this mushroom, further identification and purification of those
responsible for the observed activities is required to allow for progression into clinical trials.
1.2.2 Ganoderma lucidum
Another well-known Chinese medicinal mushroom is Ganoderma lucidum. Extracts
of this mushroom are found to possess anticancer, anti-inflammatory, and antioxidant
activity. The polysaccharide extract is known to have immunomodulating properties, while
the triterpene fractions possess cytotoxic activity67. In-vivo studies show promise in the
treatment of breast cancer67,68, colitis-associated cancer69, metastatic lung cancer70, and
colorectal cancer71. Purification of select triterpenes has been performed70,72, however,
further studies of the purified compounds are required before they are moved on to clinical
trials. Immunomodulatory properties are believed to involve TLR-433, while cytotoxic
activity is believed to be mediated by the mitochondria70.
1.2.3 Other Anti-cancerous Mushrooms
Many more mushrooms have been found to contain anti-cancerous compounds and
these remain in various stages of study (please refer to Table 2). These include some wellknown traditionally used species such as Ganoderma lucidum, Inonotus obliquus, Lentinus
edodes, Trametes versicolor, and more. With the increasing knowledge these studies yield,
one must wonder how many novel compounds must exist in the unstudied species and what
their potential may be.

8

Table 2. Some medicinal mushrooms showing anticancer activity.
Scientific
Name
Amauroderma
rude

Common
Name
-

Cancer/Cell Line

Activity

Breast Cancer (MT-1, MDA-MB231,
4T1, MDA-MB468, MCF7)

Antitumor
Antimetastatic
Pro-apoptotic
Anti-proliferative

13

Ganoderma
lucidum

Reishi

Breast cancers (MDA-MD231, SUM149)

Cytotoxic
Antimetastatic
Antitumor
Anti-proliferative
Immunomodulatory

67

Inonotus
obliquus

Chaga

Sarcoma-198
Human Colon Cancer (HT-29)
Lung Carcinoma (A-549)
Stomach Adenocarcinoma (AGS)
Breast Adenocarcinoma (MCF-7)
Cervical Adenocarcinoma (HeLa)
Hepatoma (HepG2, Hep3B)

Cytotoxic
Pro-apoptotic
Anti-proliferative

73,74

Lentinus
edodes

Shiitake

Sarcoma-180
Bowel Cancer
Liver Cancer
Stomach Cancer
Ovarian Cancer
Lung Cancer

Antitumor
Immunomodulatory
Antimetastatic
Anti-proliferative

30,31,75

Lignosus
rhinocerus

Tiger Milk
Mushroom

Human Breast Carcinoma (MCF-7)
Human Lung Carcinoma (A549)

Cytotoxic
Pro-apoptotic
Anti-proliferative

20

Panellus
serotinus

Late Fall
Oyster
Mushroom

Sarcoma-180
Colon Cancer (HT-29)
Mouse Embryonic Fibroblast
(NIH3T3)
Murine Macrophage (RAW 264.7)

Antitumor
Immunomodulatory

76

Phellinus
linteus

Black Hoof
Mushroom

Lung Cancer (LNCaP)
Breast Cancer (MDA-MB231)
Neuroblastoma (SK-N-MC)
Prostate Cancer (LNCaP)
Melanoma (B16BL6)
Benign Prostatic Hyperplasia

Immunomodulatory
Pro-apoptotic
Antiangiogenic
Antitumor
Antimetastatic
Anti-proliferative

58–61

Poria cocos

Fu Ling

Pancreatic Cancers (Panc-1, MiaPaca2, AsPc-1, BXPc-3)

Cytotoxic
Anti-invasive
Pro-apoptotic
Anti-proliferative

9

References

77

Schizophyllum
commune

Split Gill

Sarcoma-180
Carcinomas (MM-46, MH-134)
Bladder Tumor (BC-47)
Fibrosarcoma (AMC-60)
Mammary Carcinoma (A-755)
Sarcoma-37
Erlich Sarcoma
Lewis lung Carcinoma
Yoshida Sarcoma
Stomach Cancer
Neck Cancer

Immunomodulatory
Antitumor

30,49,75

Trametes
versicolor

Turkey Tail

Breast Cancer
Liver Cancer
Prostate Cancer
Lung Cancer
Stomach Cancer
Colon Cancer

Antitumor
Immunomodulatory
Cytotoxic
Pro-Apoptotic
Anti-proliferative

30,43,75

1.3 Project Goals
Mushrooms represent a major and largely untapped source of potentially powerful
new pharmaceutical natural products. The fact that there are limited studies on wild
mushrooms native to Canada, and the knowledge that many compounds derive from specific
interactions between fungi and their host species, leads to the hypothesis that novel bioactive
compounds can be found in British Columbia (BC) wild mushrooms. To this end, I set out to
study some BC wild mushrooms with the goal of finding a novel compound with growthinhibitory or immunomodulatory activity.
The overall goals of this study can be split into two experimental chapters. The first
experimental chapter (Chapter 2) describes the collection and identification of 4 mushroom
and 2 lichen species from north-central BC. This was followed by chemical extraction and
assessment of their crude extracts for growth-inhibitory and immunomodulatory activities.
The second experimental chapter (Chapter 3) describes the purification and characterization
of an immuno-stimulatory compound from the hot water extract of Phaeolepiota aurea.

10

Chapter 2: Collection, Identification, and Screening of British Columbian Wild
Mushrooms and Lichens for Anti-Proliferative and Immuno-Stimulatory Activities
2.1 Introduction
As indicated in the previous chapter, mushrooms contain a great variety of bioactive
compounds with many medicinal functions8. Some of these have made their way into
pharmaceutical practices, becoming the basis of many analogue drugs marketed today24.
However, due to the sheer number of estimated species and lack of study, many mushroom
species have not been characterized for their bioactive potential4. The majority of medicinal
mushroom research has been occurring in European and Asian countries, where a history of
traditional used has fueled interest in natural based remedies8,23. This interest has only started
to take root in western cultures and, as such, there have been few studies focusing on
medicinal mushrooms in North America23.
In this chapter, I report on the identification, extraction, and bioactive screening of
some British Columbia (BC) wild mushrooms and lichens for anti-cancerous effects:
specifically, anti-proliferation and immuno-stimulation. Lichens and fruiting bodies of
multiple fungal species were collected from their natural habitats throughout the BC interior
during the Fall 2013 – Summer 2014 seasons (Table 4). Of these, 6 species (four fungi and
two lichens) were selected for further identification and screening based upon available
biomass and lack of previous study within the laboratory and in the general literature. Herein
I describe the methods used in the identification, extraction, and screening of the selected
species. I also propose which species warrant further investigation.

11

2.2 Methods
2.2.1 Sample Collection and Identification
Four mushroom and two lichen species were collected from throughout the BC
interior (Table 4). Mushroom samples were tentatively identified morphologically using
David Arora’s field guide, Mushrooms Demystified78, and Ian Gibson’s identification
software, MatchMaker79. Lichen samples were morphologically identified using McCune’s
guide, Macrolichens of the Pacific Northwest80. Additionally, samples were genetically
identified using a PowerSoil DNA isolation kit (Mo Bio Laboratories, Carlsbad, USA), and
fungal specific primers ITS3 and NLB4. These primers were selected as the internal
transcribed spacer (ITS) region of ribosomal DNA has shown a high probability for
successful identification in a large range of fungi1.
A small sample of tissue was cut from the internal flesh of the cap when the fresh
mushroom was available. Fresh samples were stored at freezing temperatures (-20℃) until
DNA extraction was performed, using the manufacturer protocols81. Powdered mushroom
was also used from samples that had previously been dried. Approximately 0.25 grams of
mushroom sample was loaded into the PowerBead Tubes, provided with the DNA isolation
kit, and vortexed to mix. The provided Solution C1 was heated to 60℃ to dissolve
precipitates, and 60 µL of this solution was added to each of the sample tubes, once again
vortexing to disperse the sample within the solution. Samples were shaken for approximately
10 minutes, to break down the tissue samples, before the tubes were loaded into a centrifuge
and spun down at 10,000 RCF. The supernatant solution was then transferred to a clean 2 mL
Collection Tube. Two hundred and fifty microliters of Solution C2 was then added to the
supernatant solution and vortexed for 5 seconds. The samples were then incubated at 4℃ for
12

5 minutes to precipitate out the non-DNA materials. Sample tubes were then centrifuged at
10,000 RCF for one minute, to pellet the non-DNA materials. The supernatant solution was
drawn off, avoiding disruption of the pellet, and transferred again to a clean 2 mL Collection
Tube. Two hundred microliters of Solution C3 was then added to the supernatant solution,
and vortexed to mix. This solution was once again incubated at 4℃ for 5 minutes, then
centrifuged at 10,000 RCF to pellet out any additional non-DNA material. The supernatant
was drawn off and transferred to another clean 2 mL Collection Tube, avoiding the transfer
of any pelleted material. Solution C4 was shaken to mix, and 1.2 mL was added to the
sample tubes, vortexing for 5 seconds to mix. This solution was then loaded stepwise onto a
Spin Filter, 675 µL at a time, and centrifuged at 10,000 RCF for 1 minute, discarding the
flow through, until all of the solution had been filtered through. Five hundred microliters of
Solution C5 was then loaded onto the Spin Filters and centrifuged through at 10,000 RCF for
30 seconds. The flow through was discarded. Spin filters were once again centrifuged at
10,000 RCF for 1 minute to remove residual solution. The spin filters were then transferred
to a clean 2 mL Collection Tube. One hundred microliters of Solution C6 was then loaded
onto the filter membrane of the spin columns and centrifuged at 10,000 RCF for 30 seconds.
The spin filters were discarded and the flow through reserved as DNA solution81.
To determine the presence of DNA in the isolated samples, 5 µL of each isolated
sample was mixed with 2 µL of blue loading dye and run on a 1% agarose gel with a DNA
ladder. Once the samples had run for a sufficient amount of time to allow for separation,
approximately 45 minutes to 1 hour, the gel was photographed under UV and DNA bands
were identified. One microliter of the isolated DNA for each sample was mixed with 29 µL
of a provided PCR master mix containing fungal specific primers ITS3 and NLB4. These
13

samples were then thermo-cycled using the program seen in Table 3. Effectiveness of PCR
was determined with another 1% agarose gel run of the product (Figure 3). The product of
PCR was then cleaned using ethanol precipitation and sent for sequencing at the UNBC DNA
sequencing facility or the Macrogen Corporation (Maryland, USA). The obtained DNA
sequence chromatographs were loaded into sequencing software Chromas Lite82 and edited
to remove unclear segments and correct miscalled bases. The cleaned-up sequences were
then saved in FASTA format and loaded into NCBI’s BLAST83 to search for highly similar
sequences (megablast). Uncultured and environmental samples were excluded from the
search parameters.
Table 3. Thermocycler program for PCR of isolated DNA samples.
Step #
1
2
3
4
5
6
7

Temperature
95℃
95℃
52℃
72℃
Repeat Steps 2-4
72℃
4℃

Duration
5 minutes
1 minute
1 minute
1 minute
29 repetitions
5 minutes
Holding

2.2.2 Chemical Extraction
Medicinal mushroom compounds have traditionally been isolated with the use of hot
water extraction84. While it is a simple procedure, this method may lack the ability to extract
potentially active water-insoluble compounds. Multi-step extraction procedures have been
developed to target a broader range of compounds. The principle behind these extractions is
to break down the fungal cell wall, from outer to inner layers, using mild to increasingly
stronger extraction conditions84. Mizuno developed a standard four-step extraction procedure
that has been deemed by many to be reliable, and is often modified to suit a specific
extraction target84,85. The extraction procedure used in this thesis is based on a modification
14

of Mizuno’s procedure85, further optimized in Dr. Lee’s lab. Mushroom specimens were
dried at 50℃ in an oven, and then pulverized into a powder using a household blender. This
powder was then used for chemical extraction. Stepwise extraction proceeded either
manually (Figure 1), or with the use of a speed extractor (Figure 2).
As described in Figure 1, the first stage of manual extraction procedures involved
sample extraction with 80% ethanol. Powdered mushroom or lichen was loaded into a
suitable sized flask (depending on scale of extraction) and an excess of 80% ethanol solution
added. The flask was heated to 65℃ and held at this temperature for 3 hours with continuous
stirring. At the end of the 3-hour ethanol extraction, the solution was suction-filtered through
Whatman no. 2 or no. 3 filters, depending on the viscosity of the solution. The filtrate would
then be rotary-evaporated and lyophilized. This would become Extract 1 (E1) and would be
presumed to contain small molecules. The residue from ethanol extraction was transferred to
a flask, and a 50% methanol extraction was then performed. Once again, the flask was heated
to 65℃ for 3 hours with stirring. The solution was then filtered, the filtrate rotary-evaporated
and lyophilized to become Extract 2 (E2). Once again, this fraction would be considered to
contain low molecular weight compounds. The residue from stage 2 extraction was
transferred to a clean flask and topped with sterile water. The flask was heated to 65℃ for 6
hours with stirring. The solution was filtered, the filtrate rotary-evaporated and lyophilized as
Extract 3 (E3). This fraction would be considered to contain water-soluble polysaccharides.
The residue was transferred once more to a clean flask, and a 5% sodium hydroxide solution
was applied as the fourth and final step of chemical extraction. The flask was heated to 65℃
for 6 hours with stirring, before filtering the solution and rotary-evaporating the filtrate. The

15

concentrated filtrate would then be lyophilized as Extract 4 (E4). This final fraction would be
considered to contain water-insoluble polysaccharides.

Dried Mushroom Sample (Pulverized)

80% Ethanol

50% Methanol

Distilled
Water

5% Sodium
Hydroxide

65℃ for 3 Hours
Filtered
pH Neutralized
Rotary-Evaporated
Lyophilized
Stored at 4℃

65℃ for 3 Hours
Filtered
pH Neutralized
Rotary-Evaporated
Lyophilized
Stored at 4℃

65℃ for 6 Hours
Filtered
pH Neutralized
Rotary-Evaporated
Lyophilized
Stored at 4℃

65℃ for 6 Hours
Filtered
pH Neutralized
Rotary-Evaporated
Lyophilized
Stored at 4℃

Extract #1

Extract #2

Extract #3

Extract #4

Low Molecular Weight Compounds

Water-Soluble
Polysaccharides

Water-Insoluble
Polysaccharides

Figure 1. Flowchart outlining the manual chemical extraction procedure for obtaining crude extracts from
mushrooms.

Speed extraction used the same stepwise solvent sequence and yielded the same
overall extracts as the manual procedure (Figure 2). Powdered mushroom or lichen was
mixed at a 1:1 ratio with quartz sand to prevent clogging in the extraction cells. Once loaded
onto the machine, the samples were held at a temperature of 65℃ and 100 bar pressure. For
each solvent system, the samples were run through four cycles that consisted of sample
saturation with the solvent and a 15-minute holding phase, followed by a 4-minute discharge
phase where the extracts were filtered into the collection flasks. The collection flasks were

16

drained and cleaned between each solvent system. The collected filtrates were pH
neutralized, rotary-evaporated, if needed, and lyophilized to yield their respective extracts.
Dried Mushroom Sample (Pulverized)
& Quartz Sand (1:1)

85% Ethanol

50% Methanol

Distilled
Water

5% Sodium
Hydroxide

65℃, 100 bar
4 cycles
pH Neutralized
Rotary-Evaporated
Lyophilized
Stored at 4℃

65℃, 100 bar
4 cycles
pH Neutralized
Rotary-Evaporated
Lyophilized
Stored at 4℃

65℃, 100 bar
4 cycles
pH Neutralized
Rotary-Evaporated
Lyophilized
Stored at 4℃

65℃, 100 bar
4 cycles
pH Neutralized
Rotary-Evaporated
Lyophilized
Stored at 4℃

Extract #1

Extract #2

Extract #3

Extract #4

Low Molecular Weight Compounds

Water-Soluble
Polysaccharides

Water-Insoluble
Polysaccharides

Figure 2. Flowchart outlining the speed extractor procedure for obtaining crude extracts from
mushrooms. Each cycle consisted of a 15-minute holding phase followed by a 4-minute discharge
phase.

2.2.3 Biological Assays for Primary Screening
2.2.3.1 Anti-proliferation Assay
Crude extracts were tested for their effects on cell viability against human cervical
cancer, HeLa, cells with the use of MTT 3-(4,5-dimethyiazol-2-yl)-2,5-diphenyl tetrazolium
bromide assay. MTT assay is a colorimetric assay in which the tetrazolium dye, yellow in
colour, is reduced to an insoluble formazan, purple in colour, by mitochondrial reductase in
viable cells86. When dissolved in DMSO, the absorbance of the solution can be read on a
spectrophotometer and the percentage of viable cells determined relative to controls. This is a
17

common in-vitro method used for determining cytotoxic or growth-inhibitory effects against
cells86–89.
HeLa cells were purchased from the American Type Culture Collection (Rockville,
Maryland). Cells were grown in Eagles Minimal Essential Medium (EMEM) supplemented
with 10% fetal bovine serum (FBS), in a 37℃ humidified incubator supplied with 5% carbon
dioxide90. A working stock was maintained in T-25 flasks, splitting when confluent, and not
exceeding 10-15 growth cycles to reduce the chances of mutation/differentiation within the
culture. Working in a sterile Biosafety cabinet, the supernatant growth medium was poured
off. Cells were then washed with 3 mL of phosphate buffered saline (PBS), before drawing it
off and discarding it. One mL of trypsin was then added to the flask and rocked to coat the
bottom. The flask was sealed and placed in a 37℃ incubator for approximately 3 minutes,
until cells had detached, not exceeding 5 minutes. Addition of 9 mL of EMEM+FBS would
then neutralize the trypsin and provide a stock solution of cells for plating. Fifty µL of this
stock solution was aliquoted into an Eppendorf tube, and an additional 50 µL of Trypan blue
dye mixed with it. A drop of this dyed cell solution was applied to a hemocytometer and the
average cell count determined between 4 quadrants. This cell count was used to calculate the
volume of stock cell solution required to prepare a plating solution of desired concentration.
For concentration-dependent assays, a concentration of 3000 cells/well was desired. Cells
were plated at a reduced well concentration of 2000 cells/well for time-dependent assays.
The perimeter wells of a 96 well plate were filled with 200 µL of sterile water. The plating
solution was then transferred to a sterile reservoir and pipetted into all remaining wells using
a multichannel pipettor. The lid was placed on the plate, and it was incubated at 37℃ for 24
hours before treatment with mushroom extracts.
18

Crude mushroom extracts were solubilized in a suitable solvent (water, ethanol, or
methanol) at a concentration of 40 mg/mL. This solution was then filter sterilized into a
sterile Eppendorf tube through a 0.2 µm syringe filter. In a sterile biological cabinet, the
sterile solution was then used to prepare the desired treatment concentrations by dilution in
EMEM+FBS. One-hundred µL of treatment solution was pipetted into each of its designated
wells on the pre-incubated plate. Additionally, 100 µL of solvent control solutions were
treated into wells respectively. Plates were sealed and incubated at 37℃ for 48 hours for
dose-dependent assays, and for up to 7 days for time-dependent assays, treating individual
plates with MTT every 24 hours. In time-dependent assays, day 1 plates were scanned
untreated as the 0-hour starting point.
MTT dye was prepared at a concentration of 5 mg/mL in PBS. This solution was
filter sterilized through a 0.2 µm filter and stored in darkness at refrigerated temperatures
until needed. Working in a sterile biological cabinet, the stock dye solution was diluted at a 1
in 5 mixture with EMEM+FBS. This diluted solution was transferred to a sterile reservoir,
and 50 µL pipetted by multichannel pipettor into each of the treated wells on the incubated
treatment plates. These plates were then incubated at 37℃ for an additional 3 hours before
the supernatant solution was drawn off and discarded. Care was taken as to not scrape the
bottom of the wells with pipette tips, to avoid scraping off any cells/formazan. Two hundred
microliters of DMSO was then pipetted into each of the treatment wells. The plate was then
placed on a Bio-Tek Synergy 2 multi-plate reader, shaken for 5 minutes, and the absorbance
taken at 570nm. Cell viability calculations were then performed in Excel. Significance was
determined with the use of a student’s T-test (p=0.05).

19

2.2.3.2 Immuno-Modulatory Assay
Enzyme linked immunosorbent assay (ELISA) is a commonly used method for
screening immunomodulatory activity. It allows for the detection of specific cytokines
produced in response to bioactive compounds. RAW 264.7 murine macrophage cells were
used to screen for immunomodulatory response in the crude fractions collected from
chemical extraction. Cells were maintained in Dulbecco’s Modified Eagles Medium
(DMEM) supplemented with glutamine and 10% FBS, in a 37℃ incubator supplied with 5%
carbon dioxide, splitting every 2 days to prevent overgrowth and self-activation. In
preparation of treatment, raw cells were plated at a well concentration of 100,000 cells/well
on a 96 well plate. This was done in a similar manner to platting of HeLa cells, but with the
use of TrypLE and serum free DMEM. The supernatant solution was poured off of a T-75
flask of cells. Five milliliters of PBS was then added to the flask and rocked to rinse the cells.
This solution was drawn off and discarded. One and a half milliliters of TrypLE was then
added to the flask and rocked to coat the bottom. The flask was sealed and placed in a 37℃
incubator for 5-9 minutes, checking and striking the sides of the flask to dislodge adherent
cells. Care was taken not to exceed 10 minutes as cells would become active. Once cells were
sufficiently dislodged, 5-7 mL of serum free DMEM was added to the flask and mixed
repeatedly via pipettor until homogenous. This would become the stock cell solution. Thirty
microliters of the stock cell solution was mixed with 30 µL of Trypan Blue dye and applied
to a hemocytometer. The average of four quadrant counts was determined and the volume of
stock solution required for dilution was calculated. Once diluted, 200 µL of the cell solution
was transferred into each well with the use of a multichannel pipette. The plates were then
sealed and incubated at 37℃ overnight. In the morning, the supernatant medium was drawn

20

off of the cells and discarded. Wells were then washed with 300 µL of PBS. This was also
drawn off and discarded. The wells were then topped up with 100 µL of serum free DMEM
to prepare for treatment application. An additional 100 µL of treatment solutions, and their
respective controls, were added to each well, leading to a final well concentration of 1
mg/mL.
Lipopolysaccharide (LPS) was treated at a well concentration of 500 ng/mL as a
positive control, while serum free DMEM was used as a negative control for cell activity.
The plates were sealed and placed in a 37℃ incubator for 6 hours. Following incubation, 180
µL of the supernatant solution was drawn out of wells and transferred to a 96 well v-bottom
plate. The plate was then centrifuged at 200 RCF for 10 minutes to pull down any possible
cellular contaminants. One hundred and fifty microliters of the supernatant was then
transferred to a 96 well plate and labelled. This plate was then sealed with parafilm and
stored in a -80℃ freezer until ELISA was performed.
TNF-α production was assessed with the use of a Mouse TNF (Mono/Mono) ELISA
kit (BD Biosciences, San Diego, CA) and BD OptEIA Set Mouse TNF (BD Biosciences).
ELISA plates were prepared as per the manufacturer protocols. One hundred microliters of
capture antibody was plated onto a 96 well v-bottom plate, at the batch recommended
dilution in coating buffer. The plate was sealed and incubated at 4℃ overnight. The
following morning, wash buffer was prepared at a 1 in 20 dilution. The ELISA plate was then
removed from refrigeration, and the capture antibody solution discarded. The plate was then
washed 3 times with 300 µL of wash buffer, discarding between steps. The plate was then
blocked with 200 µL of assay diluent for 1 hour at room temperature. The diluent was then
discarded and the plate washed 3 times with 300 µL wash buffer. The plate was then treated
21

with 100 µL of cellular supernatant solution diluted in assay diluent, and TNF-α standards.
The plate was then sealed and incubated at room temperature for 2 hours. Following
incubation, the treatment solutions were discarded and the plate washed 5 times with 300 µL
of wash buffer. The working detector, a solution of detection antibody and horseradish
peroxidase diluted in assay diluent at the batch recommended dose, was then applied 100 µL
per well. The plate was then sealed and incubated at room temperature for one hour. After
incubation, the detector solution was discarded and the plate washed 7 times with 300 µL of
wash buffer. Substrate solution was then prepared by mixing solutions A and B at a 1:1 ratio.
This was then applied to the plate, 100 µL/well, and the plates wrapped in aluminum foil to
incubate in the dark for 30 minutes. Following the 30-minute incubation, 50 µL of stop
solution was added to the wells, turning them from blue to yellow. The plates were then
immediately scanned on a Synergy plate reader at 450 nm and 570 nm wavelengths. The
levels of TNF-α were then determined using PRISM 6 log-log regression analysis.
Significance was tested with the use of a student’s T-test (p=0.05).
2.3 Results
2.3.1 Species Identification
All 6 specimens were subjected to morphological and genetic identification. DNA
isolation and PCR amplification of samples 1, 2, and 3 was successful with the use of ITS3
and NLB4 primers. The agarose gel run of PCR products showed single bands of roughly
500 base pairs in size, suggesting that the ITS region of fungal DNA was purified. Sample #3
had the strongest band, suggesting a large amount of DNA had been amplified. Sample #1
had a moderate band, suggesting slightly less DNA was retrieved from the sample. Sample
#2 showed a weaker band, suggesting the sample may have been of lesser quality, yielding
22

less DNA product (Figure 3). DNA analysis for samples 4, 5, and 6 was performed by Dr.
Maggie Li. A summary of identification results can be seen in Table 4.

Figure 3. DNA analysis of PCR products. Five µL of PCR products were run on a 1% agarose gel.
Samples 1, 2, and 3 represent specimen samples #1, #2, and #3 respectively. For the subsequent
identification of these samples, please refer to pages 23-27. Bands can be seen around 500 base pairs,
corresponding to the sequence length expected with fungal specific primers ITS3 and NLB4.

23

2.3.1.1 Sample #1: Hericium coralloides (Scop.: Fr) Pers.

Hericium coralloides found growing on a cottonwood log in Cottonwood Island Park, Prince George,
BC (September 22, 2013).

This sample was collected from Cottonwood Island Park (Prince George, BC) on
September 22, 2013 (see Table 4). This specimen was found growing out of dead cottonwood
logs and stumps. Based on its unique morphology, this mushroom was identified to the genus
Hericium, but conclusive identification of the species remained elusive due to changes in the
classification of similar species. It was finally determined to be either H. americanum or H.
coralloides based on morphology78,79. However, according to Ginns (1984), H. americanum
only occurs in Eastern Canada and it does not occur on Populus; he refers the Western
Canadian species to H. coralloides91.
This specimen’s DNA sequences were observed to have co-amplifying sequences.
The ITS sequence was edited to 500 bases. The NLB sequence was of poor quality and was
thus edited to a sequence length of only 110 bases. A BLAST search was performed, and
both sequences showed a similar hit for Hericium coralloides (Accession: AF5064591.1, %
identity: 97% and 93%, % coverage: 100% and 100%). The NLB sequence also had a hit for
24

Clavulina cinerea (Accession: EU597083.1, % identity: 99%, % coverage: 68%), however
this was rejected due to morphological differences. A second DNA analysis was performed
by Dr. Maggie Li. The obtained sequences were edited to lengths of 506 bases for the ITS
sequence, and 502 bases for the NLB sequence. Both of these sequences showed strong
matches to H. coralloides (Accession: AF506459.1, % identity: 99% and 99%, % coverage:
100% and 94%). The identity of this specimen was concluded to be Hericium coralloides.
2.3.1.2 Sample #2: Hydnellum diabolus Banker

Hydnellum diabolus found growing in West Twin Old Growth trail near McBride, BC. Top left
represents aged specimen (November 5, 2013), while right and bottom show young fruiting bodies
(September 25, 2014).

This sample was collected from West Twin Old Growth Trail (McBride, BC) on
November 5, 2013, based on the suggestion of Dr. Hugues Massicotte. Earlier in the season,
this mushroom was seen to have red droplets collecting on its surface, and it was initially
25

identified as Hydnellum peckii. However, due to its late collection and waterlogged state of
the sample specimens, they lacked defining features, being mostly dark brown in colour with
a matted surface and debris incorporated into the fruiting bodies. A second collection was
performed in the same location on September 25, 2014. The fresher fruiting bodies were
found growing out of moss under damp conditions, in a cedar hemlock old growth forest.
Mushrooms were observed to have a maroon-coloured body, lightening to the outer
extremities, with a velvety white top. Red liquid droplets were observed on the edges and
upper surface. The fruiting bodies were toothed, and grew in a rosette shape, incorporating
debris such as needles, moss, and twigs. The flesh was firm but waterlogged at the time of
collection. Keying in Arora’s field guide led to an identification of H. peckii, however
Hydnellum diabolum (also known as H. diabolus) was also suggested to be nearly identical in
morphology, varying only by having a hairier cap in mature specimens78.
Initially, DNA analysis was performed on the first collection of this specimen. The
obtained ITS sequence was of better quality than the NLB sequence, which appeared to have
a co-amplifying sequence. Following editing, the ITS sequence consisted of 549 bases, and
the NLB sequence 243 bases. The ITS sequence yielded hits for Hydnellum geogenium
(Accession: AY631900.1, % identity: 99%, % coverage: 42%) and Hydnellum aurantiacum
(Accession: AF347113.1, % identity: 98%, % coverage: 83%). The NLB sequence suggested
multiple identities of Sarcodon imbricatus (Accession: FJ845438.1, % identity: 99%, %
coverage: 45%), Phellodon tomentosus (Accession: FJ845424.1, % identity: 99%, %
coverage: 45%), Hydnellum aurantiacum (Accession: KP406543.1, % identity: 99%, %
coverage: 44%), and Hydnellum geogenium (Accession: AY631900.1, % identity: 99%, %
coverage: 44%). Based on morphology, the Sarcodon identity was ruled out, and due to
26

colouring differences, H. aurantiacum and H. geogenium were ruled out. The identity could
not be determined further than genus Hydnellum for the sample. A second DNA analysis was
performed by Dr. Maggie Li, and obtained sequences were edited to lengths of 541 for the
ITS sequence, and 532 for the NLB sequence. Both sequences showed the strongest matches
with Hydnellum diabolus (Accession: AF351863.1, % identity: 99% and 100%, % coverage:
40% and 48%). H. geogenium was second for both sequences (Accession: AY631900.1, %
identity: 99% and 99%, % coverage: 41% and 33%). Due to morphological features at time
of collection, as well as the high BLAST score, the sample was determined to be Hydnellum
diabolus.
2.3.1.3 Sample #3: Phaeolepiota aurea (Matt. ex Fr.) Maire

Phaeolepiota aurea found growing trailside near UNBC, Prince George, BC (October 1, 2014).

This sample was collected from the UNBC campus trails (Prince George, BC) on
November 8, 2013. Fruiting bodies were frozen upon collection due to overnight freezing
temperatures. Samples were observed to have a large cap, golden yellow colour, powdery

27

surface, and a large sheath on the stem. The sample was easily identified as Phaeolepiota
aurea. This was also backed by the expert opinion of Dr. Hugues Massicotte. Additional
collections were made from the same location in the fall seasons of 2014, 2016, and 2017.
DNA sequences from the 2013 collection were edited to 404 bases for the ITS
sequence, and 399 bases for the NLB sequence. Both of the sequences showed top hits for
Phaeolepiota aurea (Accession: AM946522.1, % identity: 98% and 98%, % coverage: 75%
and 66%). DNA analysis of the subsequent collections was also performed, all showing
strong hits for P. aurea with top matches between 98-100% identity (Table 4.). Based on the
overwhelming evidence, this specimen was confidently identified as P. aurea.
2.3.1.4 Sample #4: Laetiporus sp.

Aged mushroom specimen found growing on decaying stump at Twin Falls, near Smithers, BC (May
28, 2014).

This sample was collected from Twin Falls (Smithers, BC) on May 28, 2014. The
specimen was found growing off the side of a rotten stump and appeared to be aged. The
28

fruiting body was dry and had a bone like texture and was white in colour. Due to its
degraded state, an exact identification was difficult. A matchmaker search initially yielded a
hit for Oligoporus obductus79, however this was based on the degraded morphology. Expert
opinion was sought, and the Laetiporus sp. complex was suggested by Dr. Keith Egger and
Dr. Hugues Massicotte. It was believed to be an aged specimen from the previous season’s
growth, having faded in colour and degrading with age and insect predation. Photo
comparison, as well as description of aged specimens in Arora’s field guide78, suggested this
to be a probable identification.
DNA analysis was performed by Dr. Maggie Li. Obtained sequenced were edited to
lengths of 454 bases for the ITS sequence, and 451 bases for the NLB sequence. Both
sequences showed hits for Gibellulopsis nigrescens (Accession: KX359602.1, % identity: 95
and 95%, % coverage: 100% and 100%), however this is a mold, suggesting the sample was
contaminated. A second attempt at DNA analysis yielded similar results. It was determined
that the sample was too degraded to effectively get an accurate DNA identification. Based on
the work of Burdsall and Banik (2001), this is most likely to be L. conifericola as it is the
only Western North American species likely to be found on conifers in this area92.

29

2.3.1.5 Sample #5: Letharia vulpina (L.) Hue

Letharia vulpina growing on dead branches near Beaverdam Lake, Clinton, BC (June 23, 2014).

This sample was collected from Beaverdam Lake (Clinton, BC) on June 23, 2014.
The lichen was found growing on dead conifer branches. Due to its bright yellow-green
colour and tufted appearance, this lichen was easily identified as Letharia vulpina in
McCune’s field guide80.
DNA analysis was performed by Dr. Maggie Li. Obtained sequences were edited to
444 bases for the ITS sequence, and 491 bases for the NLB sequence. Sequences were
observed to have co-amplifying sequences. A BLAST search yielded matches to Letharia
vulpina (Accession: AF228470.1, % identity: 93%, % coverage: 75%) and Letharia sp.
(Accession: EU543560.1, % identity: 95%, % coverage: 67%). A match for Letharia
columbiana (Accession: KU745817.1, % identity: 95%, % coverage: 64%) was also made.
However, based on the morphological features present, the sample identity was more likely
to be Letharia vulpina. It is believed that the co-amplifying sequences may have caused a

30

skew in results. Based on the morphological features, this sample was concluded to be
Letharia vulpina.
2.3.1.6 Sample #6: Vulpicida canadensis (Räs.) J.-E. Mattsson & M. J. Lai

Vulpicida canadensis growing on dead conifer branches near Beaverdam Lake, Clinton, BC (June 23, 2014).

This lichen sample was also collected from Beaverdam Lake (Clinton, BC) on June
23, 2014. It was found growing alongside the Letharia vulpina samples on dead conifer
branches. Due to its distinct morphology, this sample was identified as Vulpicida canadensis
in McCune’s field guide80.
DNA analysis was performed by Dr. Maggie Li. The obtained ITS sequence was
edited to 458 bases, and the NLB sequence was edited to 461 bases. Both sequences showed
strong hits for Vulpicida canadensis (Accession: AF072238.1, % identity: 99% and 99%, %
coverage: 71% and 61%). Based on the agreement between morphological analysis and DNA
analysis, this sample was concluded to be Vulpicida canadensis.

31

Table 4. A summary of fungi collected in British Columbia.

Identity
Hericium
coralloides
Hydnellum
diabolus
Phaeolepiota
aurea
Phaeolepiota
aurea
Phaeolepiota
aurea
Phaeolepiota
aurea
Laetiporus
sp.
Letharia
vulpina
Vulpicida
canadensis

Best GenBank Match
(%Identity/%Coverage)

Sample #

Date

1

Sept 22, 2013

2

Nov 5, 2013

3.1

Nov 8, 2013

3.2

Oct 1, 2014

3.3

Oct 12, 2016

3.4

Oct 13, 2017

Location in BC
Cottonwood Island Park,
Prince George
West Twin Old Growth
Trail, McBride
UNBC Campus Trails,
Prince George
UNBC Campus Trails,
Prince George
UNBC Campus Trails,
Prince George
UNBC Campus Trails,
Prince George

4

May 28, 2014

Twin Falls, Smithers

Sample contaminated

5

June 23, 2014

Beaverdam Lake, Clinton

AF228470.1 (94%/81%)

6

June 23, 2014

Beaverdam Lake, Clinton

AF072238.1 (99%/71%)

AF506459.1 (99%/100%)
AF351863.1 (100%/48%)
AM946522.1 (98%/75%)
DQ071704.2 (100%/52%)
DQ071704.2 (100%/13%)
DQ701704.2 (100%/35%)

2.3.2 Chemical Extraction
Following identification, samples were subjected to multi-stage chemical extraction,
yielding 4 crude extracts for each species. A combination of manual extraction (Figure 1) and
speed extraction (Figure 2) procedures were used depending on the characteristics of the
ground sample. While speed extraction was the quicker approach, select specimens were
observed to clog the apparatus, leading to a shift to manual extraction. The manual
procedures allowed for larger scaled extractions. Table 5 summarizes the results of these
extraction procedures.
2.3.2.1 Hericium coralloides (Scop.: Fr) Pers.
Collected specimens were dried in an oven at 50℃. Upon pulverization, the
powdered sample had the appearance of graham-cracker crumbs, or fine sawdust. A total of
75.20g of powdered sample was obtained. Of this, 32.60 g (50 mL) was mixed with 50 mL of

32

quartz sand and divided equally between 3 extraction cells of the speed extractor. Extraction
with 85% EtOH yielded an orangey-brown solution with a minor light coloured sediment
seen settled on the bottom of the collection vials. This solution had an initial pH of 6.35,
which was then neutralized with dropwise addition of 5% NaOH solution, bringing the final
pH to 7.00. The sample was then rotary-evaporated, keeping the temperature below 65℃,
and slant frozen. Lyophilization yielded a sticky, toffee-like substance (Extract #1). This
substance was re-dissolved in 15 mL of sterile water, slant frozen, and placed back on the
lyophilizer in an attempt to pull any residual moisture out of the sample. After 2 weeks of
lyophilization, the sample still yielded a sweet-smelling toffee-like substance. The final mass
was measured at 6.5718g for Extract 1 (20.15% yield).
Sequential 50% MeOH extraction of the remaining residue from EtOH extraction
yielded a darker brown, almost coffee-like, solution with a light coloured sediment. The
initial pH of this solution was 6.96, which was considered close enough to neutral, and
adjustment was not needed. The sample was rotary-evaporated, and slant frozen at -80℃
before being lyophilized. Lyophilized sample yielded dark sticky crystals with a sweet smell.
This extraction yielded 3.5075g of Extract 2 (10.76% yield).
The hot water extraction also yielded a brown tone solution. Initial pH was 6.04,
which was adjusted to pH 6.97 with the addition of 5% NaOH. The sample was then rotaryevaporated, slant frozen, and lyophilized. Dried sample was observed as fine brown coloured
crystals. A total of 1.4599 g was obtained for Extract 3 (4.48% yield).
The final extraction with 5% NaOH produced a small amount of brown coloured
solution. This had an initial pH of 7.45, which was finally adjusted to 7.06. Due to the small
amount collected, this sample was directly slant frozen and lyophilized. Post lyophilization,
33

the sample was light brown and had a fluffy texture. A second manual extraction was
performed on the residue of this extraction, to try and improve yield. The mushroom sand
mixture was combined with 800 mL of 5% NaOH and heated to 65℃ for 6 hours. The
sample was cooled and filtered through no.2 filter paper. Upon neutralization, the solution
was observed to precipitate. The sample was then rotary-evaporated, slant frozen and
lyophilized. Extract 4 had a final mass of 60.64 g (186.01% yield), suggesting that the
neutralization had added a significant amount of salt to the sample.
2.3.2.2 Hydnellum diabolus Banker
Dried samples were cleaned of as much debris as possible, however due to the nature
of the fruiting bodies, it was not possible to remove all debris. Specimens were ground to a
fine dark dust, with a total of 203.37 g of powdered mushroom obtained from the first
collection. A 38.13g portion was mixed with 50 mL quartz sand and loaded into 2 extraction
cells for speed extraction. A dark burgundy solution was obtained from 85% EtOH
extraction. This solution had a pH of 6.71, which was neutralized to 7.02 with the addition of
NaOH. The sample was rotary-evaporated, slant frozen, and lyophilized. Dried sample had
the appearance of dark crystals. A total of 7.1589 g of sample was obtained for Extract 1
(18.77% yield).
Methanol extraction yielded a dark maroon coloured solution with a pH of 7.12. This
was adjusted to 7.03 with the addition of HCl. Following concentration and lyophilization, a
mass of 2.9824 g was obtained for Extract 2 (7.82% yield).
Hot water extraction produced a dark solution. The initial pH was 7.18, which was
adjusted to pH 7.04 with the addition of HCl. The sample was rotary-evaporated, slant frozen
and lyophilized. Extract 3 yielded a recovery of 0.4848 g (1.27% yield).
34

Sodium hydroxide extraction was performed manually using the residue retrieved
from the extraction cells of the speed extractor. The mushroom and sand mixture was topped
up with 800 mL of 5% NaOH and heated to 65℃ with stirring for 6 hours. After 2 hours, it
was observed that the sample had significantly thickened. It appeared that the mushroom
particles had swelled in the solvent and appeared as gelatinous chunks in the solution. By the
3.5-hour mark, the solution had thickened enough to hold the sand in suspension. After 6
hours, the solution was removed from heat and allowed to cool overnight. Sample was then
suction filtered through no. 2 filter paper, however due to the sludgy nature of the sample, the
filter paper had to be changed multiple times during filtration. The solution was highly basic
and it was not possible to get a steady read on the pH probe at the time. The sample was
neutralized with the addition of HCl. Neutralized solution was then rotary-evaporated, slant
frozen and lyophilized. A total of 27.8070 g of dried Extract 4 was obtained (72.93% yield).
It is believed the accumulation of NaCl from pH neutralization has led to a falsely high yield.
2.3.2.3 Phaeolepiota aurea (Matt. ex Fr.) Maire
Upon grinding, the sample ended as a light coloured powder with the lightweight
consistency of fine sawdust. A total of 77.68 g of sample was ground from the first collection
of this species, with excess unprocessed specimens remaining. Fifty milliliters of powdered
mushroom was measured out and weighed at 14.01 g. This was mixed with 50mL of quartz
sand and distributed between 2 extraction cells for speed extraction. The first extract (85%
EtOH) produced a clear golden-yellow solution. The initial pH of this solution was measured
at 5.90. This was adjusted to pH 7.08 with the addition of 5% NaOH. Due to the small mass
of sample run, a second speed extraction series was set up under similar conditions, using
11.97 g of ground mushroom. The retrieved extract was neutralized from pH 6.72 to pH 7.01.
35

These were evaporated, slant frozen and lyophilized. A total mass of 3.0998 g was obtained
for Extract 1 (11.93% yield).
The second extraction with 50% MeOH also yielded a golden-yellow solution,
however this one was opaque, with a light coloured precipitate. Initially the pH measured at
7.43, but this was adjusted to 7.03 with the addition of HCl. The second extract was
neutralized from pH 7.27 to pH 7.04. Neutralized solutions were rotary-evaporated and slant
frozen. The dried samples yielded 6.2593 g of Extract 2 (24.09% yield).
Hot water extraction was performed, yielding a golden yellow solution. Due to the
small mass of sample run, a second speed extraction series was set up under similar
conditions, using 11.97 g of ground mushroom. This yielded additional extracts 1, 2, and 3.
Extract 1 was neutralized from pH 6.72 to pH 7.01, extract 2 from pH 7.27 to pH 7.04, and
extract 3 from pH 7.18 to pH 7.04. These were evaporated, slant frozen and lyophilized. A
combined mass of 2.3955 g was collected for Extract 3 (9.22% yield).
Due to clogging issues on the speed extractor, the 5% NaOH extraction was
performed manually. The residues of the prior speed extractions were combined and topped
up with 800 mL of 5% NaOH solution. This was heated to 65℃ for 6 hours with stirring.
The solution was allowed to cool overnight prior to suction filtration through a no 2. Filter
paper. Upon neutralization with HCl, a precipitate was observed to have formed, most likely
salt from the acid base reaction. The sample was rotary-evaporated, slant frozen and
lyophilized. A final mass of 58.5043 g was obtained for Extract 4 (225.19% yield). The fact
that this is larger than the starting mass suggests that additional compounds were produced,
most likely NaCl during the neutralization step.

36

2.3.2.4 Laetiporus sp.
Dried sample was pulverized, yielding a light powder with a flour like consistency.
Fifty milliliters was measured out and weighed at 17.42 g. This was mixed with 50 mL of
quartz sand and loaded into the extraction cells of the speed extractor. When 85% EtOH
extraction was performed, the sample was observed to clog the extractor. Due to this, the
residue was retrieved from the extraction cells and a manual extraction was performed. An
additional 32.58 g of powdered mushroom was added to the retrieved residue, bringing the
total mass of mushroom extracted to 50.00 g. This was topped up with 800 mL of 80% EtOH
and heated to 65℃ for 3 hours with stirring. The solution was removed from heat and
allowed to cool overnight. Suction filtration was then performed through no.2 filter paper.
Filtrate was then neutralized with the addition of HCl, from pH 9.74 to pH 7.03. Neutralized
sample was then rotary-evaporated, slant frozen, and lyophilized. A final mass of 0.9995 g
was obtained for Extract 1 (2.00% yield).
The residue retrieved from ethanol extraction was topped up with 800 mL of 50%
MeOH and heated to 65℃ for 3 hours. Once removed from heat, the solution was left to cool
overnight. Suction filtration was started with no.2 filter paper, however the filter paper was
quickly clogged by the sample. The filter was then swapped for a no. 3 filter paper and left
on suction overnight. The sample had not finished filtering by morning, and the filter was
once again swapped for filter cloth to complete the filtration step. The filtrate was neutralized
from pH 8.28 to pH 7.05 with HCl. The sample was then rotary-evaporated, slant frozen and
lyophilized. A total mass of 1.2781 g was obtained post lyophilization (2.56% yield).
The residue from the methanol extraction was combined with 800 mL of distilled
water. The solution was heated to 65℃ for 6 hours, then left overnight to cool. An attempt
37

was made to filter through a no. 3 filter paper, but this was swapped to filter cloth due to
clogging. The resulting filtrate solution was measured at pH 7.36 and was adjusted to pH
7.04 with the addition of HCl. This was then rotary-evaporated, slant frozen and lyophilized.
The retrieved mass for Extract 3 was 1.0756 g (2.15% yield).
A final 5% NaOH extraction was performed on the residue from hot water extraction.
Upon addition of 800 mL of 5% NaOH solution, the sample was observed to become viscous
in solution, creating a syrup-like consistency. The solution was heated to 65℃ for 6 hours,
and then left to cool overnight. Due to the thick nature of the solution, this sample was
filtered through filter cloth. The resulting solution was measured at pH 13.52. Upon addition
of concentrated HCl, the solution was observed to precipitate. At pH 12.88, the solution was
too thick and gelatinous for the stir bar to effectively function, thus neutralization proceeded
with manual stirring. As the solution approached pH 12.28, it had thickened significantly to
the consistency of pudding. A smaller portion of this was taken and neutralized to a final pH
of 6.96. This smaller portion was slant frozen directly, due to its consistency, and
lyophilized. A 6.6570 g mass of Extract 4 was obtained from this portion, however based on
the portion size relative to un-neutralized material, the final mass would have exceeded the
starting mass of material, suggesting this sample also had issues with salt accumulation.
2.3.2.5 Letharia vulpina (L.) Hue
Collected specimens were blended to a powder with the consistency of fine lawn
clippings. A mass of 19.20 g was mixed with 50 mL of quartz sand and loaded into the
extraction cells of the speed extractor. Due to clogging issues within the first phase of 85%
EtOH extraction, the run was stopped and the residue collected for manual extraction. An
additional 30.80g of powdered lichen was added to the residue, bringing the total mass to
38

50.00g. This was topped up with 800 mL of 80% EtOH and heated to 65℃ for 3 hours.
Sample was allowed to cool overnight and then suction filtered through a no.2 filter paper.
Filtrate was pH adjusted from pH 3.73 to pH 7.01 with the addition of NaOH. Neutralized
solution was rotary-evaporated, slant frozen and lyophilized. Extract 1 yielded 6.1440 g of
product (12.3% yield).
Residue from the prior extraction was mixed with 800 mL of 50% MeOH and heated
to 65℃. After 3 hours, the solution was removed from heat and allowed to cool overnight.
Upon suction filtration, the sample was observed to clog no.3 filter papers. The filter was
then swapped for filter cloth, and filtration proceeded. Obtained filtrate had an initial pH of
4.20, which was adjusted to pH 6.96 prior to rotary-evaporation. The concentrated sample
was then slant frozen and lyophilized. A mass of 1.5358 g was retrieved for Extract 2 (3.07%
yield)
Hot water extraction was performed on the residue from methanol extraction, with the
addition of 800 mL of distilled water. The mixture was heated to 65℃ for 6 hours, and the
solution left to cool overnight. The sample was then filtered through filter cloth. Resulting
solution was then neutralized from pH 3.97 to pH 6.97. The solution was then rotaryevaporated, slant frozen and lyophilized. Hot water extraction yielded 2.0171 g of Extract 3
(4.03% yield).
The final extraction was performed on the residue from hot water extraction. A volume of
800 mL of 5% NaOH solution was added to the residue and heated to 65℃ for 6 hours. The
solution was left to cool and then suction filtered through filter cloth, yielding a filtrate of pH
13.59. This was neutralized to pH 7.05 with the addition of concentrated HCl. The sample

39

was rotary-evaporated, slant frozen and lyophilized. Dried sample was weighed at 70.3828 g
(140.77% yield), suggesting Extract 4 contained a significant amount of salt.
2.3.2.6 Vulpicida canadensis (Räs.) J.-E. Mattsson & M. J. Lai
Dried sample was ground in a blender to a yellow-green powder. A mass of 50.02 g
was weighed out and topped up with 800 mL of 80% EtOH solution. The sample was heated
to 65℃ for 3 hours, then removed from heat and allowed to cool overnight. The cooled
solution was suction filtered through no. 2 filter paper. The filtrate was neutralized from pH
4.80 to pH 7.01 with the addition of 5% NaOH solution. The sample was then rotaryevaporated, slant frozen, and lyophilized. Dried sample obtained had a mass of 4.2083 g
(8.41% yield).
The residue of ethanol extraction was combined with 800 mL of 50% MeOH. This
was heated to 65℃ for 3 hours and left to cool overnight. Suction filtration was attempted
through no.2 and no. 3 filter papers, but ultimately had to be done through filtration cloth due
to clogging issues. The filtrate was neutralized from pH 4.66 to pH 6.99. This was then
rotary-evaporated, slant frozen and lyophilized. The final Extract 2 sample had a mass of
2.6061 g (5.21% yield).
The residue from methanol extraction was mixed with 800 mL of distilled water and
heated to 65℃ for 6 hours. Upon cooling, the solution was filtered through filtration cloth.
The filtrate was then neutralized with the addition of 5% NaOH solution, from pH 4.47 to pH
7.05. Neutralized sample was then rotary-evaporated, slant frozen and lyophilized. Extract 3
had a mass of 2.5622 g once dried (5.12% yield).

40

The hot water residue was then extracted with sodium hydroxide. The mushroom
sample was topped up with 800 mL of 5% NaOH and heated to 65℃ for 6 hours. The
resulting solution was left to cool overnight. The solution was then filtered through filtration
cloth. Filtrate solution was measured to have a pH of 13.64, which was then neutralized to
pH 7.05 with the addition of HCl. The sample was then rotary-evaporated, slant frozen and
lyophilized. The final mass retrieved was 68.1114 g of Extract 4 (136.17% yield). This
sample is believed to contain significant amounts of salt.

41

Table 5. Summary of chemical extraction results. (* yields from NaOH extractions were falsely high due to the
production of NaCl during pH neutralization)

Fungal
Sample
Hericium
coralloides

Starting
Material
(g)
32.60

Hydnellum
diabolus

38.13

Phaeolepiota
aurea

25.98

Laetiporus sp.

50.00

Letharia
vulpina

50.00

Vulpicida
canadensis

50.02

Extract
Solvent

Extraction
Method

Recovered
Mass (g)

% Yield

85% EtOH
50% MeOH
Hot Water
5% NaOH
85% EtOH
50% MeOH
Hot Water
5% NaOH
85% EtOH
50% MeOH
Hot Water
5% NaOH
80% EtOH
50% MeOH
Hot Water
5% NaOH
80% EtOH
50% MeOH
Hot Water
5% NaOH
80% EtOH
50% MeOH
Hot Water
5% NaOH

Speed
Speed
Speed
Speed/Manual
Speed
Speed
Speed
Manual
Speed
Speed
Speed
Manual
Manual
Manual
Manual
Manual
Manual
Manual
Manual
Manual
Manual
Manual
Manual
Manual

6.5718
3.5075
1.4599
60.64
7.1589
2.9824
0.4848
27.807
3.0998
6.2593
2.3955
58.5043
0.9995
1.2781
1.0756
*N/A
6.1440
1.5358
2.0171
70.3828
4.2083
2.6061
2.5622
68.1114

20.15%
10.76%
4.48%
*186.01%
18.77%
7.82%
1.27%
*72.93%
11.93%
24.09%
9.22%
*225.19%
2.00%
2.56%
2.15%
*N/A
12.30%
3.07%
4.03%
*140.77%
8.41%
5.21%
5.12%
*136.17%

2.3.3 Cell Viability MTT Assay
Crude extracts were tested against HeLa cells for growth-inhibitory activity. While
water was preferred, crude extracts were spot tested for solubility in various solvents. Crude
extracts were ultimately dissolved in water or methanol at a concentration of 20 mg/mL to
produce a stock solution for dose-dependent assays. A 40 mg/mL stock solution
concentration was adopted for time-dependent assays to reduce the amount of solvent
42

introduced into wells. These solutions were pH neutralized and filter sterilized through a 0.2
µm syringe filter prior to being diluted and applied to cells. HeLa cells were plated at a well
concentration of 3000 cells/well for dose-dependent assays. In these, multiple concentrations
were tested against cells to determine the effectiveness of the extracts to inhibit cell growth,
as well at what dose they became effective at. Based on the results of dose-dependent assays,
select extracts were chosen to proceed with time-dependent assays. HeLa cells were plated at
a lower well concentration of 2000 cells/well for inhibitory extracts, and 1500 cells/well for
extracts that showed stimulatory effects. Significance was tested with the use of a student Ttest (p=0.05).
2.3.3.1 Hericium coralloides (Scop.: Fr) Pers.
As shown in Figure 4, all extracts from H. coralloides did not appear to have any
growth-inhibitory activity. Extracts 2 and 3 appeared to stimulate HeLa cell growth, showing
significant deviation from the control at 0.5 mg/mL and 1.0 mg/mL, with a max stimulation
of 128% and 134% respectively. Extract 1 was also observed to have modest growthstimulatory activity at the same concentrations, showing a maximum stimulation of 108% at
1.0 mg/mL. Extract 4 only affected cells at 0.5 mg/mL, increasing growth by 114%, however
this effect was not carried through at higher doses, dropping back to normal growth (Figure
4).
Due to the stimulatory effects seen in the first 3 extracts, these were selected to
proceed into time-dependent MTT assay experiments. Initially, the crude extracts were tested
at a dose of 0.5 mg/mL. However, these were not observed to significantly alter cell growth
at any time points during the 7-day assay (Figure 5). A second time-dependent assay was
performed, this time at a higher dose of 1.0 mg/mL. During the seven-day assay, Extract 1
43

was not observed to stimulate or inhibit cell growth. Extract 2 deviated from the control on
day 3 and day 7, falling short of the growth of control cells. Extract 3 was seen to stimulate
over days 5 and 6, but ultimately the control cells had caught up with growth by day 7
(Figure 6). Since the extracts were not able to consistently stimulate or inhibit cells during
the 7-day period, it is concluded that H. coralloides had no significant effect on the growth of

C e ll V ia b ility (%

o f C o n tr o l)

HeLa cells.

150

E1
E2
100

E3
E4

50

0
0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

C o n c e n tr a tio n (m g /m L )

Figure 4. Dose-dependent MTT-assay of H. coralloides crude extracts. HeLa cells were plated at a
density of 3000 cells/well. Treatment was applied at well concentrations of 0.01, 0.5, 0.1 0.5, and 1.0
mg/mL for 48 hours prior to MTT application. Cell viability was standardized against the solvent
control (H2O). Error bars reflect the standard error of the mean (SEM).

44

o f C o n tr o l)
C e ll V ia b ility (%

120

H 2O
100

(2 .5 % )

E 1 (0 .5 m g /m L )

80

E 2 (0 .5 m g /m L )

60

E 3 (0 .5 m g /m L )

40
20
0
0

50

100

150

T im e (H o u r s )

C e ll V ia b ility (%

o f C o n tr o l)

Figure 5. Time-dependent MTT-assay of H. coralloides crude extracts. Cells were plated at a density
of 1500 cells/well and were treated with crude extracts at 0.5 mg/mL. Cell viability was tested against
the day 7 control (H2O). Error bars are SEM.

120

H 2O
100

(5 % )

E 1 (1 .0 m g /m L )

80

E 2 (1 .0 m g /m L )

60

E 3 (1 .0 m g /m L )

40
20
0
0

50

100

150

T im e (H o u r s )

Figure 6. Time-dependent MTT-assay of H. coralloides crude extracts. Cells were plated at a density
of 1500 cells/well and were treated with crude extracts at 1.0 mg/mL. Cell viability was tested against
the day 7 control (H2O). Error bars reflect SEM.

2.3.3.2 Hydnellum diabolus Banker
Growth-inhibitory activity was observed in the crude extracts of H. diabolus. The
strongest inhibition was observed with Extract 2, dropping cell viability to 70.8% when
45

treated at 0.5 mg/mL, and 26.2% at 1.0 mg/mL. Close behind this was Extract 1, which again
showed significant activity at concentrations greater than 0.5 mg/mL, dropping cell viability
by up to 31.9%. Extract 3 showed less inhibition, deviating from the control at 0.5 and 1.0
mg/mL, reducing cell growth to only 51.2% of the control. The final extract was only
effective at the higher dose, reducing cell viability to 88.9% in treatments of 1.0 mg/mL

C e ll V ia b ility (%

o f C o n tr o l)

(Figure 7).

150

E1
E2
100

E3
E4

50

0
0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

C o n c e n tr a tio n (m g /m L )

Figure 7. Dose-dependent MTT-assay of H. diabolus crude extracts. HeLa cells were plated at a
density of 3000 cells/well. Treatments were applied at well concentrations of 0.01, 0.05, 0.1, 0.5, and
1.0 mg/mL for 48 hours prior to MTT application. Cell viability was standardized against the solvent
control (H2O). Error bars reflect SEM.

Based on the dose-dependent results, extracts 1 and 3 were selected as candidates for
time-dependent assay. Both were initially treated at a concentration of 0.75 mg/mL, as this
was roughly where 50% inhibition was seen in the dose-dependent graph. Both extracts were
observed to significantly decrease cell viability within 24 hours of treatment. Extract 3
dropped cell viability to 3.69% at the 24-hour mark, and the cells were never able to recover,
finishing at 2.93% viability on day 7. Extract 1 also dropped viability to 11.6% within 24

46

hours, again holding the cells at this low value for the duration of the assay, finishing at
9.77% viability on day 7 (Figure 8). A second lower dose run was set up, this time treating
cells with only 0.1 mg/mL of extracts 1 and 3. Cells were less affected by the lower dose,
with only extract 3 significantly inhibiting growth in days 3-6. However, inhibition was not
significant on day 7, suggesting the cells had recovered. Extract 1 did not deviate from
normal cell growth until day 6, whereby it was observed to stimulate cell growth, finishing at
118% of the control at day 7 (Figure 9). In conclusion, these results show that extracts 1 and

C e ll V ia b ility (%

o f C o n tr o l)

3 of H. diabolus are growth-inhibitory at higher concentrations.

100

H 2O

(3 .7 5 % )

E 1 (0 .7 5 m g /m L )
E 3 (0 .7 5 m g /m L )
50

0
0

50

100

150

T im e (H o u r s )

Figure 8. Time-dependent MTT assay of growth-inhibitory extracts from H. diabolus. HeLa cells were
plated at 2000 cells/well and treated with 0.75 mg/mL of crude extracts. Cell viability was tested
against the day 7 control (H2O). Error bars reflect SEM.

47

o f C o n tr o l)
C e ll V ia b ility (%

H 2O

120

(0 .5 % )

E 1 (0 .1 m g /m L )

100

E 3 (0 .1 m g /m L )

80
60
40
20
0
0

50

100

150

T im e (H o u r s )

Figure 9. Time-dependent MTT-assay of crude extracts isolated from H. diabolus. HeLa cells were
plated at a density of 2000 cells/well and treated with crude extracts at 0.1 mg/mL. Cell viability was
determined against the day 7 solvent control (H2O). Error bars reflect SEM.

2.3.3.3 Phaeolepiota aurea (Matt. ex Fr.) Maire
Dose-dependent assay showed both stimulatory and strong inhibitory activity in P.
aurea extracts. Extract 1 showed strong inhibition, deviating from the control at
concentrations as low as 0.05 mg/mL. Maximum inhibition was seen at 1.0 mg/mL, with cell
viability dropping to 8.27%. Extract 2 showed mild inhibition throughout all concentrations,
however it appeared irregular, dropping to a cell viability of 75.9% at doses of 0.1 mg/mL
before rising back up to 90.7% viability at dose of 1.0 mg/mL. Mild inhibition was also seen
at all concentrations in extract 4, which showed a gradual decline in viability to 65.7% at 1.0
mg/mL treatment. Extract 3 was observed to be stimulatory, starting with a decline to 72.9%
in 0.05 mg/mL treatments before jumping up to a final stimulation of 135% in 1.0 mg/mL
treatments (Figure 10).

48

o f C o n tr o l)
C e ll V ia b ility (%

150

E1
E2
100

E3
E4

50

0
0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

C o n c e n tr a tio n (m g /m L )

Figure 10. Dose-dependent MTT-assay of P. aurea crude extracts. HeLa cells were plated at a density
of 3000 cells/well. Treatment was applied at well concentrations of 0.01, 0.5, 0.1 0.5, and 1.0 mg/mL
for 48 hours prior to MTT application. Cell viability was standardized against the solvent controls (E1:
MeOH, E2: H2O, E3: H2O, E4: H2O). Error bars reflect the SEM.

Due to their stronger effects, extracts 1 and 3 were selected to proceed into timedependent assays. Extract 1 was initially treated at a dose of 0.5 mg/mL and was observed to
show significant inhibition within 24 hours of treatment. Viability was only 13.1% at 24
hours, and subsequently dropped to only 2.57% at the end of the assay (Figure 11). A second
lower dose run was performed, treating the cells with only 0.1 mg/mL of extract 1. Again, a
significant decline in cells was observed at the 24-hour mark. Viability remained lower for
the subsequent days, until day 7, whereby the deviation was no longer significant. This
suggests that the cells were able to overcome the effects of the lower dose treatment within
144 hours (Figure 12). Extract 3 was treated at a dose of 0.5 mg/mL to determine its
stimulatory effect over time. While significant activity was observed within 24 hours, it was
growth-inhibitory instead of stimulatory. By day 7, treated cells were at 65.4% of the control
(Figure 13). Since this was not as effective as extract 1 at the same dose level, it was not
investigated further.
49

o f C o n tr o l)
C e ll V ia b ility (%

100

M e O H (2 .5 % )
E 1 (0 .5 m g /m L )

50

0
0

50

100

150

T im e (H o u r s )

C e ll V ia b ility (%

o f C o n tr o l)

Figure 11. Time-dependent MTT-assay of P. aurea crude fractions. HeLa cells were plated at a
density of 2000 cells/well and treated at 0.5 mg/mL. Cell viability was determined against the day 7
solvent control (MeOH). Error bars reflect SEM.

100

M e O H (0 .5 % )
E 1 (0 .1 m g /m L )

50

0
0

50

100

150

T im e (H o u r s )

Figure 12. Time-dependent MTT-assay of P. aurea crude fractions. HeLa cells were plated at a
density of 2000 cells/well and treated at 0.1 mg/mL. Cell viability was determined against the day 7
solvent control (MeOH). Error bars reflect SEM.

50

o f C o n tr o l)
C e ll V ia b ility (%

100

H 2O

(2 .5 % )

E 3 (0 .5 m g /m L )

50

0
0

50

100

150

T im e (H o u r s )

Figure 13. Time-dependent MTT-assay of P. aurea crude fraction. HeLa cells were plated at a density
of 1500 cells/well and treated at 0.5 mg/mL. Cell viability was determined against the day 7 solvent
control (H2O). Error bars reflect SEM.

Because of its potent growth-inhibitory activity, Extract 1 was further assessed in a
dose-dependent manner against multiple cancer cell lines. SK-OV 3 (ovarian cancer), MCF-7
(breast cancer), H441 (lung cancer), HCT 16 (colon cancer), HeLa (cervical cancer), and
HepG2 (liver cancer) cells were all treated with Extract 1. It was effective against all tested
lines in a dose-dependent manner, dropping the viability below 50% for all cell lines but
H441 at a dose of 0.5 mg/mL. All cell lines were dropped to below 20% viability at 1.0
mg/mL with Extract 1 (Figure 14).

51

o f C o n tr o l)
C e ll V ia b ility (%

150

S K O V 3
M C F -7
100

H 441
H C T 116
H eLa

50

H ep G 2

0
0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

C o n c e n tr a tio n (m g /m L )

Figure 14. Multiple cell line dose-dependent MTT-assay of P. aurea crude ethanol extract. Cells were
plated at a density of 3000 cells/well. Treatment was applied at well concentrations of 0.01, 0.5, and
1.0 mg/mL for 48 hours prior to MTT application. Cell viability was standardized against the solvent
controls. Error bars reflect the SEM.

2.3.3.4 Laetiporus sp.
Dose-dependent MTT assay show that crude extracts from Laetiporus sp. have
growth-inhibitory activity Extract 1 was observed to show the strongest inhibition, dropping
cell viability from treatment concentrations as low as 0.01 mg/mL. Cell viability had dropped
to under 6% at concentrations greater than 0.5 mg/mL. Extracts 2 and 3 showed similar
activity, again dropping cell viability around 50% at doses of 1.0 mg/mL. Overall, extract 4
was not observed to affect cell growth (Figure 15).
As the top inhibitory extract, extract 1 proceeded into time-dependent assays. Initially
the extract was treated at a dose of 0.5 mg/mL, which yielded significant deviation from the
control within 24 hours. Cell viability had dropped to 4.28% of the day 7 control and
remained at this low value for the duration of the assay. On day 7, cell viability was
measured at 2.38% of the control (Figure 16). A lower dose run was performed using a

52

treatment concentration of 0.1 mg/mL. Again, extract 1 caused significant deviation from the
control within 24 hours of treatment, dropping cell viability to as low as 38.7% in day 6
readings, before rising back up to 52.0% viability in day 7 (Figure 17). In summary, Extract 1

C e ll V ia b ility (%

o f C o n tr o l)

from Laetiporus sp. was found to be a potent growth-inhibitor.

150

E1
E2
100

E3
E4

50

0
0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

C o n c e n tr a tio n (m g /m L )

Figure 15. Dose-dependent MTT-assay of Laetiporus sp. crude extracts. HeLa cells were plated at a
density of 3000 cells/well. Treatments were applied at well concentrations of 0.01, 0.05, 0.1, 0.5, and
1.0 mg/mL for 48 hours prior to MTT application. Cell viability was standardized against the solvent
controls (E1: MeOH, E2: H2O, E3: H2O, E4: H2O). Error bars reflect SEM.

53

o f C o n tr o l)
C e ll V ia b ility (%

100

M e O H (2 .5 % )
E 1 (0 .5 m g /m L )

50

0
0

50

100

150

T im e (H o u r s )

C e ll V ia b ility (%

o f C o n tr o l)

Figure 16. Time-dependent MTT-assay of crude extracts isolated from Laetiporus sp.. HeLa cells
were plated at a density of 2000 cells/well and treated with E1 at 0.5 mg/mL. Cell viability was
determined against the day 7 solvent control (MeOH). Error bars reflect SEM.

100

M e O H (0 .5 % )
E 1 (0 .1 m g /m L )

50

0
0

50

100

150

T im e (H o u r s )

Figure 17. Time-dependent MTT-assay of E1 from Laetiporus sp.. HeLa cells were plated at a density
of 2000 cells/well and treated with E1 at 0.1 mg/mL. Cell viability was determined against the day 7
solvent control (MeOH). Error bars reflect SEM.

2.3.3.5 Letharia vulpina (L.) Hue
Dose-dependent assay of L. vulpina crude extracts showed evidence of growthinhibitory activity. Extract 1 was observed to significantly decrease cell viability at
54

concentrations of 0.1 mg/mL and greater, dropping cell viability to as low as 5.60% of the
control. The remaining three extracts all showed similar inhibition trends, dropping cell
viability between 53.6-67.1% of the control in 1.0 mg/mL treatments (Figure18). While
methanol was selected as the initial solvent for extract 1, spot testing had also shown that it
was somewhat soluble in water as well. Another dose-dependent assay was performed to see
if activity would be retained using water as the solvent. A similar trend was observed, with
significant declines in cell viability being seen in concentrations of 0.5 mg/mL or higher,
ultimately dropping to 12.3% viability in 1.0 mg/mL dosing. Interestingly, the extract was
observed to stimulate cell growth at lower doses, peaking at 149% cell viability in the 0.1
mg/mL treatment (Figure 19). This suggests that the bioactive compounds found in this
extract are water soluble. Water was subsequently used as a solvent for the time-dependent

C e ll V ia b ility (%

o f C o n tr o l)

assays.

150

E1
E2
100

E3
E4

50

0
0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

C o n c e n tr a tio n (m g /m L )

Figure 18. Dose-dependent MTT-assay of crude extracts isolated from L. vulpina. HeLa cells were
plated at a density of 3000 cells/well. Treatments were applied at well concentrations of 0.01, 0.05,
0.1, 0.5, and 1.0 mg/mL for 48 hours prior to MTT application. Cell viability was standardized against
the solvent controls (E1: MeOH, E2: H2O, E3: H2O, E4: H2O). Error bars reflect SEM.

55

o f C o n tr o l)
C e ll V ia b ility (%

200

150

100

50

0
0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

C o n c e n tr a tio n (m g /m L )

Figure 19. Dose-dependent MTT-assay of crude extract E1 from L. vulpina. HeLa cells were plated at
a density of 3000 cells/well. Treatments were applied at well concentrations of 0.01, 0.05, 0.1, 0.5, and
1.0 mg/mL for 48 hours prior to MTT application. Cell viability was standardized against the solvent
control. Error bars reflect SEM.

Extract 1, showing the strongest growth-inhibitory activity, was selected to proceed
into time-dependent assays. An initial treatment concentration of 0.5 mg/mL was used. Cell
viability had significantly dropped below that of the control within 24 hours of treatment,
dropping to final values of 3.42% on day 7 (Figure 20). A second assay, using a treatment
concentration of 0.1 mg/mL, was performed. The lower dose treatment showed significant
deviation from the control on days 2 through 5, having a higher growth rate than the control.
This trend ended by day 6 (Figure 21). This evidence suggests that extract 1 may be
stimulatory at lower doses, and inhibitory at higher doses. However, further experiments
would need to be performed to confirm this unexpected finding.

56

o f C o n tr o l)
C e ll V ia b ility (%

100

H 2O

(2 .5 % )

E 1 (0 .5 m g /m L )

50

0
0

50

100

150

T im e (H o u r s )

C e ll V ia b ility (%

o f C o n tr o l)

Figure 20. Time-dependent MTT-assay of crude extract E1 from L. vulpina. HeLa cells were plated at
a density of 2000 cells/well and treated with E1 at 0.5 mg/mL. Cell viability was determined against
the day 7 solvent control (H2O). Error bars reflect SEM.

100

H 2O

(0 .5 % )

E 1 (0 .1 m g /m L )

50

0
0

50

100

150

T im e (H o u r s )

Figure 21. Time-dependent MTT-assay of E1 from L. vulpina. HeLa cells were plated at a density of
2000 cells/well and treated with E1 at 0.5 mg/mL. Cell viability was determined against the day 7
solvent control (H2O). Error bars reflect SEM.

2.3.3.6 Vulpicida canadensis (Räs.) J.-E. Mattsson & M. J. Lai
Dose-dependent MTT assay of the V. canadensis crude extracts show that this lichen
contains growth-inhibitory activity. The strongest inhibition was seen with extract 1,
57

significantly deviating from the control at all treatment concentrations. A maximum
inhibition was seen in treatment concentrations of 0.5 mg/mL, dropping cell viability to
6.16%. Extracts 2 and 4 showed similar trending, coming out in the middle with cell
viabilities of 70.2% and 54.9% at the highest treatment concentration. The hot water extract
(extract 3) was not observed to significantly affect cell growth in any of the treatment

C e ll V ia b ility (%

o f C o n tr o l)

concentrations used (Figure 22).

150

E1
E2
100

E3
E4

50

0
0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

C o n c e n tr a tio n (m g /m L )

Figure 22. Dose-dependent MTT-assay of crude extracts isolated from V. canadensis. HeLa cells were
plated at a density of 3000 cells/well. Treatments were applied at well concentrations of 0.01, 0.05,
0.1, 0.5, and 1.0 mg/mL for 48 hours prior to MTT application. Cell viability was standardized against
the solvent control. Error bars reflect SEM.

Time-dependent assays were performed on extract 1 due to its strong inhibitory
activity. An initial assay was performed with a treatment concentration of 0.5 mg/mL. The
sample was observed to significantly affect cell growth within 24 hours of treatment. At day
7, the cell viability was at 2.57% of the control (Figure 23). A lower concentration of 0.1
mg/mL was used in a second time-dependent assay. Cell growth began to deviate from the
control at the 24-hour mark and continued to do so throughout the duration of the assay. Day

58

7 viability was measured at 44.4% of the control, suggesting this extract has the ability to
inhibit cells at lower doses (Figure 24). In summary, Extract 1 from V. canadensis was found

C e ll V ia b ility (%

o f C o n tr o l)

to have potent growth-inhibitory activity.

100

M e O H (2 .5 % )
E 1 (0 .5 m g /m L )

50

0
0

50

100

150

T im e (H o u r s )

C e ll V ia b ility (%

o f C o n tr o l)

Figure 23. Time-dependent MTT-assay of E1 from V. canadensis. HeLa cells were plated at a density
of 2000 cells/well and treated with E1 at 0.5 mg/mL. Cell viability was determined against the day 7
solvent control (MeOH). Error bars reflect SEM.

100

M e O H (0 .5 % )
E 1 (0 .1 m g /m L )

50

0
0

50

100

150

T im e (H o u r s )

Figure 24. Time-dependent MTT-assay of E1 from V. canadensis. HeLa cells were plated at a density
of 2000 cells/well and treated with E1 at 0.1 mg/mL. Cell viability was determined against the day 7
solvent control (MeOH). Error bars reflect SEM.

59

2.3.4 Immuno-stimulatory activity
ELISA was performed on the supernatants of treated Raw 264.7 cells to determine
which extracts stimulated TNF-α production. Initially cells were treated for 3 and 6-hour
periods to determine which timeframe allowed enough activity to be seen on ELISA plates.
The 6-hour incubation time was subsequently adopted for all of the later experiments. LPS
was used as a positive control, to ensure cells were active, and to give a reference point for
active fractions. All extracts were initially treated at a dose of 1.0 mg/mL, however for some
cytotoxic extracts the concentration used were dropped to 0.1 mg/mL to ensure results were
not the product of cell lysis. All extracts were subjected to three assays to ensure results were
not the product of cell variability. Significance was tested with a student’s T-Test (p=0.05).
2.3.4.1 Hericium coralloides (Scop.: Fr) Pers.
In the first ELISA experiment, H. coralloides crude extracts E2 and E3 showed
modest stimulatory activity. At both the 3 and 6-hour time points, these extracts were found
to significantly deviate from the water control. Extract 4 was only significant after 6 hours of
treatment. This being said, the top stimulatory activity, seen in E3 (821.25 pg/mL), was only
15.10% of the LPS positive control (Figure 25). Repeat assays found similar results, with
extracts 2, 3, and 4 deviating from the control. E3 remained the top stimulator of TNF-α,
stimulating at a rate of 21-25% of LPS respectively (Figures 26-27).

60

3 H our
4000

6 H our

3000

2000

1000

4
E

3
E

2
E

E

2 O

L

H

P

M
E
D

M

1

0

S

C o n c e n tr a tio n (p g /m L )
T N F -

5000

6000

4000

2000

4
E

3
E

2
E

1
E

2 O

P
L

E
M
D

H

S

0

M

T N F -

C o n c e n tr a tio n (p g /m L )

Figure 25. ELISA 1 results for crude extracts of H. coralloides. Raw 264.7 cells were plated at a
density of 100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive
control. Extracts were applied at a well concentration of 1.0 mg/mL and the supernatant solution was
collected after incubations of 3 and 6 hours. Error bars represent SEM.

Figure 26. ELISA 2 results for crude extracts of H. coralloides. Raw 264.7 cells were plated at a
density of 100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive
control. Extracts were applied at a well concentration of 1.0 mg/mL and the supernatant solutions were
collected after 6 hours of treatment. Error bars represent SEM.

61

6000

4000

2000

L

4
E

3
E

2
E

1
E

H

P

M
E
D

M

2 O

0

S

C o n c e n tr a tio n (p g /m L )
T N F -

8000

Figure 27. ELISA 3 results for crude extracts of H. coralloides. Raw 264.7 cells were plated at a
density of 100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive
control. Extracts were applied at a well concentration of 1.0 mg/mL and the supernatant solutions were
collected after 6 hours of treatment. Error bars represent SEM.

2.3.4.2 Hydnellum diabolus Banker
Extracts 2, 3 and 4 from Hydnellum diabolus was observed to significantly stimulate
TNF-α production. Results were significant in both the 3 and 6-hour treatment timeframes.
The top stimulator was E2, stimulating TNF-α production at 33.31% of the LPS positive
control (Figure 28). E1 (0% of LPS) and E3 (27.72% of LPS) were observed to have
cytotoxic effects on cells. Due to this, these extracts were treated at 0.1 mg/mL in the next
assay, to determine if cytokine readings were the product of cell lysis, or actual stimulation.
A second ELISA was performed, and all extracts were observed to deviate significantly from
the control. Cytotoxic E1 and E3 were both observed to stimulate TNF-α production at their
lowered treatment concentrations, however, cell death was still observed in treatment wells.
E1 now stimulated at 23.27% and E3 at 30.39% of LPS (Figure 29). This suggests that there
may be competing compounds within the extracts. E2 (32.83% of LPS) was observed to have
some adverse effects on cells during this assay, and it was subsequently dropped to a
62

treatment concentration of 0.1 mg/mL. This would also allow for comparison between the
first 3 extracts. As seen in Figure 30, E3 is the most stimulatory extract at the lowered dose
of 0.1 mg/mL, stimulating at 21.25% of LPS. E1 was second most stimulatory at 17.79% of
LPS and E2 came third at 21.25% of LPS. E2 was observed to be the least cytotoxic to Raw

5000

3 H our
4000

6 H our

3000

2000

1000

4
E

3
E

2
E

1
E

2 O

P
L

E
M
D

H

S

0

M

T N F -

C o n c e n tr a tio n (p g /m L )

cells in the lower dose assay.

Figure 28. ELISA 1 results for crude extracts of H. diabolus. Raw 264.7 cells were plated at a density
of 100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive control.
Extracts were applied at a well concentration of 1.0 mg/mL and the supernatant solution was collected
after incubations of 3 and 6 hours. Error bars represent SEM.

63

4000

3000

2000

1000

L

4
E

3
E

2
E

1
E

H

P

M
E
D

M

2 O

0

S

C o n c e n tr a tio n (p g /m L )
T N F -

5000

6000

4000

2000

4
E

3
E

2
E

1
E

2 O

P
L

E
M
D

H

S

0

M

T N F -

C o n c e n tr a tio n (p g /m L )

Figure 29. ELISA 2 results for crude extracts of H. diabolus. Raw 264.7 cells were plated at a density
of 100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive control. E1
and E3 were treated at 0.1 mg/mL due to cytotoxic activity against the cells. E2 and E4 were treated at
1.0 mg/mL. The supernatant solutions were collected after 6 hours of treatment. Error bars represent
SEM.

Figure 30. ELISA 3 results for crude extracts of H. diabolus. Raw 264.7 cells were plated at a density
of 100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive control. E1,
E2, and E3 were treated at concentrations of 0.1 mg/mL due to cytotoxic activity against cells. E4 was
treated at 1.0 mg/mL. The supernatant solutions were collected after 6 hours of treatment. Error bars
represent SEM.

64

2.3.4.3 Phaeolepiota aurea (Matt. ex Fr.) Maire
In the initial ELISA experiments, crude extracts 2, 3 and 4 of P. aurea were found to
have significant stimulatory activity. Top production of TNF-α was seen in the 6-hour
treatment with E3, causing stimulation at rates 76.17% of LPS. E2 was the second most
stimulatory, showing 21.67% the stimulation of LPS. E4 only stimulated at 11.19% of LPS
(Figure 31). E1 was observed to kill cells and was thus diluted to 0.1 mg/mL in subsequent
assays. However, E1 still caused cell death in the lower dose treatments. The second and
third ELISA tests confirmed E3 as the most potent stimulator (86-98% of LPS), with E2
close behind (59-77% of LPS, Figures 32-33). Due to its highly potent immuno-stimulatory
activity which is as strong as the positive control LPS, E3 was selected as the source for the
purification and characterization of immuno-stimulatory compound(s) described in detail in

5000

3 H our
4000

6 H our

3000

2000

1000

3

4
E

E

E

2

1
E

e

O

H

2 O

M

P
L

E
M
D

H

S

0

M

T N F -

C o n c e n tr a tio n (p g /m L )

Chapter 3.

Figure 31. ELISA 1 results for crude extracts of P. aurea. Raw 264.7 cells were plated at a density of
100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive control. E1 was
dissolved in MeOH and all remaining extracts were dissolved in water. Extracts were applied at a well
concentration of 1.0 mg/mL and the supernatant solution was collected after incubations of 3 and 6
hours. Error bars represent SEM.

65

4000

3000

2000

1000

D

4
E

3
E

2
E

1
E

H
O

M

e

L

H

P

M
E
M

2 O

0

S

C o n c e n tr a tio n (p g /m L )
T N F -

5000

8000

6000

4000

2000

4
E

3
E

2
E

1
E

M

e

O

H

2 O

P
L

E
M
D

H

S

0

M

T N F -

C o n c e n tr a tio n (p g /m L )

Figure 32. ELISA 2 results for crude extracts of P. aurea. Raw 264.7 cells were plated at a density of
100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive control. E1,
dissolved in MeOH, was treated at a concentration of 0.1 mg/mL due to cytotoxic activity against cells.
E2, E3, and E4, dissolved in water, were treated at a concentration of 1.0 mg/mL. The supernatant
solutions were collected after 6 hours of treatment. Error bars represent SEM.

Figure 33. ELISA 3 results for crude extracts of P. aurea. Raw 264.7 cells were plated at a density of
100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive control. E1,
dissolved in MeOH, was treated at a concentration of 0.1 mg/mL due to cytotoxic activity against cells.
E2, E3, and E4, dissolved in water, were treated at a concentration of 1.0 mg/mL. The supernatant
solutions were collected after 6 hours of treatment. Error bars represent SEM.

66

2.3.4.4 Laetiporus sp.
In the first ELISA experiment with Laetiporus sp. crude extracts, LPS failed to
stimulate cells. This was attributed to a bad batch of LPS solution. However, trends were still
seen in the extract data, with E2, E3 and E4 all deviating from their solvent controls. The
most potent extract was E3, followed by E2, and then E4. Accurate comparisons could not be
made due to the low value of LPS (Figure 34). E1 was observed to kill cells at the 1.0 mg/mL
concentration and was dropped to 0.1 mg/mL for subsequent assays, however, this extract
still caused cell death at the lower dosing. In the subsequent two experiments, all extracts
were observed to significantly deviate from the control. The stimulation now seen in E1,
while low, suggests that there are competing compounds within this extract. E3 remained the
most potent stimulator, exceeding the stimulation seen in LPS (117-126% of LPS). E2
remained a close second, also exceeding LPS (115-119% of LPS). E4 was also significant,
stimulating at values 51-60% of LPS (Figures 35-56). Due to their consistently potent
activity, E2 and E3 are strong candidates for future investigation into finding potential novel
immuno-stimulatory compounds.

67

3000

3

H our

6

H our

2000

1000

4
E

3
E

2
E

E

O

H

2 O

D

M

e

L

H

P

M
E
M

1

0

S

C o n c e n tr a tio n (p g /m L )
T N F -

4000

8000

6000

4000

2000

4
E

3
E

2
E

1
E

e

O

H

2 O

M

P
L

E
M
D

H

S

0

M

T N F -

C o n c e n tr a tio n (p g /m L )

Figure 34. ELISA 1 results for crude extracts of Laetiporus sp.. Raw 264.7 cells were plated at a
density of 100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive
control. E1 was dissolved in MeOH and E2, E3, E4 were dissolved in water. Extracts were applied at a
well concentration of 1.0 mg/mL and the supernatant solution was collected after incubations of 3 and
6 hours. Error bars represent SEM.

Figure 35. ELISA 2 results for crude extracts of Laetiporus sp.. Raw 264.7 cells were plated at a
density of 100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive
control. E1, dissolved in MeOH, was treated at a concentration of 0.1 mg/mL due to cytotoxic activity
against the cells. E2, E3, and E4 were dissolved in water and treated at a concentration of 1.0 mg/mL.
The supernatant solutions were collected after 6 hours of treatment. Error bars represent SEM.

68

6000

4000

2000

4
E

E

3

*

*
E

2

1
E

H
O
e

M

H

L

P

M
E
M
D

2 O

0

S

C o n c e n tr a tio n (p g /m L )
T N F -

8000

Figure 36. ELISA 3 results for crude extracts of Laetiporus sp.. Raw 264.7 cells were plated at a
density of 100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive
control. E1, dissolved in MeOH, was treated at a concentration of 0.1 mg/mL due to cytotoxic activity
against the cells. E2, E3, and E4 were dissolved in water and treated at a concentration of 1.0 mg/mL.
The supernatant solutions were collected after 6 hours of treatment. Error bars represent SEM. (* value
based off one sample well due to equipment limitations, actual value expected to be larger)

2.3.4.5 Letharia vulpina (L.) Hue
The initial ELISA experiment with L. vulpina crude extracts showed stimulatory
trends with extracts 3 and 4. Due to a bad batch of LPS solution, the positive control was not
observed to strongly stimulate TNF-α production. Due to this, only observations were made
indicating extract 3 stimulated stronger than extract 4 (Figure 37). E1 was cytotoxic at the
treatment dose of 1.0 mg/mL and was dropped to 0.1 mg/mL for all further assays, however,
cell death was still observed in treatment wells. The same data trends were seen in the
subsequent two ELISA experiments, with E3 and E4 showing significant stimulation. E3
remained the most potent stimulator, showing values at 35-41% of LPS. E4 stimulated at 817% of LPS (Figures 38-39).

69

3 H our
400

6 H our

300

200

100

D

4
E

E

3

2
E

E

M

e

O

H

2 O

L

H

P

M
E
M

1

0

S

C o n c e n tr a tio n (p g /m L )
T N F -

500

5000

4000

3000

2000

1000

4
E

3
E

2
E

1
E

e

O

H

2 O

M

P
L

E
M
D

H

S

0

M

T N F -

C o n c e n tr a tio n (p g /m L )

Figure 37. ELISA 1 results for crude extracts of L. vulpina. Raw 264.7 cells were plated at a density of
100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive control. E1 was
dissolved in MeOH while E2, E3, and E4 were dissolved in water. Extracts were applied at a well
concentration of 1.0 mg/mL and the supernatant solution was collected after incubations of 3 and 6
hours. Error bars represent SEM.

Figure 38. ELISA 2 results for crude extracts of L. vulpina. Raw 264.7 cells were plated at a density of
100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive control. E1,
dissolved in MeOH, was treated at a concentration of 0.1 mg/mL due to cytotoxic activity against the
cells. E2, E3, and E4 were dissolved in water and treated at a concentration of 1.0 mg/mL. The
supernatant solutions were collected after 6 hours of treatment. Error bars represent SEM.

70

6000

4000

2000

4
E

3
E

2
E

1
E

H
O

M

e

H

L

P

M
E
M
D

2 O

0

S

C o n c e n tr a tio n (p g /m L )
T N F -

8000

Figure 39. ELISA 3 results for crude extracts of L. vulpina. Raw 264.7 cells were plated at a density of
100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive control. E1,
dissolved in MeOH, was treated at a concentration of 0.1 mg/mL due to cytotoxic activity against the
cells. E2, E3, and E4 were dissolved in water and treated at a concentration of 1.0 mg/mL. The
supernatant solutions were collected after 6 hours of treatment. Error bars represent SEM.

2.3.4.6 Vulpicida canadensis (Räs.) J.-E. Mattsson & M. J. Lai
The initial ELISA experiment of V. canadensis crude extracts found stimulatory
activity in extracts 2, 3, and 4. E4 was the most potent, followed by E2 and E3. E1 was
observed to be cytotoxic at treatment concentration 1.0 mg/mL and was therefore dropped to
0.1 mg/mL in subsequent assays. Cell death was still observed in E1 treatments at the
lowered dose. Due to the lack of strong stimulation in LPS, attributed to a bad batch of
solution, comparison values were not made for the first ELISA. Experiments 2 and 3 were
more successful, showing E2 to be the most stimulatory at 28-47% of LPS. This was
followed by E4 at 18-27% stimulation, and E3 at 15-20% stimulation (Figures 41-42).

71

3 H our
6 H our

600

400

200

4
E

E

3

2
E

E

M

e

O

H

2 O

L

H

P

M
E
M

1

0
S

C o n c e n tr a tio n (p g /m L )
T N F -

800

6000

4000

2000

4
E

3
E

2
E

1
E

e

O

H

2 O

M

P
L

E
M
D

H

S

0

M

T N F -

C o n c e n tr a tio n (p g /m L )

Figure 40. ELISA 1 results for crude extracts of V. canadensis. Raw 264.7 cells were plated at a
density of 100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive
control. Extracts were applied at a well concentration of 1.0 mg/mL and the supernatant solution was
collected after incubations of 3 and 6 hours. Error bars represent SEM.

Figure 41. ELISA 2 results for crude extracts of V. canadensis. Raw 264.7 cells were plated at a
density of 100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive
control. E1, dissolved in MeOH, was treated at a concentration of 0.1 mg/mL due to cytotoxic activity
against the cells. E2, E3, and E4 were dissolved in water and treated at a concentration of 1.0 mg/mL.
The supernatant solutions were collected after 6 hours of treatment. Error bars represent SEM.

72

6000

4000

2000

4
E

3
E

2
E

1
E

H
O
e

M

H

L

P

M
E
M
D

2 O

0

S

C o n c e n tr a tio n (p g /m L )
T N F -

8000

Figure 42. ELISA 3 results for crude extracts of V. canadensis. Raw 264.7 cells were plated at a
density of 100,000 cells/well. LPS was treated at a well concentration of 500 ng/mL as a positive
control. E1, dissolved in MeOH, was treated at a concentration of 0.1 mg/mL due to cytotoxic activity
against the cells. E2, E3, and E4 were dissolved in water and treated at a concentration of 1.0 mg/mL.
The supernatant solutions were collected after 6 hours of treatment. Error bars represent SEM.

2.4 Discussion
The initial proceedings of this study have yielded many interesting results.
Identification of mushroom samples has proven the effectiveness of the ITS region for
mushroom identification, as well as its limitations. While fresh collections show an ease in
identification, aged specimens can be a challenge in both morphological and DNA
identification, being prone to varying features as well as contamination, as seen in sample #4.
As samples are collected from the environment, contaminating spores and molds can be an
issue when proceeding with DNA analysis, especially since they would also be the target of
fungal specific primers. Once identified, the history of the species studied could be
determined.
Hericium coralloides has not been subject to intense study. Previously, studies have
found its extracts to contain antioxidant activity93–95. Erinacin E, isolated from this species,
73

has proven to be a highly selective agonist of the kappa opioid receptor, and it was suggested
it could show similar effects as morphine, without the side effects75. A novel laccase was
isolated in 2012, which showed significant activity against HIV-1 reverse transcriptase, but
did not affect cancer cells when tested for anti-proliferative effects96. Additionally, isolated
corallocins A-C were found to induce nerve growth factor (NGF) in human astrocytes97.
Corallocin B was also observed to show anti-proliferative effects against MCF-7 and KB-3-1
cancer cell lines97. A relative of H. coralloides, H. erinaceus, is well-known in traditional
Chinese medicine, and has been studied extensively in recent years. Commonly known as
Lion’s Mane, H. erinaceus has been found to contain many bioactive polysaccharides,
proteins, lectins, phenols and terpenoids98–100; hericenones and erinacines isolated from this
fungus have been shown to stimulate NGF98,99. Multiple studies have attributed a vast array
of biological activities to H. erinaceus, including antitumor, immunomodulatory,
neuroprotective, neuroregenerative, anti-oxidant, anti-hyperglycemia, anti-fatigue, antiaging, hepatoprotective, and gastroprotective effects99,100. Due to these effects, it has been
used in the treatment of cancers, hepatic disorders, Alzheimer’s and Parkinson’s disease,
dementia, schizophrenia, to improve sleep, to reduce menopause symptoms, as well as many
other applications98–100. Due to its phylogenetic relationship, one may expect to find similar
compounds within H. coralloides.
While Hydnellum diabolus has been studied for ecological purposes, very little
research has been performed in regards to its potential bioactivities. The only study directly
related to bioactivity in this species was the isolation of the anticoagulant atromentin from
ethanolic extracts; its activity was comparable to that of heparin101. Additional p-terphenyl
derivatives have been isolated from a variety of other Hydnellum species102–105. Of these,
74

identified bioactivity was found to be α-glucosidase inhibitors104. Moderate antioxidant
activity has been seen in other species106, however, very few studies have looked at
bioactivity of the isolated compounds. The limited bioactivity studies in this and related
species makes it an obvious candidate for future research.
Similarly, there has been relatively little study done on Phaeolepiota aurea. At once
thought to be edible, tests have found this species to contain trace amounts of hydrogen
cyanide107, and it is no longer recommended as a food source. Many studies pertain to the
taxonomy and ecology of this species, but as of our study, only one has approached it in
regards to isolation and identification of bioactive compounds. Two lectins have been
isolated from fruiting bodies extracted in saline solution. These were found to be Nacetylgalactosamine specific, and showed a preference for type A erythrocytes in
agglutination assays108. As this species is the only one in its genus, it has no related species
showing bioactivity. This species has high potential since its medical potential is
uncharacterized.
While the species identity of the Laetiporus specimen remains unknown, some
members of this genus have been well studied. Of particular interest is L. sulphureus, a wellknown edible mushroom. Known as “chicken of the woods”, it is a highly prized food in
many cultures. Traditionally, it has been used for treating pyretic diseases, coughs, gastric
cancer and rheumatism109,110. Studies have found it to be a rich source of polysaccharides,
proteins, polysaccharide-protein complexes, triterpenoids, organic acids, benzofurans,
flavonoids, coumarins and nitrogen containing compounds109,111. These compounds have
elicited an array of activities, including antioxidant, antimicrobial, anti-proliferative, food
preserving, hepatoprotective, anti-inflammatory, immuno-stimulatory, and anti-diabetic109–
75

112

. Overall, L. sulphureus is well characterized for bioactivity, however novel compounds

are still being identified from this species113. Alternatively, as the specimen was well aged
and was noted to have mold contaminants in DNA analysis, one could expect that activity
may arise from compounds contributed by such contaminants. Gibellulopsis nigrescens does
not appear to be characterized for bioactivity, and as such, its isolation may lead to novel
findings. Due to the condition of the specimen, an exact species identity is required before
pursuing the Laetiporus specimen for further characterization.
Letharia vulpina obtains its bright colour from vulpinic acid; known to be toxic, this
lichen has been used as a poison in wolf bait80,114. Acetone extracts of this lichen have shown
antibacterial effects, being effective against multiple strains of bacteria, including
methicillin-resistant Staphylococcus aureus115,116. Extracts have also proven cytotoxic to
Burkitt’s lymphoma (Raji) cells117. Vulpinic acid has in itself been proven to show antiproliferative effects against Hep-G2, NS20Y, and HUVEC cells. Interestingly, it was more
cytotoxic to the cancer cells than normal cells, and showed a stronger anti-angiogenic activity
in normal cells118. Vulpinic acid also shows photoprotective abilities119. While many studies
have explored the ecology of this species, few involve its bioactive compounds, leaving this a
species of interest for future investigation.
Vulpicida canadensis also contains vulpinic acid, as well as usnic and pinastric
acids120. Its extracts have shown antioxidant, and neuroprotective activities120. Cytotoxic
activity was observed against Raji117, Hep-G2, and MCF-7 cells120. Acetone extracts also
showed antibacterial properties against multiple bacterial strains116. This lichen has yet to be
characterized for any further bioactivities.

76

In this chapter, the bioassay results show support for previously discovered activities,
as well as some novel findings. MTT assay results show a strong trend for ethanol extracts
being the most cytotoxic. H. diabolus, P. aurea, Laetiporus sp., L. vulpina, and V. canadensis
all showed strong anti-proliferative activity within E1, suggesting all contain bioactive small
molecules. Of these, only Laetiporus sp. and V. canadensis were observed to retain
significant activity in the low dose time-dependent assays, suggesting the compounds in their
ethanol extracts are more potent, or compose a larger percentage of the fraction respectively.
However, cytotoxic activity has already been reported in these two species. Additionally, the
growth inhibitory effects of L. vulpina have been reported in acetone extracts. The antiproliferative activity observed in P. aurea and H. diabolus appears to be novel findings, as
they have not been previously studied for growth-inhibitory effects.
The results from ELISA experiments showed that all mushrooms studied contain
some immuno-stimulatory activity. This is not unexpected, as it is suggested that all
mushrooms could contain immuno-stimulatory compounds, as they generally contain βglucans, which are components of the cell wall of fungal cells84. The stimulatory compounds
are part of the structure of mushrooms, not just secondary compounds. As such, the strongest
stimulation was often observed in E3, the hot water extract, suspected to contain water
soluble polysaccharides. The strong immuno-stimulatory effects seen in Laetiporus sp.
support previous findings in this genus. The immuno-stimulatory effects observed in all other
species studied appear to be novel at the time of this study. However, of these novel findings,
P. aurea remains of the highest interest due to its stimulation at levels comparable to LPS.
An unexpected finding was that some cytotoxic extracts were observed to stimulate
growth in their lower dose treatments. L. vulpina’s ethanol extract was observed to have a
77

brief stimulatory spike in its dose-dependent MTT assay, initially thought to be due to cell
variance. However, upon low dose time-dependent assay, cells were observed to grow at a
rate greater than the control for a large portion of the assay. This may suggest there are
competing compounds within the extracts and raises the concern that some bioactivity may
be masked by other compounds. Alternatively, one may also suggest that the compound of
interest is only toxic at higher doses, being relatively benign in small amounts. This can also
be seen in the results from ELISA experiments, with the cytotoxic E1 extracts showing some
stimulatory effects when diluted.
Due to the production of salt when neutralizing the highly alkaline sodium hydroxide
extracts (E4), the mass collected exceeded the starting mass of material extracted. As dialysis
was not adopted at this time, a large portion of the mass used to make solutions would have
been caused by this salt, and therefore less of the crude extract made it into treatment
solutions. This could have caused an underestimation of the activity of the E4 extracts within
the bioassays. These extracts could show stronger activity upon removal of the salt for
accurate concentration applications in the assays.
By pursuing extracts with stronger observed activity, one could assume that there is
an active compound in larger quantity, and thus be easier to purify in larger amounts.
Alternatively, there could be small amounts of a far more potent compound, in which case
purification would require a larger scale approach to isolate enough compound for further
analysis. Based on the strength of the above results, the novelty of findings, and due to
availability of raw material, P. aurea E3 extract was selected for further studies in an attempt
to purify, characterize and potentially identify the compound responsible for immunostimulatory activity. The anti-proliferative activity of E1 extract of P. aurea was not pursued
78

because it could contain hydrogen cyanide107 which potentially could have given rise to the
cytotoxic activity.

79

Chapter 3: Purification and Characterization of an Immuno-Stimulatory Compound
from Phaeolepiota aurea
3.1 Introduction
Based on the literature search of the mushroom species as well as the results of the
biological evaluation of their crude extracts, in the previous chapter, I decided to further
investigate the immuno-stimulatory activity of the species Phaeolepiota aurea. Prior to our
study, the immuno-stimulatory activity of P. aurea has never been described. Herein, I
provide the purification scheme for the isolation of a large molecular weight compound with
immuno-stimulatory activity from P. aurea. I also provide an initial characterization of the
immuno-stimulatory compound.
3.2 Methods
3.2.1 Collection, Identification, and Re-extraction of Phaeolepiota aurea
Prior to purification attempts, a fresh batch of P. aurea E3 extract was prepared. A
second batch of P. aurea fruiting bodies was collected from the same location on the UNBC
campus trails in October 2014. The samples were morphologically and DNA identified as
outlined in Chapter #2. Fruiting bodies were dried in an oven and ground to a powder with a
flour like consistency. The powdered mushroom was extracted using the manual extraction
method outlined in Figure 1.
One hundred grams of powdered mushroom was mixed with 1600 mL of 80% EtOH.
The mixture was heated to 65℃ for 3 hours then left to cool overnight. The solution was then
suction filtered through a no. 2 filter paper. The filtrate was rotary-evaporated and slant
frozen at -80℃ before being lyophilized.

80

The residue from ethanol extraction was then transferred to a clean flask and topped
up with 1600 mL of 50% MeOH. This was heated to 65℃ for 3 hours and allowed to cool
overnight. This was then filtered through a no.2 filter and required no neutralization prior to
rotary-evaporating, slant freezing at -80℃, and lyophilizing.
The residue from methanol extraction was transferred to a clean flask and topped up
with 1600 mL of distilled water. The mixture was then heated to 65℃ for 6 hours and
allowed to cool overnight. The solution was suction-filtered through no.2 filter papers,
changing the filter three times due to clogging. The filtrate was then neutralized from pH
5.44 to pH 7.00 with the addition of 5% NaOH. This was then rotary-evaporated, slant frozen
at -80℃, and lyophilized. The second batch of extract yielded 4.5183 g of P. aurea E3
(4.51% yield) and was used in polysaccharide targeted purification when the first batch of
hot water extract was depleted.
3.2.2 Column Chromatography
Purification was performed on the crude hot water extracts E3 collected from the
fruiting bodies of P. aurea. Activity of semi-purified samples was assessed using ELISA
during all purification steps to ensure that the activity had not been lost. Initially, sizeexclusion chromatography (Sephadex LH-20) was used to separate the crude extract into
fractions. Active fractions were then tested for carbohydrate and protein content. Following
this, a new approach to purification was attempted based on the belief that the active
compound contained polysaccharides. Precipitation methods (see pages 87-88) were then
applied to the crude hot water extract to remove contaminants from the sample. Following
this, the precipitated material was subjected to ion-exchange chromatography (DEAE
Sephadex A-50). The eluted material was then run on a high-resolution chromatography
81

column (Sephacryl S500 HR). Active fractions were then subjected to carbohydrate and
protein quantification, as well as enzyme digestion studies to determine whether the active
components of the compound were protein and/or carbohydrate. Active fractions were finally
pooled and lyophilized for further characterization studies.
3.2.2.1 Size-Exclusion Chromatography
Sephadex LH-20 was used as a preliminary separation agent in an attempt to estimate
the size of active compounds. With an exclusion limit of 4-5 kDa, it is commonly used for
the purification of natural products targeting low molecular weight compounds121. A smallscale size exclusion column was prepared for preliminary analysis. Sephadex LH-20 was
prepared as per the manufacturer’s protocol121. Media was swelled in an excess of sterile
water, overnight, at room temperature. A 75% packed media and 25% water slurry was made
by drawing off the excess supernatant water and swirling the settled media into suspension.
This slurry was poured into a modified serological pipette and packed with water via gravity
drip. After running over 3 bed volumes of water through the column, its final packed volume
was 30.4 mL.
Phaeolepiota aurea E3 crude extract was prepared at a concentration of 100 mg/mL
in sterile water. This solution was pH neutralized and filter sterilized through a 0.2 µm
syringe filter. Six-hundred microliters (2% of bed volume) of the sterile solution was loaded
onto the column. The sample was run with sterile water via gravity drip (approximately 21.76
mL/hour). A total of forty 1.5 mL fractions were collected by running 2 bed volumes of
water through the column. The column was flushed with a minimum of two bed volumes (60
mL) of water prior to running the next batch of sample. A total of 3 runs were performed

82

using the small-scale column. All collected fractions were stored at 4℃ until ELISA and
MTT assays were performed (as per protocols on pages 17-22).
A large-scale column was prepared in a similar fashion. Sephadex LH-20 media was
swelled in an excess of sterile water. The swelled media and additional sterile water was
degassed to prep for column packing. A 75% slurry was made with the degassed media and
poured into a C16/70 column. The column was then packed at a flow rate of 2.5 mL/min,
resulting in a final bed volume of approximately 128.68 mL. A 100 mg/mL solution of P.
aurea crude extract E3 dissolved in water was prepared as above, and 2.2 mL was loaded
onto the column. The column was run at a flow rate of 1.0 mL/min with degassed sterile
water. Fractions were collected every 10 minutes with the use of an auto-collector, for a total
of 26 fractions (approximately 2 bed volumes). The column was flushed with a minimum of
2 bed volumes prior to running more sample. The large-scale column was run a second time
under the same experimental conditions. Collected fractions were stored at 4℃ for further
analysis.
3.2.2.2 Ion-Exchange Chromatography
DEAE Sephadex A50 is a weak anion exchange media for use in ion-exchange
chromatography. Under the correctly buffered conditions, negatively charged compounds
will bind to the media, allowing positively charged compounds to flow through. The bound
compounds could then be removed from the column by flushing it with a buffer containing
NaCl. The chloride ions displace the bound ions from the media, allowing them to be eluted.
This allows for the separation of compounds by their net charge. As DEAE Sephadex A50 is
less ridged in structure and is prone to variability in volume relative to pH and ionic strength,
it has been suggested that this media is better suited to a batch method approach for
83

separation122. Batch method involves equilibrating the media in a buffer of choice, and then
stirring it in with the solution to be treated. Once the slurry reaches equilibrium, it can be
filtered and washed with more buffer. The filtrate can then be applied to a new media bed to
capture any additional compounds. The filtered media would then be stirred in with elution
buffer to release the bound compounds and filtered to retrieve the product122.
Initially a small scale approach was used to test various buffer systems for their
ability to allow the active compound to bind to the media.123. This was done by equilibrating
DEAE Sephadex A50 in one of 6 buffer systems: N-methylpiperazine, piperazine, bis-TRIS,
triethanolamine, 1,3-diaminopropane, and water. Buffer was changed a minimum of 3 times
during the swelling of the media. Equilibrated media was then mixed with an equal volume
of 1 mg/mL post-Sevag extract (see precipitation methods, page 84-85) dissolved in loading
buffer. This was left for two hours to bind. Treatments were then spun down at 200 x g and
the supernatant solution drawn off as the flow through. The media beds were then washed
with an equal volume of loading buffer, and again spun down and the supernatant drawn off
as the wash. Media beds were then treated with elution buffer (1 M NaCl in loading buffer)
for 30 minutes before being spun down and the supernatant collected as the elution. Solutions
were stored at 4℃. The collected samples were pH neutralized prior to performing ELISA.
A two-stage batch method approach was taken when experiments were scaled up.
Treatment was initially applied to a primary media bed and the filtrate from this was then
applied to a secondary media bed to bind any compound that may have been in excess.
DEAE Sephadex A50 media was swelled in a 0.2 M NaCl overnight, to prevent the beads
from swelling too quickly and breaking122. This solution was drawn off the following day
and the media was topped up with an excess of pure milli-Q water. The water was changed
84

on the swelling media an additional 3 times during swelling. The swelled media was then
suction filtered with a bottle top filter (Filtropur BT50 0.2, 500 mL) and divided between two
flasks. One media bed was topped up with excess milli-Q water and set aside. The other was
topped up to the top of media bed with milli-Q water, and a solution of post-Sevag sample
was applied. This was swirled into the media and left to bind for 2 hours. The media was then
filtered and rinsed with fresh milli-Q water. Once filtered, the media was transferred into a
flask and topped up with an excess of 1-2 M NaCl solution. This was left for 1 hour before
the media bed was filtered and rinsed with additional NaCl solution. The media bed was
transferred to a flask and topped up with excess milli-Q water to begin re-swelling. The
elution was set aside.
The water was filtered off the secondary media bed, and the filtrate from the primary
media bed was applied to it. This was left to bind excess compound for an hour before the
secondary media bed was filtered and transferred to a flask. It was then topped up with 1-2 M
NaCl solution for 30 minutes. The media bed was then filtered and rinsed with excess NaCl
solution. The filtered media was transferred to a flask and topped up with excess milli-Q
water to begin re-swelling the media. The elution was combined with the elution from the
primary media bed.
The combined elutions were rotary-evaporated to reduce their volume. The
concentrated elution was then subject to dialysis to remove the NaCl. Concentrated solution
was poured into 3500 MWCO Snakeskin Dialysis Tubing and tied off. The dialysis tubes
were suspended in an excess of milli-Q water with stirring. This was stored at 4℃ for the
duration of dialysis. Water was changed a minimum of 5 times depending on salt
concentrations of tubes (dependent on scale of experiment). Dialyzed samples were then
85

rotary-evaporated to workable volumes, slant frozen and lyophilized. Dried sample was then
subject to ELISA and further purification.
3.2.2.3 High-Resolution Size-Exclusion Chromatography using Sephacryl S-500
This portion of the purification was performed with the help of Mr. Sebastian
Mackedenski. A HiPrep 26/60 Sephacryl S-500 HR column was obtained from GE
Healthcare. This column has a fractionation range of 4 x 104 – 2 x 107 Da and is intended for
the separation of polysaccharides and macromolecules. The column was loaded onto an
AKTA PURE chromatography system. The column was equilibrated with degassed sterile
water. Dextran standards were run to determine the size separation of the column. The
column was flushed with more water prior to performing a sample run. One hundred
milligrams of post DEAE sample was dissolved in 2.0 mL of sterile water. The solution was
centrifuged at 10,000 x g for 10 minutes to pellet out any insoluble material. The supernatant
solution was drawn off and loaded into a 2 mL injection loop on the AKTA PURE system.
This was then loaded onto the column by the system. The column was run at a flow rate of
1.3 mL/min, collecting a total of fifty 10 mL fractions with the use of a fraction collector.
Following the water run, the column was flushed with a 150 mM NaCl solution. This was run
at a flow rate of 1.3 mL/min, collecting an additional fifty 10 mL fractions. Collected
fractions were stored in a 4℃ cooler until used in further assays. The column was reequilibrated with sterile water prior to running more sample. A total of 4 runs were
performed on the high-resolution column. The three most active fractions of each run were
pooled, lyophilized and sent for structural characterization. The most active fractions seen in
ELISA were sent for cytokine analysis using Mouse Cytokine Array/Chemokine Array 32-

86

Plex (Eve Technologies Corporation, Calgary) after treatment on mouse macrophage Raw
264.7 cells.
3.2.3 Carbohydrate Quantification
Initially, a total carbohydrate colorimetric assay kit (BioVision, USA) was used to
determine the carbohydrate content of post-column fractions. The assay was performed based
on the manufacturer’s protocol. Glucose standards were prepared via dilution in distilled
water in their respective wells on a 96-well plate (0, 4, 8, 12, 16, 20 µg/well), for a total of 30
µL/well. Samples were then loaded in duplicate onto the plate at a volume of 30 µL/well.
Concentrated sulfuric acid (H2SO4) was added, 150 µL, to each of the standard and sample
wells. The plate was shaken for one minute on a plate shaker, and then incubated at 90℃ for
15 minutes. Following incubation, 30 µL of developer was added to each well and the plate
was shaken for 5 minutes at room temperature. The absorbance was then read at 490 nm on a
Synergy plate reader. A standard curve was plotted and the carbohydrate concentration of
samples was calculated.
Later in the study, a phenol-sulfuric carbohydrate assay was adopted for carbohydrate
analysis. Glucose standards were prepared in distilled water (0-1000 µg/mL). Fifty
microliters of standard or sample was loaded onto a 96-well plate in triplicate. To this, 50 µL
of a 6% phenol solution was added, followed by an additional 125 µL of concentrated
H2SO4. The plate was then incubated at room temperature for 30 minutes before the
absorbance was read at 490 nm using a plate reader. A standard curve was plotted and the
concentration of carbohydrate in each of the samples was calculated.

87

3.2.4 Protein Quantification
A Pierce BCA Protein Assay Kit (Thermo Scientific, USA) was used to determine the
protein content of post-column fractions. The assay was performed as according to the
manufacturer protocol124. BSA standards were prepared by dilution in water (0-2000 µg/mL).
The BCA working reagent was made by mixing 50 parts of BCA Reagent A with 1 part of
BCA Reagent B. Twenty-five microliters of either standard or sample was pipetted into the
wells of a 96 well plate, in duplicate. To this, 200 µL of BCA working reagent was added.
The plate was shaken for 30 seconds on a plate shaker and then covered and incubated at
37℃ for 30 minutes. Following incubation, the plate was allowed to cool to room
temperature before the absorbance was read at 562 nm using a plate reader. A standard curve
was plotted and the protein content of the samples was calculated.
3.2.5 Precipitation Methods
When dealing with hot water extracts, a common starting point for purification is the
use of precipitation to help remove impurities from the sample, often used in the isolation of
polysaccharides84. Two methods of precipitation were initially compared; ethanol
precipitation and the Sevag method125.
For the ethanol precipitation, a 40 mg/mL solution of crude E3 was prepared. This
was mixed with 5 volumes (or greater) of 100% EtOH and placed in a -20℃ freezer for an
hour to facilitate precipitation. The precipitate solution was then centrifuged at 3000 RPM for
10 minutes to form a pellet. The supernatant solution was drawn off and the pellet was
washed with chilled 100% EtOH. The EtOH was drawn off and the pellet was resuspended in
a small amount of sterile water before being slant frozen at -80℃ and lyophilized.

88

Sevag method involved shaking the sample with a chloroform solution. Crude E3 was
dissolved in water to make a 40 mg/mL solution. A 1/5 butanol-chloroform solution was also
prepared. Equal parts of E3 and butanol chloroform solution were mixed and shaken
vigorously for 5-10 minutes. The samples were then centrifuged at 3000 RPM for 10 minutes
to separate the organic and aqueous layers. The aqueous supernatant solution was drawn off,
being careful not to disturb the interphase, and transferred to a new tube. This was topped up
with an equal part of fresh butanol-chloroform solution and shaken again for 5 – 10 minutes
before being centrifuged and the supernatant collected. This was repeated for a total of 3
chloroform shakes (or until little to no denatured protein was observed in the interphase).
After the final shake, the supernatant solution was drawn off and subject to ethanol
precipitation as per the preceding procedure. Dried precipitates were then subject to ELISA
and further purification.
3.2.6 Enzyme Digestion
In order to determine the active component of the immuno-stimulatory compound of
P. aurea, an enzyme digestion was performed on the bioactive fractions collected from
Sephacryl-S500 run. It was expected that the activity would be reduced or eliminated if the
active component was digested. Three enzymes were used for this purpose: cellulase,
amyloglucosidase, and proteinase-K (Sigma Aldrich). Cellulase and amyloglucosidase target
polysaccharides, with cellulase hydrolyzing endo-1,4-β-D-glucosidic linkages and
amyloglucosidase hydrolyzing α-D-glucosidic bonds. Proteinase-K is a non-specific protein
digester, often used to remove protein contaminants from DNA samples. This enzyme is
known to cleave peptide bonds adjacent to carboxyl groups.

89

Post-Sephacryl 500 fraction, 2 mg/mL Krestin (PSK), and sterile water samples were
aliquoted into clean Eppendorf tubes, 250 µL each. A 109 mg/mL cellulase solution was
prepared by dissolving 50 mg cellulase in 458 µL of sterile water (120 units/mL). A 5
mg/mL solution of amyloglucosidase was prepared by dissolving the contents of a 5 mg vial
in 1.0 mL of sterile water (approximately 225 units/mL). Proteinase-K was available in
solution (600 units/mL). Cellulase was treated into samples at two dose levels; 1 unit and 3
units. This was done by adding 8.33 µL or 25 µL of the prepared solution. Amyloglucosidase
was treated into samples at doses of 0.3 unit and 1 unit, by adding 6.6 µL or 22.2 µL of the
prepared enzyme solution. Proteinase-K was treated at a ratio of 1:50 and 1:10, as suggested
by Mr. Sebastian Mackedenski. Five microliters or 25 µL of the enzyme solution was added
into sample vials, equaling doses of 3 or 15 units. Twenty-five microliters of sterile water
was added to the controls to mimic the dilution effect of treatments. All treatments were then
placed in a heating block and heated to 37℃ for 2 hours. Following this, the samples were
brought up to 80℃ for 30 minutes, to denature the enzymes. Samples were then stored at 4℃
until ELISA was performed.
A higher dose repeat of cellulase and amyloglucosidase digestion was performed
using the same procedure. In this experiment, both enzymes were treated at doses of 10 and
30 units. Post digestion samples were stored at 4℃ until ELISA was performed.
3.3 Results
3.3.1 Sephadex LH-20
Based on the bed volume of the small scale Sephadex LH-20 column, and an assumed
35% exclusion volume (based on previous researcher’s findings), the exclusion volume
would consist of fractions LH1-7. A front of activity was observed at this limit, with both
90

immuno-stimulatory and cytotoxic activity seen in the early stages of column elution. As
shown in Figure 43, the stimulatory activity was observed at the front of elution and seen to
taper off slowly over the column run, with maximum stimulation being observed in fractions
LH7 and LH9-11. Cytotoxic activity was prominent at the exclusion limit, with fraction LH8
killing Raw cells in the first run, and fractions LH7-8 killing cells in the second run (Figure
44). These trends were reproduceable amongst column runs, however minor shifts in the
fractions were observed due to loading error, as the column was manually loaded and ran
(Figure 44). MTT assay was performed, encompassing the cytotoxic fractions of column runs
1-3. As seen in Figure 45 (representative data), fractions LH7-9 were cytotoxic against HeLa
cells. Based on these observations, it was suspected that the bioactive compound(s) of

1500

1000

500

0

H

2
L O
H
L 5
H
L 7
L H
H 9
L 1
H 1
L 1
H 3
L 1
H 5
L 1
H 7
L 1
H 9
L 2
H 1
L 2
H 3
L 2
H 5
L 2
H 7
L 2
H 9
L 3
H 1
L 3
H 3
L 3
H 5
L 3
H 7
3
9

T N F -

C o n c e n tr a tio n (p g /m L )

interest is relatively large in size.

Figure 43. ELISA of post-Sephadex LH20 fractions from the first small column run. Raw 264.7 cells
were plated at a density of 100,000 cells/well. Fractions were treated at a 50% dilution in wells. LPS
was treated at a well concentration of 500 ng/mL as a positive control (data not shown). Fraction 8 was
observed to kill cells. Error bars represent SEM.

91

R un #1
2000

R un #2

1500

1000

500

4

5

6

1

1

1

L

L

H

3
1
L

H

2
H
L

H

1

1

L

H

0

1
H

L

L

L

H

1

9

8

H

H
L

L

H

7

6

5

H

H

L

H

2 O

0

L

C o n c e n tr a tio n (p g /m L )
T N F -

2500

150

100

50

H
L 8
L H9
H
L 10
H
L 11
H
L 12
H
L 13
H
L 14
H
L 15
H
L 16
H
L 17
H
L 18
H
L 19
H
2
0

7

H

L

L

5

6

H

L

H

H

L

H

L

L

4

0

3

C e ll V ia b ility (%

o f C o n tr o l)

Figure 44. ELISA of post-Sephadex LH20 fractions, comparing small column runs 1 and 2. Raw
264.7 cells were plated at a density of 100,000 cells/well. Fractions were treated at a 50% dilution in
wells. LPS was treated at a well concentration of 500 ng/mL as a positive control (data not shown).
Fraction 7 was observed to kill cells in run #2, and fraction 8 was observed to be cytotoxic in both
runs. Error bars represent SEM.

Figure 45. MTT assay of post-Sephadex LH-20 small column run #3. HeLa cells were plated at a
density of 3000 cells/well. Fractions were 50% diluted in the treatment wells. Plates were scanned after
48 hours of treatment. Error bars represent SEM.

92

A scaled-up column was used in an attempt to achieve better separation of the
compounds. It was calculated to have its exclusion volume occurring within fractions LH1-5.
Once again, cytotoxic activity was observed to elute close to this exclusion value. Again, a
spread in cytotoxic activity was observed with run #2, which was believed to be caused by a
headspace formed during loading of the column. Interestingly, it appears that there may be
two compounds causing cytotoxic activity, with drops in cell viability being seen around
fraction LH5 and then again at fraction LH8 (Figure 46). ELISA found a stimulatory spike
between fractions LH7-10 for the first large column run, which shifted to LH8-10 for the
second column run. Peak stimulation was observed in fraction LH8 of both runs (Figure 47).
The shift was again believed to be due to loading error or may show that flushing the column

R un #1
100

C e ll V ia b ility (%

R un #2

50

8
L H
H 9
L 10
H
L 11
H
L 12
H
L 13
H
L 14
H
L 15
H
L 16
H
L 17
H
L 18
H
L 19
H
2
0

7

H

L

6

H

L

5

H

L

L

4

H

3

H

L

H

L

C

ru

d

e

E

3

0

L

o f C o n tr o l)

with water alone may not be removing all residues from the media.

Figure 46. MTT assay of post-Sephadex LH-20 large column runs 1 and 2. HeLa cells were plated at a
density of 3000 cells/well. Fractions were 50% diluted in the treatment wells. Plates were scanned after
48 hours of treatment. Error bars represent SEM.

93

R un #1
20000

R un #2

15000

10000

5000

6
1

H

4

5
1

H

L

L

3

1

1

H

L

L

H

1

2

1

1

H

H

L

L

9

0

1

H

H

L

L

7

8

H

6

H

L

L

L

H

5

4
L

H

3

H

3
E

H

L

H
C

ru

d

e

2 O

0

L

C o n c e n tr a tio n (p g /m L )
T N F -

25000

Figure 47. ELISA of post-Sephadex LH20 fractions, comparing large column runs 1 and 2. Raw 264.7
cells were plated at a density of 100,000 cells/well. Fractions were treated at a 50% dilution in wells.
LPS was treated at a well concentration of 500 ng/mL as a positive control (data not shown). Fractions
LH5 and LH6 were observed to kill cells in both runs. Error bars represent SEM.

3.3.2 Carbohydrate and Protein Quantification
Large column Sephadex LH-20 fractions were subject to carbohydrate and protein
quantification of the active fractions. Fractions LH5-10 were tested in an attempt to
determine the makeup of the fractions, and thus, what compounds may be responsible for
activity. The strongest stimulatory activity was seen in fraction LH8 (Figure 47), which
corresponded to the highest carbohydrate content seen in the active fractions (Figure 48), as
well as the second strongest peak in protein content (Figure 49). Likewise, the strongest
cytotoxic activity was observed in fraction LH5 (Figure 46), which was observed to have the
highest protein concentration of the tested fractions (Figure 49). In order to determine the
effects of protein content on immuno-stimulatory activity, active fractions were heated to
95℃ for 40 minutes in an attempt to denature protein within the samples. Heat treatment was
only observed to significantly affect fraction LH8 of the first column run, causing a mild
94

drop in TNF-α production relative to the untreated fraction, but not completely eliminating
activity (Figure 50). Due to this, it was believed that the fractions still contained a mixture of
bioactive compounds. As the activity was not completely removed with heat, and due to the
high carbohydrate content, it was suspected that the stimulatory activity seen in fraction LH8
was partially caused by carbohydrates. The fact that heat treatment was not observed to affect
fraction LH7 or LH9 also suggests the bioactive compound is not entirely made up of
protein. It was decided that a more targeted approach would be taken to purify the bioactive

3
>

2

0

0
3
>

2

R un #1

0

0

4000

R un #2

3000

2000

1000

1

0

9
L

L

H

H

8
H
*L

L

H

7

6
H
L

H

5

0

L

C a r b o h y d r a t e C o n c e n t r a t io n (  g /m L )

compound(s) from the crude P. aurea extract.

Figure 48. Total Carbohydrate Assay Kit results for large column Sephadex LH-20 runs 1 and 2.
Fraction 8 of both runs exceeded the bounds of the Synergy 2 plate reader and would be greater than
3200 µg/mL in concentration based on the maximum absorbance of the machine. Error bars represent
SEM.

95

R un #1
R un #2
400

200

2

3

4

1

1

1
H

L

L

H

1
1
L

L

L

H

H

0

9

1

H
L

H

7

H
L

L

L

H

H

6

5

4
L

L

H

H

H
L

8

0

3

P r o te in C o n c e n tr a tio n ( g /m L )

600

20000

U n tre a te d
H e a t T re a te d

15000

10000

5000

2
H
L

L

H

2

-9

-8

-5

L

H

2

1
H
L

L

H

1

-9

-8

-7

L

H

1
H
L

1

3
E

C

ru

d

e

H

-5

0
2 O

T N F -

C o n c e n tr a tio n (p g /m L )

Figure 49. Pierce BCA Protein Assay Kit results for Sephadex LH-20 large column runs 1 and 2. Error
bars represent SEM.

Figure 50. Effects of heat on immuno-stimulatory activities seen in post-Sephadex LH-20 fractions.
Active fractions were heated to 95℃ for 40 minutes. Error bars represent SEM.

96

3.3.3 Precipitation Methods
A Sevag purification method was optimized and compared against simple ethanol
precipitation. Sevag purification was observed to effectively remove contaminants, showing
little to no interphase material after 3-4 chloroform shakes with equal volumes of 40 mg/mL
crude extract E3. Sevag method produced yields between 37%-52% of starting material
while ethanol precipitation alone yielded approximately 50% of starting material. The yield
was seen to improve slightly with the use of a larger EtOH volume ratio during the
precipitation steps. Post-Sevag precipitates were observed to show significant deviation from
the crude E3 activity, having a slightly higher stimulatory value than its crude predecessor
(Figure 51). Ethanol precipitation alone did not significantly improve the activity of crude
E3. A dose-dependent assay also confirmed that Sevag precipitation was more effective than
ethanol precipitation alone, showing significantly higher activity at all tested concentrations,
whereas ethanol precipitates were only significantly higher at doses of 0.05 and 0.5 mg/mL
(Figure 52). Due to its higher stimulatory values which suggests its effectiveness as a
purification step, the Sevag method was adopted as the first step in the purification of the
immuno-stimulatory compound(s) from crude extract E3 of P. aurea.

97

3000

2000

1000

P

P

P
g
a
v
e
S

E

C

tO

ru

H

d

e

P

E

2 O

H

T

T

0

3

C o n c e n tr a tio n (p g /m L )
T N F -

4000

T N F - C o n c e n tr a tio n (p g /m L )

Figure 51. ELISA of precipitates from E3 purification: comparing Sevag method with ethanol
precipitation. Raw 264.7 cells were plated at a density of 100,000 cells/well. Treatments were applied
at well concentrations of 1.0 mg/mL. LPS was treated at a well concentration of 500 ng/mL as a
positive control (data not shown). Error bars represent SEM.

8000

H 2O
C ru d e E 3

6000

E tO H P P T
4000

S evag P P T

2000

0
0 .0

0 .5

1 .0

C o n c e n tr a tio n (m g /m L )

Figure 52. Concentration-dependent ELISA of precipitates from E3 purification: comparing Sevag
method with ethanol precipitation. Raw 264.7 cells were plated at a density of 100,000 cells/well.
Treatments were applied at well concentrations of 0.05, 0.1, 0.5, and 1.0 mg/mL. LPS was treated at a
well concentration of 500 ng/mL as a positive control (data not shown). Error bars represent SEM.

98

3.3.4 DEAE Sephadex A50
Small scale DEAE Sephadex ion-exchange chromatography was effectively used to
choose a buffer system for the binding of bioactive compounds from Sevag-purified E3. The
flow through collections were tested against the initial treatment solutions to see if there was
a decrease in activity, suggesting that the active compounds had bound to the media. Samples
were tested at a 1/8 dilution in wells to reduce buffer related toxicity and morphological
effects seen in higher dose treatments. As shown in Figure 53, bis-TRIS was the only buffer
that did not show significant deviation from the treatment solution in its flow through
collection. All other buffers used showed some binding to different extents. The most
effective system for binding of active compound was water, showing 82% decreased activity
between treatment and flow through collections, suggesting that the bioactive compound(s)
bound to the media when water was used. The second most effective buffer was 1,3diaminopropane, decreasing activity by 56% in its flow through collection (Figure 53). Due
to its higher binding capacity, lack of toxicity, and ease of use, the water system was selected
for large scale DEAE Sephadex A50 purification. Sodium chloride elution collected from the
column was dialyzed to remove the salt before the sample was added to cells. Comparison of
the water collections show that this system was effective in binding immuno-stimulatory
compound, with the elution collection showing significantly higher activity than the flow
through. However, the activity from the elution was statistically similar to that of the
treatment solution (Figure 54).

99

C o n tro l
T re a tm e n t
10000

F lo w T h r o u g h
W ash

5000

T N F -

2 O

H

e
a

ia
-D
1

,3

th
e
T

ri

p
ro
p
o
in

m

o
n
a

e
-M
N

n

L
C
-H
e
in

la

P

m

ip

B

e

is

ra

-T

z

R

in

e
in
z
a
ir
ip
th

y

lp

IS

0

e

C o n c e n tr a tio n (p g /m L )

15000

Figure 53. ELISA of post-DEAE Sephadex A50 collections, comparing the effectiveness of different
buffer systems. Controls are 20 mM buffer solution. Treatment solutions consisted of 1.0 mg/mL of
post-Sevag precipitate dissolved in buffer. Raw 264.7 cells were plated at a density of 100,000
cells/well. Solutions were applied to cells at a 1/8 dilution in wells (approximately 0.125 mg/mL). LPS
was treated at a well concentration of 500 ng/mL as a positive control (data not shown). Error bars
represent SEM.

100

7500

5000

2500

n

h
lu
E

W

a

ti

o

s

h
g
u
h

F

lo

w

T

T

re

C

a

o

tm

n

e

tr

n

o

t

l

0

ro

C o n c e n tr a tio n (p g /m L )
T N F -

10000

Figure 54. ELISA of post-DEAE Sephadex A50 using water as a buffer system. Treatment solution
consisted of 1.0 mg/mL of post-Sevag precipitate dissolved in water. Flow through and wash solutions
were treated directly from their collections. Elution was dialyzed, lyophilized, and re-dissolved in
water equal to the volume of solution originally applied to the media bed. Raw 264.7 cells were plated
at a density of 100,000 cells/well. Solutions were applied to cells at a 1/8 dilution in wells
(approximately 0.125 mg/mL). LPS was treated at a well concentration of 500 ng/mL as a positive
control (data not shown). Error bars represent SEM.

Large scale DEAE Sephadex A50 proceeded with similar results. The dialyzed
elutions from large batch applications yields between 25-36% of the starting material.
Interestingly, treating larger bed volumes with smaller amounts of post-Sevag precipitate
caused smaller percent yields. As colour was still seen on the media following elution, it is
suspected that a portion of the compound was not removed with 1 M NaCl elution. Elution
buffer concentration was therefore increased to 2 M NaCl. However, this did not
significantly improve yields in larger batch applications, and some colour still remained in
the media. Eluted compound did not show any significant difference in activity from the
post-Sevag precipitate (Figure 55).

101

4000

2000

S

E
o
P

e

v

s

t

a

g

D

P

H

P

A

T

E

0
2 O

T N F -

C o n c e n tr a tio n (p g /m L )

6000

Figure 55. ELISA of post-DEAE Sephadex A50 using water as a buffer system. Raw 264.7 cells were
plated at a density of 100,000 cells/well. Solutions were applied to cells at a treatment concentration of
0.5 mg/mL. LPS was treated at a well concentration of 500 ng/mL as a positive control (data not
shown). Error bars represent SEM.

3.3.5 Sephacryl S500 HR
Elutions from the DEAE Sephadex A50 column were dialyzed and freeze dried. The
powder was then used for high resolution size exclusion chromatography. In the first column
run, a single significant peak of activity occurred within the fractions comprising 263-323
mL elution volume. Of these the top three active fractions occurred between the elution
volumes of 273-293 mL, corresponding to a compound sizing between 270 and 410 kDa
(Figure 56). Carbohydrate and protein quantification of the active fractions found that the
peak concentrations of these compounds corresponded with the peak of TNF-α production. It
was noted that the shape of the active peak corresponded well with the shape of the protein
peak, suggesting that protein may be a key component in activity (Figure 57). However, due
to the strong presence of carbohydrate within the fractions, it was suspected that the active
compound may be a carbohydrate-protein complex. The most active fraction, collected at
102

elution volume 283 mL, was subsequently used in enzyme digestion analysis in order to
determine which component may be responsible for activity. Upon washing the column with
0.15 M NaCl solution, a second peak was detected by the UV detector of the AKTA PURE
system. It was decided that the NaCl wash would be collected following the second column

T N F -

10000

10000

S e p h a c ry l s 5 0 0 H R
D e x tra n S ta n d a rd s

8000
1000
6000

4000
100

S iz e (K D a )

C o n c e n tr a tio n (p g /m L )

run to test this peak for activity.

2000

0

10
0

100

200

300

400

500

V o lu m e (m L )

Figure 56. ELISA of fractions collected from Sephacryl S500 HR column chromatography. PostDEAE Sephadex A50 purified samples were loaded onto Sephacryl S-500 column. Fractions collected
were applied to Raw cells in 96-well plates at a 50% dilution. LPS was treated at a well concentration
of 500 ng/mL as a positive control (data not shown). Error bars represent SEM. In separate Sephacryl
S500 runs, Dextran Standards were loaded and the fractions collected were analyzed for presence of
the standards.

103

8000
2000
6000

4000
1000
2000

0
240

0
260

280

300

320

C o n c e n tr a tio n ( g /m L )

T N F - C o n c e n tr a tio n (p g /m L )

3000

C a r b o h y d r a te a n d P r o te in

10000

T N F - a C o n c n e t r a t io n
C a rb o h y d ra te C o n te n t
P r o t e in C o n t e n t ( u g / m L )

340

V o lu m e (m L )

Figure 57. TNF-α production of the first Sephacryl S500 HR column run in relation to carbohydrate
and protein content. Raw 264.7 cells were plated at a density of 100,000 cells/well. Eluted fractions
were applied to wells at a 50% dilution. LPS was treated at a well concentration of 500 ng/mL as a
positive control (data not shown). Error bars represent SEM.

A second run of the Sephacryl S500 HR column was performed under similar
conditions to the first run. Following a 483 mL elution of the column with water, the elution
buffer was then switched to 0.15 M NaCl solution, collecting wash fractions to determine if
active compounds had bound to the column. Again, a single significant active peak was
observed within the water elution fractions, occurring between 283-333 mL elution. The top
three active fractions occurred between 283-303 mL. The NaCl wash fractions yielded a
single active peak as well, occurring between elution volumes of 776-796 mL (Figure 58),
suggesting that some active compound may be binding to the column under water elution
conditions. Carbohydrate and protein analysis of the active fractions found the presence of
both compounds within the active fractions, again with the general shape of the protein peak
being slightly more relevant to that of activity (Figure 59). The NaCl wash fractions also had
a significant amount of carbohydrate and protein within their active peak (Figure 60),
suggesting the second peak may be caused by a carbohydrate-protein complex.

104

10000

S w itc h to 0 .1 5 M

N aC l

N a C l (0 .1 5 M )

5000



T N F - C o n c e n tr a tio n (p g /m L )

H 2O

0
0

200

400

600

800

1000

V o lu m e (m L )

1500

10000
8000

1000

6000
4000

500

2000
0
240

0
260

280

300

320

C o n c e n tr a tio n ( g /m L )

T N F -

12000

C a r b o h y d r a te a n d P r o te in

C o n c e n tr a tio n (p g /m L )

Figure 58. ELISA of post-Sephacryl S500 HR column chromatography fractions, with NaCl wash
data, for the second column run. Column was eluted with 483 mL of water, and then washed with 0.15
M NaCl solution. Eluted fractions were applied to Raw cells in 96-well plates at a 50% dilution. LPS
was treated at a well concentration of 500 ng/mL as a positive control (data not shown). Error bars
represent SEM.

T N F -  C o n c n e t r a t io n
C a rb o h y d ra te C o n te n t
P r o t e in C o n t e n t

340

V o lu m e (m L )

Figure 59. TNF-α production of the second Sephacryl S500 HR column run in relation to carbohydrate
and protein content. Eluted fractions were applied to Raw cells in 96-well plates at a 50% dilution.
LPS was treated at a well concentration of 500 ng/mL as a positive control (data not shown). Error bars
represent SEM.

105

C o n c e n tr a tio n (p g /m L )

6000
100
4000
50
2000

0
720

0
740

760

780

800

C o n c e n tr a tio n ( g /m L )

T N F -

150

C a r b o h y d r a te a n d P r o te in

8000

T N F -  C o n c e n t r a t io n
C a rb o h y d ra te C o n te n t
P r o t e in C o n t e n t

820

V o lu m e (m L )

Figure 60. TNF-α production of the second Sephacryl S500 HR column NaCl wash in relation to
carbohydrate and protein content. Eluted fractions were applied to Raw cells in 96-well plates at a 50%
dilution. LPS was treated at a well concentration of 500 ng/mL as a positive control (data not shown).
Error bars represent SEM.

Similar trends were observed in the third run of the Sephacryl S500 HR column.
Significant activity was seen between elution volumes of 273-343 mL. The top three
fractions were observed between 283-303 mL and contained the highest carbohydrate and
protein contents. Again, the shape of the active peak corresponded with the shape of the
protein peak (Figure 61). The NaCl wash of the column showed a significant peak of activity
between 766-826 mL. This appeared to closely correlate with the carbohydrate content,
however, a spike in protein was also observed in the most active fraction (Figure 62).

106

8000

3000

6000
2000
4000
1000

T N F -

2000

0

C o n c e n tr a tio n ( g /m L )

C o n c e n tr a tio n (p g /m L )

4000

0

200

250

300

350

C a r b o h y d r a te a n d P r o te in

10000

T N F -  C o n c e n t r a t io n
C a rb o h y d ra te C o n te n t
P r o t e in C o n t e n t

400

V o lu m e (m L )

250

200
4000
150

100
2000
50

0

0
760

780

800

C a r b o h y d r a te a n d P r o te in

6000

C o n c e n tr a tio n ( g /m L )

T N F - C o n c e n tr a tio n (p g /m L )

Figure 61. TNF-α production of the third Sephacryl S500 HR column run in relation to carbohydrate
and protein content. Eluted fractions were applied to Raw cells in 96-well plates at a 50% dilution.
LPS was treated at a well concentration of 500 ng/mL as a positive control (data not shown). Error bars
represent SEM.

T N F -  C o n c e n t r a t io n
C a rb o h y d ra te C o n te n t
P r o t e in C o n t e n t

820

V o lu m e (m L )

Figure 62. TNF-α production of the third Sephacryl s500 HR column NaCl wash in relation to
carbohydrate and protein content. Eluted fractions were applied to Raw cells in 96-well plates at a 50%
dilution. LPS was treated at a well concentration of 500 ng/mL as a positive control (data not shown).
Error bars represent SEM.

107

A fourth run was performed on the Sephacryl S500 HR column to increase the amount
of post column fraction for subsequent analysis. Activity peaks were observed between 273323 mL during the water run (Figure 63), and 766-826 mL for the NaCl run (Figure 64). Of
these, the top three fractions occurred between 283-303 mL in the water run, and 776-806 mL
in the NaCl run. The top three fractions of each water run were pooled, lyophilized, and
subjected to further chemical and biological characterization. The top two fractions of each

T N F - C o n c e n tr a tio n (p g /m L )

NaCl run were also pooled, dialyzed, lyophilized and kept for future characterization.

10000

8000

6000

4000

2000

0
200

250

300

350

400

V o lu m e (m L )

Figure 63. TNF-α production of the fourth Sephacryl S500 HR column run. Raw 264.7 cells were
plated at a density of 100,000 cells/well. Eluted fractions were applied to wells at a 50% dilution. LPS
was treated at a well concentration of 500 ng/mL as a positive control (data not shown). Error bars
represent SEM.

108

T N F - C o n c e n tr a tio n (p g /m L )

5000

4000

3000

2000

1000

0
740

760

780

800

820

V o lu m e (m L )

Figure 64. TNF-α production of the fourth Sephacryl S500 HR column NaCl wash. Raw 264.7 cells
were plated at a density of 100,000 cells/well. Eluted fractions were applied to wells at a 50% dilution.
LPS was treated at a well concentration of 500 ng/mL as a positive control (data not shown). Error bars
represent SEM.

3.3.6 Enzyme Digestion
Enzyme digestions were performed on the most active post-Sephacryl S500 HR
fraction from the first column run. The first series of digestions were performed with lower
doses of cellulase and amyloglucosidase, calculated from the manufacturer suggested activity
of the enzyme relative to the carbohydrate content of the sample. A higher dose repeat was
performed for cellulase and amyloglucosidase to observe if higher concentrations could
remove the immuno-stimulatory activity. Immediately apparent was that cellulase stimulated
TNF-α in its water controls, potentially masking its activity on the samples tested (Figures 65
and 66). By subtracting the enzyme control values from the treated sample values, it was
possible to see the effects of enzyme activity that could be masked by such stimulation.
A PSK solution was tested alongside the post-Sephacryl fraction as a positive control.
PSK activity was completely removed by proteinase-K digestion at both treatment
109

concentrations used (Figure 67). Cellulase was also able to reduce PSK activity in a dosedependent manner, completely removing activity in the 10 and 30 unit doses (Figure 67 and

8000

H2O
PSK

6000

4000

2000

m

m

A

A

u
1

-K
e

-K
P

ro

te

in

a

s

e
s
a

in
te

ro

y

P

5

u
.0
3

1
e

s
o
c

lu
g
lo

u
.0

u
0
a
id

id
s
o

c
lu
g

lo
y

s

s
a

s
la
u
ll

e
C

.3

u
3

e

e
s
la
u

ll
e
C

.0

.0
1

e
H
+

l
o
tr

e

t
a

o
tr
n
o
C
o
C

n

u

0

l

T N F -  C o n c e n t r a t io n ( p g /m L )

68). Amyloglucosidase was only effective at its highest tested dose of 30 units (Figure 68).

Figure 65. Effect of proteinase K, cellulose and amyloglucosidase on the immuno-stimulatory activity
of PSK. Two-hundred and fifty microliters of PSK solution was treated with either 1 or 3 units of
cellulase, 0.3 or 1 unit of amyloglucosidase, or 3 or 15 units of proteinase-K. Raw 264.7 cells were
plated at a density of 100,000 cells/well. Eluted fractions were applied to wells at a 1/4 dilution. LPS
was treated at a well concentration of 500 ng/mL as a positive control (data not shown). Error bars
represent SEM.

110

H2O
P o s t- S e p h a c r y l

20000

10000

m

m

A

A

u
1

-K
e
s
a

in

P

ro

te

in
te

ro

y

P

5

u
3

-K
e
s
a

s
o
c

lu
g
lo

.0

.0
1
e

s
a
id

id
s
o

c
lu
g

lo
y

u

u
0

s
a

s
la
u
ll

e
C

.3

u
3

e

e
s
la
u

ll
e
C

.0

.0
1

e
H
+

l
o

e

t
a

o
tr
n
o
C

tr
n
o
C

u

0

l

T N F -  C o n c e n t r a t io n ( p g /m L )

30000

Figure 66. Effect of cellulase, amyloglucosidase and proteinase-K on the immuno-stimulatory activity
of bioactive compound(s) from P. aurea. Two-hundred and fifty microliters of post-sephacryl S500
fraction was treated with either 1 or 3 units of cellulase, 0.3 or 1 unit of amyloglucosidase, or 3 or 15
units of proteinase-K. Raw 264.7 cells were plated at a density of 100,000 cells/well. Eluted fractions
were applied to wells at a 1/4 dilution. LPS was treated at a well concentration of 500 ng/mL as a
positive control (data not shown). Error bars represent SEM.

111

6000

4000

2000

m

m

A

A

u
1

-K
e
s
a

in

P

ro

te

in
te

ro

y

P

5

u
3

-K
e
s
a

s
o
c

lu
g
lo

.0

.0
1
e

s
a
id

id
s
o

c
lu
g

lo
y

u

u
0

s
a

s
la
u
ll

e
C

.3

u
3

e

e
s
la
u

ll
e
C

.0

.0
1

e
H
+

l
o
tr

e

t
a

o
tr
n
o
C
n
o
C

u

0

l

T N F -  C o n c e n t r a t io n ( p g /m L )

8000

Figure 67. Effect of cellulase, amyloglucosidase and proteinase-K, on the immuno-stimulatory activity
of PSK. Two-hundred and fifty microliters of PSK solution was treated with either 1 or 3 units of
cellulase, 0.3 or 1 unit of amyloglucosidase, or 3 or 15 units of proteinase-K. Raw 264.7 cells were
plated at a density of 100,000 cells/well. Eluted fractions were applied to wells at a 1/4 dilution. LPS
was treated at a well concentration of 500 ng/mL as a positive control (data not shown). Error bars
represent SEM.

112

3000

2000

1000

0

0

0

3

1

e

e

s

s

a

a

id

id
y
m
A

A

m

y

lo

lo

g

g

lu

lu

c

c

o

o

s

s

ll
e
C

C

e

ll

u

u

la

la

s

s

e

e

n
o
C

u

u

u
0
3

1

tr

0

o

u

l

T N F - C o n c e n tr a tio n (p g /m L )

4000

Figure 68. Effect of cellulase or amyloglucosidase on the immuno-stimulatory activity of PSK. Twohundred and fifty microliters of PSK solution was treated with either 10 or 30 units of cellulase or
amyloglucosidase. Raw 264.7 cells were plated at a density of 100,000 cells/well. Eluted fractions
were applied to wells at a 1/4 dilution. LPS was treated at a well concentration of 500 ng/mL as a
positive control (data not shown). Error bars represent SEM.

The post-Sephacryl S500 HR fraction was tested under the same conditions as the
PSK control. The most effective enzyme in reducing activity was proteinase-K, showing
reductions in activity at both dose levels (Figure 69). This would suggest that activity may be
protein-dependent. A slight reduction in activity was seen with cellulase digestion at a 3 unit
dose (Figure 69), becoming more pronounced in the higher doses of 10 and 30 units (Figure
70). This suggests the presence of β-glucans, which are also contributing to the activity.
Amyloglucosidase was not observed to significantly affect activity in any dose level tested
(Figures 69 and 70). As activity can be reduced to relatively low levels by both proteinase-K
and cellulase, it is suspected that the bioactive compound could be a β-glucan-protein
complex, requiring both components to be functional for optimum activity.
113

20000

10000

m

m

A

A

u
1

-K
e
s
a

in

P

ro

te

in
te

ro

y

P

5

u
3

-K
e
s
a

s
o
c

lu
g
lo

.0

.0
1
e

s
a
id

id
s
o

c
lu
g

lo
y

u

u
0

s
a

s
la
u
ll

e
C

.3

u
3

e

e
s
la
u

ll
e
C

.0

.0
1

e
H
+

l
o
tr

e

t
a

o
tr
n
o
C
n
o
C

u

0
l

T N F -  C o n c e n t r a t io n ( p g /m L )

30000

Figure 69. Effect of cellulase, amyloglucosidase and proteinase-K on the immuno-stimulatory
compound(s) from P. aurea. Two-hundred and fifty microliters of post-sephacryl fraction was treated
with either 1 or 3 units of cellulase, 0.3 or 1 unit of amyloglucosidase, or 3 or 15 units of proteinase-K.
Raw 264.7 cells were plated at a density of 100,000 cells/well. Eluted fractions were applied to wells at
a 1/4 dilution. LPS was treated at a well concentration of 500 ng/mL as a positive control (data not
shown). Error bars represent SEM.

114

20000

15000

10000

5000

0

0

0

3

1

e

e

s

s

a

a

id

id
y
m
A

A

m

y

lo

lo

g

g

lu

lu

c

c

o

o

s

s

ll
e
C

C

e

ll

u

u

la

la

s

s

e

e

n
o
C

u

u

u
0
3

1

tr

0

o

u

l

T N F - C o n c e n tr a tio n (p g /m L )

25000

Figure 70. Effect of cellulase or amyloglucosidase on the immuno-stimulatory compound(s) from P.
aurea. Two-hundred and fifty microliters of post-sephacryl fraction was treated with either 10 or 30
units of cellulase or amyloglucosidase. Raw 264.7 cells were plated at a density of 100,000 cells/well.
Eluted fractions were applied to wells at a 1/4 dilution. LPS was treated at a well concentration of 500
ng/mL as a positive control (data not shown). Error bars represent SEM.

3.3.7 Mouse Cytokine Array/Chemokine Array 32-Plex
Results of cytokine analysis following treatment onto Raw 264.7 cells were obtained
from Eve Technologies Corporation (Calgary). As seen in Table 5, the post-Sephacryl
fractions were able to stimulate the production of many cytokines. It appears that the
strongest stimulation was seen in pro-inflammatory cytokines, suggesting that the
compounds stimulate the innate immune system. TNF-α production was again confirmed and
suggests an inflammatory response, as it is known to be an immune cell regulator affiliated
with systemic inflammation126. The strongest stimulation was seen in the macrophage
inflammatory proteins (MIP-1α, MIP-1β, and MIP-2). These are known chemotactic agents
in the recruitment and activation of leukocytes and hematopoietic cells in response to injury
115

or infection127–129. Granulocyte colony-stimulating factor (G-CSF) and granulocytemacrophage colony-stimulating factor (GM-CSF) were also induced and are involved in the
proliferation and maturation of neutrophils, macrophages, and eosinophils130. Stimulation of
interferon gamma-induced protein 10 (IP-10) is promising, as this cytokine has been
affiliated with antitumor activity due to its antiangiogenic effects131. It is also a
chemoattractant that promotes the adhesion of T cells to endothelial cells132. Monocyte
chemoattractant protein 1 (MCP-1) is another recruitment protein, attracting monocytes,
memory T cells and dendritic cells to sites of inflammation133,134, supporting the theory of
pro-inflammatory activity in the tested compounds. Interleukin 6 (IL-6) was also moderately
stimulated and is involved in acute phase immune response, showing pro-inflammatory
activities135. While other cytokines were mildly induced, the strength of the ones described
suggest that P. aurea contains compounds that stimulate the innate immune system.
Purification attempts appear to have isolated pro-inflammatory compounds.

116

117
IL-15
3.71
30.44
6.81
14.30

MCP-1
7.07
2255.44
10.42
128.02

H2O Control (Run #2)
Post-Sephacryl (Run #2)
H2O Control (Run #3)
Post-Sephacryl (Run #3)

Post-Sephacryl (Run #3)

H2O Control (Run #2)
Post-Sephacryl (Run #2)
H2O Control (Run #3)

MIP-1α
20.89
7390.43
63.32
5012.63

MIP-1β
22.62
206462.27
62.03
> 206462.27

RANTES
< 0.60
87.13
< 0.60
12.73

IL-1α
1.89
38.45
12.88
19.63

Th2 response
IL-5
IL-9
< 0.55 15.62
3.10
34.99
< 0.55 16.00
< 0.55 18.31

GM-CSF
< 0.77
1798.18
3.28
93.48

IL-4
0.09
0.26
0.12
0.22

G-CSF
0.41
3899.41
1.63
1329.37

IL-12 (p70)
1.93
18.08
7.88
14.87

Eotaxin
< 0.38
4.71
< 0.38
1.22

KC
< 0.24
22.40
0.44
3.51

IP-10
0.05
2441.49
0.59
432.04

H2O Control (Run #2)
Post-Sephacryl (Run #2)
H2O Control (Run #3)
Post-Sephacryl (Run #3)

MIP-2
19.35
14795.86
32.18
3810.28

Th1 response
TNF-α
IL-2
IL-10
< 0.81
0.64 1.42
639
6.15 8.42
2.67
1.40 1.69
473.59 2.28 6.94

IFNγ
< 0.58
5.89
< 0.58
0.58

IL-1β
1.88
16.23
6.72
7.61

LIF
0.17
99.26
< 0.17
10.23

LIX
11.38
195.86
7.73
4.70

Th17 response
IL-17
IL-6
< 0.10
< 0.67
3.21
1554.15
< 0.10
< 0.67
2.13
48.20

Table 6. Cytokine/Chemokine concentrations (pg/mL) obtained from Eve Technologies of post-column fractions treated onto Raw 264.7
cells. Samples represent the 293 mL fractions of Sephacryl s500 HR column runs #2 and #3, and their water controls.

3.4 Discussion
The initial purification attempts on P. aurea hot water extracts appear to show the
presence of multiple bioactive compounds. While primarily immuno-stimulatory, this extract
was also found to contain cytotoxic compounds, which could be separated using Sephadex
LH20 size exclusion method directly from the crude extract. Based on the protein content of
the cytotoxic fraction, one would suspect the compound is primarily protein based and larger
than 4000 kDa due to its early elution from the LH-20 columns. By proceeding with the
Sevag method, a known deproteinating step125,136,137, the cytotoxic activity was eliminated,
supporting the idea that the anti-proliferative compound found in the crude extract is proteinbased. Since the choice was made to pursue the immuno-stimulatory activity in the extract,
the anti-proliferative compound(s) was not further studied and may be a target for future
characterization. Subjecting the immuno-stimulatory fractions to heat did not eliminate its
activity, suggesting that its active compound(s) may not be completely protein dependent.
Due to this, and the high carbohydrate content of the immuno-stimulatory fractions, the
switch to a more carbohydrate targeted approach was taken.
The purification scheme used has been successfully applied to the isolation of
bioactive polysaccharides from mushrooms. Following hot water extraction, crude extracts
were subjected to deproteination, such as Sevag method, and then applied to various
chromatography columns to separate the polysaccharides into homogenous fractions. This is
done commonly with ion-exchange chromatography, followed by size exclusion
methods84,137,138. Following Sevag method deproteinization, the P. aurea crude
polysaccharides were then applied to an ion-exchange media equilibrated in water. The use
of water as a buffer has been successful used in the separation of antitumor
118

polysaccharides138. By using the batch method in experiments, difficulties arising from
variances in volume due to the ionic strength of the elution buffer were avoided. It was also
easier to upscale experiments, allowing for larger volumes, and thus larger quantities of
crude polysaccharides to be processed at a time. As yellowing of the media was observed, it
was suspected that not all of the bound sample was retrieved with elution. This may have
affected the binding of active compounds in subsequent experiments, as media was reequilibrated in water and re-used. The effects of this may be mitigated by the two-stage
approach used, as the secondary media bed would offer more binding opportunity. As yields
were observed to slightly decrease when the bed volume to sample ratio was increased, it is
not likely that the binding capacity of the media was exceeded, but that the media was
perhaps trapping compounds following elution. Even with 2 M NaCl in elution buffer, the
yields were not increased, suggesting the compound may have an affinity for the media.
The purified sample collected from DEAE media was not observed to show any
increase in activity relative to the starting material. This suggests that the DEAE ionexchange chromatography step may not be effective in purifying the bioactive compound(s)
so much as portioning the starting material. The post-Sevag extract may be relatively pure,
and there may be little or no contaminants to be removed with ion-exchange
chromatography. Upon proceeding into size-exclusion chromatography, only one activity
peak was observed, suggesting one active compound, or a group of similarly sized
compounds was retrieved following elution from the DEAE media. Again, binding to the
media was observed with the Sephacryl S500 column, as a secondary front of activity was
collected with the NaCl wash. It is unclear if this is a portion of the same compound or a
separate compound all together. As all active fractions were observed to have both
119

carbohydrate and protein, it is suspected that the activity may be linked to a carbohydrateprotein complex. The phenol-sulfuric carbohydrate method adopted for the later experiments
was observed to be more variable than the assay kit that was initially used. Triplicates were
not observed to consistently change colour, causing larger standard error values. However, it
did allow for a general determination of which fractions contained the most carbohydrate.
Enzyme digestion of the purified sample has given some hints as to what components
are required for its immuno-stimulatory activity. The dramatic decrease with proteinase-K
digestion suggested that protein component is important for its activity. As the samples were
deproteinated during the chloroform shakes of the Sevag method, it is believed that protein in
the sample may be covalently bound to a carbohydrate, which may have protected it from
denaturation. The decrease in activity with cellulase digestion also supports the idea of
carbohydrate being a contributor to its activity. As cellulase cleaves β-linkages, and due to
the common presence of β-glucans in mushrooms, it is suspected that the bioactive
compound(s) has polysaccharide component composed of β-glucan. Indeed, recent β-glucan
assay experiments in Dr. Lee’s laboratory on the purified sample from P. aurea show that it
has glucans consisting of 86% β-glucan and 14% α-glucan. Since both Proteinase K and
cellulase significantly decrease its activity, it can be concluded that both the protein and
carbohydrate components are required for the immuno-stimulatory activity.
The pro-inflammatory stimulation could be another sign of β-glucan presence. βglucans forming helical structures have been found to stimulate TNF-α production in
macrophages. The conformation of these polysaccharides has been proven to be a relevant
part of their activity139. The degree of side branching from the main chain of the carbohydrate
was seen to affect the conformation and thus activity. β-glucans with a higher degree of side
120

branching tend to form tighter helical structures, and were therefore more active33,139. It is
thought that these glucans are recognized and bound by Dectin-1 receptors, which then
initiate an innate immune response. Downstream effects include the release of cytokines IL12, IL-6, IL-10, and TNF-α, ultimately leading to phagocytosis and a pro-inflammatory
response33. As β-glucans do not naturally occur in the human body, it is generally thought
that they are the body’s recognition molecule for fungal and bacterial contaminants. The
response is therefore the body’s natural way of dealing with fungal and bacterial infection33.
Further detailed structural characterization is required to determine the exact identity
of the bioactive molecule responsible for immuno-stimulatory activity. While the evidence
points to the presence of protein and β-glucans, results from structural analysis have not been
obtained at this time. Once the structure is identified, a more specific purification approach
can be made. This could eliminate unnecessary steps, as well as improve product yields.

121

Chapter 4: General Discussion
4.1 Introduction
The primary goal of this study is to initiate exploration of some wild mushrooms and
lichens native to northern British Columbia for bioactivities that are relevant to anticancer
therapies. The first objective involved collection, identification, extraction and screening of
crude extracts from four mushrooms and two lichens for growth-inhibitory and immunostimulatory activities. The second objective involved the purification and characterization of
an immuno-stimulatory compound from a selected mushroom species.
The results from this study show that indeed wild mushrooms and lichens of northern
BC do contain growth-inhibitory and immuno-stimulatory compounds. This study has
revealed many new findings in regards to fungi and lichens from northern BC which is
primarily due to the lack of previous study. This exposes a need for more research into the
bioactivity of natural products from BC. It would appear that the growth-inhibitory effects
arise from the fungi’s own defense mechanisms, where production of such adverse
compounds would be beneficial to deter predation, whereas the immunological responses
arise from the host’s defense mechanisms, where the ability to detect fungal infectants would
benefit its own survival. One could assume that since the human body evolved in a world of
natural compounds, it is naturally geared to identify these compounds and elicit a response.
Due to this, one may argue that we are more likely to find bioactive compounds from natural
sources, rather than synthetically produced compounds with no natural origin. It is the body’s
own defenses that have caused the immunological responses seen from mushroom
compounds, and by boosting its defenses, a wide array of illnesses can be affected. The

122

mushroom compounds basically prime the immune system, which can then target other
pathogens, leaving them strong candidates for overall health promotion140.
4.2 Sample Identification and Screening for Growth-Inhibitory and Immunostimulatory Activities
Sample #1 was identified as Hericium coralloides. While not widely studied, this
species is related to the well-known Hericium erinaceus, which is commonly used in
traditional Chinese medicine. Results show that this mushroom contains mild immunostimulatory activity in regards to TNF-α production. This was primarily seen in hot water
extracts. It was not observed to show any growth-inhibitory effects against HeLa cells.
Interestingly, mild growth-stimulatory activity was initially observed, but this effect was not
persistent over time. Due to its relation to a well characterized species and its unexpected
growth-stimulatory activity, this mushroom warrants further investigation.
Sample # 2 was identified as Hydnellum diabolus. Species from this genus have not
been well characterized in terms of bioactivity, being primarily studied for their morphology,
taxonomy, and conservation141–146. One study had isolated an anticoagulant, atromentin, from
this species101, but otherwise it remains unstudied for other biological effects. Growthinhibitory effects were observed in the ethanol and hot water extracts of Hydnellum diabolus.
In addition, mild stimulation of TNF-α was seen in the hot water extract. To our knowledge,
this study is the first to report such bioactivities from this species. Future investigations such
as purification and identification of its bioactive constituents are clearly warranted.
Sample #3 was identified as Phaeolepiota aurea. This monotypic genus has not been
previously studied for immunological or growth-inhibitory effects. One study had previously
isolated two lectins from this mushroom108, however no further attempts to derive natural
123

products have been made. The cytotoxicity MTT assays show growth-inhibitory activity in
the ethanol and hot water extracts of this mushroom. A strong stimulation of TNF-α
production was seen in the methanol and hot water extracts, with hot water extracts showing
slightly stronger activity. These are novel findings for this species, and as such, an attempt
was made to isolate the immuno-stimulatory compound from its hot water extract. The work
of this study is described in Chapter 3 of this thesis.
Sample #4 was identified as Laetiporus sp.. Some species within this genus are prized
as food and have been used in traditional medicine. Laetiporus sulphureus is particularly well
characterized for its bioactivity109–113,147,148. Growth-inhibitory effects were found in the
ethanol, methanol, and hot water extracts, and strong stimulation of TNF-α production was
observed in the methanol and hot water extracts of this specimen. These findings support
previously identified bioactivity109,112,147 within this genus; however, a confirmed species
identity is needed to proceed with further analysis of this specimen.
Samples #5 and #6 were identified as Letharia vulpina and Vulpicida canadensis. As
these lichen species are known to naturally occur together, both have been similarly studied
and are known to contain vulpinic acid115,116,118–120. The ethanol extracts of both species were
found to show growth-inhibitory activity. Stimulation of TNF-α production was seen in the
hot water extract of L. vulpina and the methanol extract of V. canadensis. While the cytotoxic
activity has already been reported117,118,120, it appears that the immuno-stimulatory activity is
a novel finding for these species. As such, these species could be targets for further
purification, identification and characterization of the responsible compounds.

124

4.3 Purification and Characterization of an Immuno-Stimulatory Compound from
Phaeolepiota aurea.
Multiple experiments show that the crude extracts of P. aurea exhibited more than
one bioactivity. The hot water extract was shown to contain both cytotoxic and immunostimulatory compounds. Results show that the cytotoxic compound(s) can be separated using
size exclusion chromatography and are thought to be primarily protein in composition.
Immuno-stimulatory activity appeared to correlate with the carbohydrate and protein content
seen in the bioactive fractions. As the immuno-stimulatory activity was not completely
removed with heat, carbohydrates were thought to be a major component of the immunostimulatory compound(s) and a more polysaccharide targeted approach was adopted for
subsequent purification steps. Cytotoxic activity exhibited by a protein component was first
removed from the crude extracts with deproteination steps (Sevag method), and the crude
polysaccharide mixture was subjected to ion-exchange and high-resolution size-exclusion
chromatography. While immuno-stimulatory activity was not seen to significantly increase
with purification using DEAE ion exchange chromatography, the activity was not lost and
remained similar to that of the crude polysaccharide material. It is possible that the activity
may be arising from a population of polysaccharide compounds, and purification attempts are
just sub-fractionating them. The compound may also be relatively pure following
deproteination, becoming more dilute with each purification attempt as material is being lost
to media binding. Once the exact structure of the bioactive compound is known, a more
direct purification scheme can be determined, which can improve the yields obtained from
crude mixtures.

125

The immuno-stimulatory compound purified from the last step of chromatography
Sephacryl S-500 appears to be made up of a polysaccharide-protein complex. The postSephacryl S-500 column fractions showing the strongest activity were also the ones showing
the highest carbohydrate and protein concentrations. As the immuno-stimulatory activity
could be dramatically reduced with the use of proteinase-K and cellulase, it is concluded that
both protein and β-glucans are responsible for the activity. Indeed, a recent study
(unpublished) in Dr. Lee’s laboratory show that the purified compound has 22% w/w of
glucans in which 86% are β–glucans and 14% are α–glucans. Based on the fact that the
deproteination Sevag method was used during purification and the results from proteinase-K
and cellulase enzyme experiments, it is likely the immuno-stimulatory compound is a
polysaccharide-protein complex linked by N- or/and O-linked glycosidic bonds Future
studies are clearly required to corroborate this.
The cytokine and chemokine analyses support the conclusion that the compound of
interest is a stimulant of the innate immune system. The strongest stimulation was seen in
cytokines affiliated with a pro-inflammatory response and leukocyte recruitment. The
compound stimulates the host into an immunological response used to defend against
infection. This correlates to the response seen from β-glucans, which are suggested to be the
immune system’s fungal recognition molecule32. This further supports the notion that the
immuno-stimulatory compound contains β-glucan.
4.4 Final Conclusions and Future Directions
The tested wild mushrooms and lichens of British Columbia have proven themselves
to be bio-factories of immuno-stimulatory and growth-inhibitory compounds. Due to limited
studies on the bioactivities of forest fungi worldwide, many novel bioactivities were found in
126

the fungal species native to BC. This shows a need for further study into naturally occurring
compounds found in the wild mushrooms of western culture.
Attempts to purify active compounds from the hot water extracts of P. aurea found
the presence of multiple bioactive compounds. Immuno-stimulatory activity was linked to βglucan and protein within the final purified sample. It is believed that the immunostimulatory compound is composed of polysaccharide-protein complex in which the
polysaccharide component contains β–glucan. The compound was found to stimulate the
innate immune system and caused the release of pro-inflammatory cytokines.
Future studies must be undertaken to elucidate the structure of the immunostimulatory compound. Monosaccharide analysis can be performed with the use of gas
chromatography-mass spectrometry (GC-MS) to determine the monosaccharide composition
and their linkages in the polysaccharide component of the compound49,149–151. Due to the
presence of protein in the purified sample, experiments must be undertaken to determine if it
is covalently linked to the polysaccharide. A β-elimination assay will aid in identifying olinked glycans152, while the use of enzyme N-glycosidase F (PNGase F) can determine the
presence of N-linked glycans153. By identifying the active components of the compound, a
more targeted isolation scheme may be determined. Once the active compound is purified to
homogeneity, nuclear magnetic resonance (NMR) analysis can then be performed to
determine its complete structure84,154–159.
Screening of the 6 fungal species from British Columbia has found numerous active
extracts to pursue. As many of the activities observed are novel findings for their species,
purification of any of these active fractions may lead to the identification of novel bioactive
compounds. These may be of use in pharmacological or natural therapies and warrant future
127

investigation. Purification attempts have led to the partial identification of an immunostimulatory compound from P. aurea, which was found to stimulate the innate immune
system. Due to the immuno-boosting properties, this compound, once identified, can be used
in the treatment of a variety of ailments, including cancer, by stimulating the body to defend
itself. This is the method of action of many anticancer mushroom compounds in use and
clinical trials today84,140,160.

128

References
1. Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W,
Fungal Barcoding Consortium. Nuclear ribosomal internal transcribed spacer (ITS) region as
a universal DNA barcode marker for Fungi. Proceedings of the National Academy of
Sciences of the United States of America. 2012;109(16):1–6. doi:10.1073/pnas.1117018109
2. El Enshasy H, Elsayed EA, Aziz R, Wadaan MA. Mushrooms and truffles: Historical
biofactories for complementary medicine in Africa and in the middle East. Evidence-based
Complementary and Alternative Medicine. 2013;2013. doi:10.1155/2013/620451
3. Mizuno M, Nishitani Y. Immunomodulating compounds in Basidiomycetes. J. Clin.
Biochen. Nutr. 2013;52(May):202–207. doi:10.3164/jcbn.13
4. Hawksworth DL. Mushrooms: The extent of the unexplored potential. International
Journal of Medicinal Mushrooms. 2001;3(4):5. doi:10.1615/IntJMedMushr.v3.i4.50
5. Popovi V, Davidovi S, Stevanovi M, Stojkovi D. Mycotherapy of cancer : An update on
cytotoxic and antitumor activities of mushrooms , bioactive principles and molecular
mechanisms of their action. Current Topics in Medicinal Chemistry. 2013;13(21):2791–
2806.
6. Guggenheim AG, Wright KM, Zwickey HL. Immune modulation from five major
mushrooms: Application to integrative oncology. Integrative Medicine. 2014;13(1):32–44.
7. Stamets P, Zwickey H. Medicinal mushrooms: Ancient remedies meet modern science.
Integrative medicine (Encinitas, Calif.). 2014;13(1):46–7.
8. Wasser S. Medicinal mushroom science: Current perspectives, advances, evidences, and
challenges. Biomedical Journal. 2014;37(6):345. doi:10.4103/2319-4170.138318
9. Capasso L. 5300 years ago, the Ice Man used natural laxatives and antibiotics. Lancet.
1998;352(9143):1864. doi:10.1016/S0140-6736(05)79939-6
10. Oyetayo O. Medicinal uses of mushrooms in Nigeria: towards full and sustainable
exploitation. African Journal of Traditional, Complementary and Alternative Medicines.
2011;8(3). doi:10.4314/ajtcam.v8i3.65289
11. Zhang L, Ravipati AS, Koyyalamudi SR, Jeong SC, Reddy N, Smith PT, Bartlett J,
Shanmugam K, Münch G, Wu MJ. Antioxidant and anti-inflammatory activities of selected
medicinal plants containing phenolic and flavonoid compounds. Journal of Agricultural and
Food Chemistry. 2011;59(23):12361–12367. doi:10.1021/jf203146e
12. Ajith T. Indian medicinal mushrooms as a source of antioxidant and antitumor agents.
Journal of Clinical Biochemistry and Nutrition. 2007;40(2):74–81. doi:10.3164/jcbn.40.74
13. Jiao C, Xie YZ, Yang X, Li H, Li XM, Pan HH, Cai MH, Zhong HM, Yang BB.
Anticancer activity of Amauroderma rude. PLoS ONE. 2013;8(6).
doi:10.1371/journal.pone.0066504
14. Tang YJ, Zhong JJ. Modeling the kinetics of cell growth and ganoderic acid production
129

in liquid static cultures of the medicinal mushroom Ganoderma lucidum. Biochemical
Engineering Journal. 2004;21(3):259–264. doi:10.1016/j.bej.2004.06.008
15. Choi HS, Cho HY, Yang HC, Ra KS, Suh HJ. Angiotensin I-converting enzyme inhibitor
from Grifola frondosa. Food Research International. 2001;34(2–3):177–182.
doi:10.1016/S0963-9969(00)00149-6
16. Mizuno T, Zhuang C. Maitake, Grifola frondosa pharmacological effects. Food Reviews
International. 1995;11(1):135–149. doi:10.1080/87559129509541024
17. Cui Y, Kim DS, Park KC. Antioxidant effect of Inonotus obliquus. Journal of
Ethnopharmacology. 2005;96(1–2):79–85. doi:10.1016/j.jep.2004.08.037
18. Huang NL. Inonotus obliquus. Edible Fungi of China. 2002;21:7–8.
19. Bisen PS, Baghel RK, Sanodiya BS, Thakur GS, Prasad GBKS. Lentinus edodes : A
macrofungus with pharmacological activities. Current Medicinal Chemistry.
2010;17(22):2419–2430.
20. Lee ML, Tan NH, Fung SY, Tan CS, Ng ST. The antiproliferative activity of sclerotia of
Lignosus rhinocerus (tiger milk mushroom). Evidence-based Complementary and
Alternative Medicine. 2012;2012:5–9. doi:10.1155/2012/697603
21. Zhu T, Kim S-H, Chen C-Y. A medicinal mushroom: Phellinus linteus. Current
medicinal chemistry. 2008;15(13):1330–1335. doi:10.2174/092986708784534929
22. Tibuhwa DD. Folk taxonomy and use of mushrooms in communities around Ngorongoro
and Serengeti National Park, Tanzania. Journal of Ethnobiology and Ethnomedicine.
2012;8(1):36. doi:10.1186/1746-4269-8-36
23. Sullivan R, Smith JE, Rowan NJ. Medicinal mushrooms and cancer therapy: translating a
traditional practice into Western medicine. Perspectives in biology and medicine.
2006;49(2):159–170. doi:10.1353/pbm.2006.0034
24. Aly AH, Debbab A, Proksch P. Fifty years of drug discovery from fungi. Fungal
Diversity. 2011;50:3–19. doi:10.1007/s13225-011-0116-y
25. Dewick PM. Medicinal natural products : a biosynthetic approach. Wiley; 2002.
26. Endo A, Kuroda M, Tsujita Y. ML-236A, ML-236B, and ML-236C, new inhibitors of
cholesterogensis produced by Penicillium citrinum. The Journal of Antibiotics.
1976;29(12):1346–1348. doi:10.7164/antibiotics.29.1346
27. Negishi S, Huang ZC, Hasumu K, Murakawa S, Endo A. Productivity of monacolin K
(mevinolin) in the genus Monascus (in Japanese). Hakko Kogaku Kaishi. 1986;64(6):584–
590. doi:10.1016/0385-6380(86)90091-9
28. Ondeyka JG, Helms GL, Hensens OD, Goetz MA, Zink DL, Tsipouras A, Shoop WL,
Slayton L, Dombrowski AW, Polishook JD, Ostlind DA, Tsou NN, Ball RG, Singh SB.
Nodulisporic acid A, a novel and potent insecticide from a Nodulisporium sp. isolation,
structure determination, and chemical transformations. Journal of the American Chemical
Society. 1997;119(38):8809–8816. doi:10.1021/ja971664k
130

29. Hensens OD, Ondeyka JG, Dombrowski AW, Ostlind DA, Zink DL. Isolation and
structure of nodulisporic acid A1and A2, novel insecticides from a Nodulisporium sp.
Tetrahedron Letters. 1999;40(30):5455–5458. doi:10.1016/S0040-4039(99)01064-3
30. Lemieszek M, Rzeski W. Anticancer properties of polysaccharides isolated from fungi of
the Basidiomycetes class. Wspolczesna Onkologia. 2012;16(4):285–289.
doi:10.5114/wo.2012.30055
31. Zhang Y, Li S, Wang X, Zhang L, Cheung PCK. Advances in lentinan: Isolation,
structure, chain conformation and bioactivities. Food Hydrocolloids. 2011;25(2):196–206.
doi:10.1016/j.foodhyd.2010.02.001
32. Lull C, Wichers HJ, Savelkoul HFJ. Antiinflammatory and immunomodulating properties
of fungal metabolites. Mediators of Inflammation. 2005;2005(2):63–80.
doi:10.1155/MI.2005.63
33. Chan GC, Chan WK, Sze DM. The effects of β -glucan on human immune and cancer
cells. Journal of Hematology & Oncology. 2009;2:1–11. doi:10.1186/1756-8722-2-25
34. Price LA, Wenner CA, Sloper DT, Slaton JW, Novack JP. Role for toll-like receptor 4 in
TNF-alpha secretion by murine macrophages in response to polysaccharide Krestin, a
Trametes versicolor mushroom extract. Fitoterapia. 2010;81(7):914–919.
doi:10.1016/j.fitote.2010.06.002
35. Xia Y, Vetvicka V, Yan J, Hanikýrová M, Mayadas T, Ross GD. The beta-glucanbinding lectin site of mouse CR3 (CD11b/CD18) and its function in generating a primed state
of the receptor that mediates cytotoxic activation in response to iC3b-opsonized target cells.
Journal of immunology (Baltimore, Md. : 1950). 1999;162(4):2281–90.
doi:10.4049/jimmunol.1203243
36. Maehara Y, Tsujitani S, Saeki H, Oki E, Yoshinaga K, Emi Y, Morita M, Kohnoe S,
Kakeji Y, Yano T, Baba H. Biological mechanism and clinical effect of protein-bound
polysaccharide K (KRESTIN®): Review of development and future perspectives. Surgery
Today. 2012;42(1):8–28. doi:10.1007/s00595-011-0075-7
37. Meng X, Liang H, Luo L. Antitumor polysaccharides from mushrooms: A review on the
structural characteristics, antitumor mechanisms and immunomodulating activities.
Carbohydrate Research. 2016;424:30–41. doi:10.1016/j.carres.2016.02.008
38. Standish LJ, Wenner CA, Sweet ES, Bridge C, Nelson A, Martzen M, Novack J,
Torkelson C. Trametes versicolor mushroom immune therapy in breast cancer. Journal of the
Society for Integrative Oncology. 2008;6(3):122–128. doi:10.2310/7200.2008.0016
39. Jiménez-Medina E, Berruguilla E, Romero I, Algarra I, Collado A, Garrido F, GarciaLora A. The immunomodulator PSK induces in vitro cytotoxic activity in tumour cell lines
via arrest of cell cycle and induction of apoptosis. BMC cancer. 2008;8:78.
doi:10.1186/1471-2407-8-78
40. Ooi VEC, Liu F. Immunomodulation and anti-cancer activity of polysaccharide- protein
complexes. Current Medicinal Chemistry. 2000;7:715–729. doi:10.2174/0929867003374705
41. Kidd PM. The use of mushroom glucans and proteoglycans in cancer treatment.
131

Alternative Medicine Review. 2000;5(1):4–27.
42. Nio Y, Tsubono M, Tseng CC, Morimoto H, Kawabata K, Masai Y, Shiraishi T, Imai S,
Ohgaki K, Tobe T. Immunomodulation by orally administered protein-bound polysaccharide
PSK in patients with gastrointestinal cancer. Biotherapy. 1992;4(2):117–128.
doi:10.1007/BF02171756
43. Engel AL, Sun GC, Gad E, Rastetter LR, Strobe K, Yang Y, Dang Y, Disis ML, Lu H.
Protein-bound polysaccharide activates dendritic cells and enhances OVA-specific T cell
response as vaccine adjuvant. Immunobiology. 2013;218(12):1468–1476.
doi:10.1016/j.imbio.2013.05.001
44. Fisher M, Yang L-X. Anticancer effects and mechanisms of polysaccharide-K (PSK):
implications of cancer immunotherapy. Anticancer Research. 2002;22(3):1737–54.
45. Fritz H, Kennedy DA, Ishii M, Fergusson D, Fernandes R, Cooley K, Seely D.
Polysaccharide K and Coriolus versicolor extracts for lung cancer: A systematic review.
Integrative Cancer Therapies. 2015;14(3):201–211. doi:10.1177/1534735415572883
46. Bohn JA, BeMiller JN. (1→3)-β-d-Glucans as biological response modifiers: a review of
structure-functional activity relationships. Carbohydrate Polymers. 1995;28(1):3–14.
doi:10.1016/0144-8617(95)00076-3
47. Ina K, Kataoka T, Ando T. The use of lentinan for treating gastric cancer. Anti-cancer
agents in medicinal chemistry. 2013;13(5):681–8. doi:10.2174/1871520611313050002
48. Rice PJ, Kelley JL, Kogan G, Ensley HE, Kalbfleisch JH, Browder IW, Williams DL.
Human monocyte scavenger receptors are pattern recognition receptors for (1-->3)-beta-Dglucans. Journal of Leukocyte Biology. 2002;72(1):140–6.
49. Zhang Y, Kong H, Fang Y, Nishinari K, Phillips GO. Schizophyllan: A review on its
structure, properties, bioactivities and recent developments. Bioactive Carbohydrates and
Dietary Fibre. 2013;1(1):53–71. doi:10.1016/j.bcdf.2013.01.002
50. Norisuye T, Yanaki T, Fujita H. Triple helix of a Schizophyllum commune polysaccharide
in aqueous solution. Journal of Polymer Science: Polymer Physics Edition. 1980;18(3):547–
558. doi:10.1002/pol.1980.180180314
51. Komatsu N, Okubo S, Kikumoto S, Kimura K, Saito G, Sakai S. Host-mediated
antitumor action of schizophyllan, a glucan produced by Schizophyllum commune. Japanese
Journal of Cancer Research. 1969;60(2):137–144. doi:10.20772/cancersci1959.60.2_137
52. Suzuki M, Arika T, Amemiya K, Fujiwara M. Cooperative role of T lymphocytes and
macrophages in anti-tumor activity of mice pretreated with schizophyllan (SPG). The
Japanese Journal of Experimental Medicine. 1982;52(2):59–65.
53. Sakagami Y, Mizoguchi Y, Shin T, Seki S, Kobayashi K, Morisawa S, Yamamoto S.
Effects of an anti-tumor polysaccharide, schizophyllan, on interferon-γ and interleukin 2
production by peripheral blood mononuclear cells.pdf. Biochemical and Biophysical
Research Communications. 1988;155(2):650–655. doi:10.1016/S0006-291X(88)80544-8
54. Tsuchiya Y, Igarashi M, Inoue M, Kumagai K. Cytokine-related immunomodulating
132

activities of an anti-tumor glucan, sizofiran (SPG). Journal of Pharmacobio-Dynamics.
1989;12(10):616–625. doi:10.1248/bpb1978.12.616
55. Kimura Y, Tojima H, Fukase S, Takeda K. Clinical evaluation of sizofilan as assistant
immunotherapy in treatment of head and neck cancer. Acta Oto-Laryngologica.
1994;114(S511):192–195. doi:10.3109/00016489409128330
56. Nakao I, Uchino H, Orita K, Kaido I, Kimura T, Goto Y, Kondo T, Takino T, Taguchi T,
Nakajima T, Fujimoto S, Miyazaki T, Miyoshi A, Yachi A, Yoshida K, Ogawa N, Furue H.
Clinical evaluation of schizophyllan (SPG) in advanced gastric cancer--a randomized
comparative study by an envelope method. Gan-To-Kagaku-Ryoho. 1983;10(4 Pt 2):1146–
1159.
57. Miyamoto N, Mochizuki S, Sakurai K. Designing an immunocyte-targeting delivery
system by use of beta-glucan. Vaccine. 2018;36(1):186–189.
doi:10.1016/j.vaccine.2017.11.053
58. Sliva D. Medicinal mushroom Phellinus linteus as an alternative cancer therapy (review).
Experimental and Therapeutic Medicine. 2010;1(3):407–411. doi:10.3892/etm_00000063
59. Zhu T, Guo J, Collins L, Kelly J, Xiao ZJ, Kim SH, Chen CY. Phellinus linteus activates
different pathways to induce apoptosis in prostate cancer cells. British Journal of Cancer.
2007;96(4):583–590. doi:10.1038/sj.bjc.6603595
60. Sliva D, Jedinak A, Kawasaki J, Harvey K, Slivova V. Phellinus linteus suppresses
growth, angiogenesis and invasive behaviour of breast cancer cells through the inhibition of
AKT signalling. British journal of cancer. 2008;98(8):1348–1356.
doi:10.1038/sj.bjc.6604319
61. Kim YN, Kim MS, Chun SS, Choi JH. Effect of Phellius linteus water extract on benign
prostatic hyperplasia. Nutrition Research and Practice. 2013;7(3):172–177.
doi:10.4162/nrp.2013.7.3.172
62. Jeon T Il, Jung CH, Cho JY, Park DK, Moon JH. Identification of an anticancer
compound against HT-29 cells from Phellinus linteus grown on germinated brown rice.
Asian Pacific Journal of Tropical Biomedicine. 2013;3(10):785–789. doi:10.1016/S22211691(13)60156-2
63. Lee JW, Baek SJ, Bae WC, Park JM, Kim YS. Antitumor and antioxidant activities of the
extracts from fruiting body of Phellinus linteus. Mycobiology. 2006;34(4):230–235.
64. Collins L, Zhu T, Guo J, Xiao ZJ, Chen CY. Phellinus linteus sensitises apoptosis
induced by doxorubicin in prostate cancer. British Journal of Cancer. 2006;95(3):282–288.
doi:10.1038/sj.bjc.6603277
65. Yu T, Ganapathy S, Shen L, Peng B, Kim S-H, Makriyannis A, Chen C. A lethal synergy
induced by Phellinus linteus and camptothecin11 in colon cancer cells. Oncotarget.
2018;9(5):6308–6319. doi:10.18632/oncotarget.23918
66. Hsieh MJ, Chien SY, Chou YE, Chen CJ, Chen J, Chen MK. Hispolon from Phellinus
linteus possesses mediate caspases activation and induces human nasopharyngeal carcinomas
cells apoptosis through ERK1/2, JNK1/2 and p38 MAPK pathway. Phytomedicine.
133

2014;21(12):1746–1752. doi:10.1016/j.phymed.2014.07.013
67. Suarez-Arroyo IJ, Rosario-Acevedo R, Aguilar-Perez A, Clemente PL, Cubano LA,
Serrano J, Schneider RJ, Martínez-Montemayor MM. Anti-tumor effects of Ganoderma
lucidum (Reishi) in inflammatory breast cancer in in vivo and in vitro models. PLoS ONE.
2013;8(2). doi:10.1371/journal.pone.0057431
68. Jiang J, Slivova V, Harvey K, Valachovicova T, Sliva D. Ganoderma lucidum suppresses
growth of breast cancer cells through the inhibition of Akt/NF-kappaB signaling. Nutr
Cancer. 2004;49:209–216. doi:10.1207/s15327914nc4902_13
69. Sliva D, Loganathan J, Jiang J, Jedinak A, Lamb JG, Terry C, Baldridge LA, Adamec J,
Sandusky GE, Dudhgaonkar S. Mushroom Ganoderma lucidum prevents colitis-associated
carcinogenesis in mice. PLoS ONE. 2012;7(10):1–13. doi:10.1371/journal.pone.0047873
70. Tang W, Liu JW, Zhao WM, Wei DZ, Zhong JJ. Ganoderic acid T from Ganoderma
lucidum mycelia induces mitochondria mediated apoptosis in lung cancer cells. Life
Sciences. 2006;80(3):205–211. doi:10.1016/j.lfs.2006.09.001
71. Xie JT, Wang CZ, Wicks S, Yin JJ, Kong J, Li J, Li YC, Yuan CS. Ganoderma lucidum
extract inhibits proliferation of SW 480 human colorectal cancer cells. Experimental
Oncology. 2006;28(1):25–29. doi:25/490 [pii]
72. Zhu M, Chang Q, Wong LK, Chong FS, Li RC. Triterpene antioxidants from Ganoderma
lucidum. Phytotherapy Research. 1999;13(6):529–531. doi:10.1002/(SICI)10991573(199909)13:6<529::AID-PTR481>3.0.CO;2-X
73. Chung MJ, Chung C-K, Jeong Y, Ham S-S. Anticancer activity of subfractions
containing pure compounds of Chaga mushroom (Inonotus obliquus) extract in human cancer
cells and in Balbc/c mice bearing Sarcoma-180 cells. Nutrition research and practice.
2010;4(3):177–182. doi:10.4162/nrp.2010.4.3.177
74. Youn M-J, Kim J-K, Park S-Y, Kim Y, Kim S-J, Lee JS, Chai KY, Kim H-J, Cui M-X,
So HS, Kim K-Y, Park R. Chaga mushroom (Inonotus obliquus) induces G0/G1 arrest and
apoptosis in human hepatoma HepG2 cells. World journal of gastroenterology : WJG.
2008;14(4):511–7. doi:10.3748/wjg.14.511
75. Lindequist U, Niedermeyer THJ, Jülich WD. The pharmacological potential of
mushrooms. Evidence-based Complementary and Alternative Medicine. 2005;2(3):285–299.
doi:10.1093/ecam/neh107
76. Kim JH, Lee JS, Lee KR, Shim MJ, Lee MW, Shin PG, Cheong JC, Yoo YB, Lee TS.
Immunomodulating and antitumor activities of Panellus serotinus polysaccharides.
Mycobiology. 2012;40(3):181–188. doi:10.5941/MYCO.2012.40.3.181
77. Cheng S, Eliaz I, Lin J, Sliva D. Triterpenes from Poria cocos suppress growth and
invasiveness of pancreatic cancer cells through the downregulation of MMP-7. International
Journal of Oncology. 2013;42(6):1869–1874. doi:10.3892/ijo.2013.1902
78. Arora D. Mushrooms demystified: A comprehensive guide to the fleshy fungi. Ten Speed
Press; 1986. doi:ISBN 0898151694
134

79. Gibson I, Gibson E, Kendrick B. MatchMaker: mushrooms of the Pacific Northwest.
Canadian Forest Service. 1999.
80. McCune B, Geiser L, Sharnoff SD, Sharnoff S, Mikulin AG. Macrolichens of the Pacific
Northwest. Oregon State University Press; 2009.
81. MOBIO laboratories. PowerSoil ® DNA Isolation Kit. 2014:1–24.
82. Technelysium. Chromas Lite. Technelysium Pty Ltd. 2012.
83. National Center for Biotechnology Information. BLAST: Basic Local Alignment Search
Tool. [accessed 2018 Apr 2]. https://blast.ncbi.nlm.nih.gov/Blast.cgi
84. Zhang M, Cui SW, Cheung PCK, Wang Q. Antitumor polysaccharides from mushrooms:
a review on their isolation process, structural characteristics and antitumor activity. Trends in
Food Science and Technology. 2007;18(1):4–19. doi:10.1016/j.tifs.2006.07.013
85. Mizuno T. The extraction and development of antitumor-active polysaccharides from
medicinal mushrooms in Japan (Review). International Journal of Medicinal Mushrooms.
1999;1(1):9–29. doi:10.1615/IntJMedMushrooms.v1.i1.20
86. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to
proliferation and cytotoxicity assays. Journal of Immunological Methods. 1983;65(1–2):55–
63. doi:10.1016/0022-1759(83)90303-4
87. Berridge M V, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology : New
insights into their cellular reduction. Biotechnology Annual Review. 2005;11(05):127–152.
doi:10.1016/S1387-2656(05)11004-7
88. Ferrari M, Fornasiero MC, Isetta AM. MTT colorimetric assay for testing macrophage
cytotoxic activity in vitro. Journal of Immunological Methods. 1990;131(2):165–172.
doi:10.1016/0022-1759(90)90187-Z
89. Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Journal of
Immunological Methods. 1986;89(2):271–277. doi:10.1016/0022-1759(86)90368-6
90. Smith A, Javed S, Barad A, Myhre V, Li WM, Reimer K, Massicotte HB, Tackaberry
LE, Payne GW, Egger KN, Lee CH. Growth-inhibitory and immunomodulatory activities of
wild mushrooms from north-central British Columbia (Canada). International Journal of
Medicinal Mushrooms. 2017;19(6):485–497. doi:10.1615/IntJMedMushrooms.v19.i6.10
91. Ginns J. Hericium in North America: cultural characteristics and mating behaviour. Can.
J. Bot. 1985;63:1551–1563.
92. Burdsall H, Banik MT. The genus Laetiporus in North America. Harvard Papers in
Botany. 2001;6(1):43–55.
93. Ren L, Hemar Y, Perera CO, Lewis G, Krissansen GW, Buchanan PK. Antibacterial and
antioxidant activities of aqueous extracts of eight edible mushrooms. Bioactive
Carbohydrates and Dietary Fibre. 2014;3(2):41–51. doi:10.1016/j.bcdf.2014.01.003
94. Heleno SA, Barros L, Martins A, Queiroz MJRP, Morales P, Fernández-Ruiz V, Ferreira
ICFR. Chemical composition, antioxidant activity and bioaccessibility studies in phenolic
135

extracts of two Hericium wild edible species. LWT - Food Science and Technology.
2015;63(1):475–481. doi:10.1016/j.lwt.2015.03.040
95. Vamanu E, Voica A. Total phenolic analysis, antimicrobial and antioxidant activity of
some mushroom tinctures from medicinal and edible species, by in vitro and in vivo tests.
Scientific Bulletin. Series F. Biotechnologies. 2017;XXI:318–324.
96. Zou Y-J, Wang H-X, Ng T-B, Huang C-Y, Zhang J-X. Purification and characterization
of a novel laccase from the edible mushroom Hericium coralloides. The Journal of
Microbiology. 2012;50(1):72–78. doi:10.1007/s12275-012-1372-6
97. Wittstein K, Rascher M, Rupcic Z, Löwen E, Winter B, Köster RW, Stadler M.
Corallocins A-C, nerve growth and brain-derived neurotrophic factor inducing metabolites
from the mushroom Hericium coralloides. Journal of Natural Products. 2016;79(9):2264–
2269. doi:10.1021/acs.jnatprod.6b00371
98. Thongbai B, Rapior S, Hyde KD, Wittstein K, Stadler M. Hericium erinaceus, an
amazing medicinal mushroom. Mycological Progress. 2015;14(10):91. doi:10.1007/s11557015-1105-4
99. Sokół S, Golak-Siwulska I, Sobieralski K, Siwulski M, Górka K. Biology, cultivation,
and medicinal functions of the mushroom Hericium erinaceum. Acta Mycologica.
2016;50(2). doi:10.5586/am.1069
100. He X, Wang X, Fang J, Chang Y, Ning N, Guo H, Huang L, Huang X, Zhao Z.
Structures, biological activities, and industrial applications of the polysaccharides from
Hericium erinaceus (Lion’s Mane) mushroom: A review. International Journal of Biological
Macromolecules. 2017;97:228–237. doi:10.1016/j.ijbiomac.2017.01.040
101. Khanna JM, Malone MH, Euler KL, Brady LR. Atromentin. Anticoagulant from
Hydnellum diabolus. Journal of Pharmaceutical Sciences. 1965;54(7):1016–1020.
doi:10.1002/jps.2600540714
102. Liu JK. Natural terphenyls: Developments since 1877. Chemical Reviews.
2006;106(6):2209–2223. doi:10.1021/cr050248c
103. Montfort ML, Tyler VE, Brady LR. Isolation of aurantiacin from Hydnellum caeruleum.
Journal of Pharmaceutical Sciences. 1966;55(11):1300–1302. doi:10.1002/jps.2600551127
104. Wang SM, Han JJ, Ma K, Jin T, Bao L, Pei YF, Liu HW. New α-glucosidase inhibitors
with p-terphenyl skeleton from the mushroom Hydnellum concrescens. Fitoterapia.
2014;98:149–155. doi:10.1016/j.fitote.2014.07.019
105. Quang DN, Hashimoto T, Hitaka Y, Tanaka M, Nukada M, Yamamoto I, Asakawa Y.
Thelephantins I-N: p-terphenyl derivatives from the inedible mushroom Hydnellum
caeruleum. Phytochemistry. 2004;65(8):1179–1184. doi:10.1016/j.phytochem.2004.02.018
106. Hashimoto T, Quang DN, Kuratsune M, Asakawa Y. Hydnellins A and B, nitrogencontaining Terphenyls from the mushrooms Hydnellum suaveolens and Hydnellum
geogerirum. Chemical & Pharmaceutical Bulletin. 2006;54(6):912–914.
doi:10.1248/cpb.54.912
136

107. Stijve T. Phaeolepiota aurea, a beautiful and mysterious mushroom. Australasian
Mycologist. 2002;21(1):24–28.
108. Kawagishi H, Wasa T, Murata T, Usui T, Kimura A, Chiba S. Two N-acetyl-Dgalactosamine-specific lectins from Phaeolepiota aurea. Phytochemistry. 1996;41(4):1013–
1016.
109. Khatua S, Ghosh S, Acharya K. Laetiporus sulphureus (Bull.: Fr.) Murr. as food as
medicine. Pharmacogn J. 2017;9(6):s1–s15.
110. Grienke U, Zöll M, Peintner U, Rollinger JM. European medicinal polypores - A
modern view on traditional uses. Journal of Ethnopharmacology. 2014;154(3):564–583.
doi:10.1016/j.jep.2014.04.030
111. Acharya K, Ghosh S, Khatua S, Mitra P. Pharmacognostic standardization and
antioxidant capacity of an edible mushroom Laetiporus sulphureus. Journal fur
Verbraucherschutz und Lebensmittelsicherheit. 2016;11(1):33–42. doi:10.1007/s00003-0150977-1
112. Petrović J, Stojković D, Reis FS, Barros L, Glamočlija J, Ćirić A, Ferreira ICFR,
Soković M. Study on chemical, bioactive and food preserving properties of Laetiporus
sulphureus (Bull.: Fr.) Murr. Food Funct. 2014;5(7):1441–1451. doi:10.1039/C4FO00113C
113. Wang Y, Wu B, Shao J, Jia J, Tian Y, Shu X, Ren X, Guan Y. Extraction, purification
and physicochemical properties of a novel lectin from Laetiporus sulphureus mushroom.
LWT - Food Science and Technology. 2018;91:151–159. doi:10.1016/j.lwt.2018.01.032
114. Spiteller P. Chemical defence strategies of higher fungi. Chemistry - A European
Journal. 2008;14(30):9100–9110. doi:10.1002/chem.200800292
115. Shrestha G, Thompson A, Robison R, St. Clair LL. Letharia vulpina, a vulpinic acid
containing lichen, targets cell membrane and cell division processes in methicillin-resistant
Staphylococcus aureus. Pharmaceutical Biology. 2016;54(3):413–418.
doi:10.3109/13880209.2015.1038754
116. Shrestha G, Raphael J, Leavitt SD, St Clair LL. In vitro evaluation of the antibacterial
activity of extracts from 34 species of North American lichens. Pezzuto Pharm Biol.
2014;52(10):1262–1266. doi:10.3109/13880209.2014.889175
117. Shrestha G, El-Naggar AM, St. Clair LL, O’Neill KL. Anticancer activities of selected
species of North American lichen extracts. Phytotherapy Research. 2015;29(1):100–107.
doi:10.1002/ptr.5233
118. Koparal AT. Anti-angiogenic and antiproliferative properties of the lichen substances ()-usnic acid and vulpinic acid. Zeitschrift fur Naturforschung - Section C Journal of
Biosciences. 2015;70(5–6):159–164. doi:10.1515/znc-2014-4178
119. Varol M, Türk A, Candan M, Tay T, Koparal AT. Photoprotective activity of vulpinic
and gyrophoric Acids toward ultraviolet B-induced damage in human keratinocytes.
Phytotherapy Research. 2016;30(1):9–15. doi:10.1002/ptr.5493
120. Fernández-Moriano C, Divakar PK, Crespo A, Gómez-Serranillos MP. Neuroprotective
137

activity and cytotoxic potential of two Parmeliaceae lichens: Identification of active
compounds. Phytomedicine. 2015;22(9):847–855. doi:10.1016/j.phymed.2015.06.005
121. GE Healthcare. Sephadex LH-20. GE Healthcare. 2006:1–11.
122. Pharmacia Biotech. Ion exchnage chromatography: Sephadex ion exchange media.
Pharmacia Biotech. 1997:1–8.
123. Williams A, Frasca V. Ion-exchange chromatography. In: Current Protocols in Protein
Science. John Wiley & Sons, Inc.; 1999. p. 8.2.1-8.2.30. doi:10.1002/0471140864.ps0802s15
124. Thermo Scientific. User Guide: Pierce BCA Protein Assay Kit. Pierce Biotechnology.
2011;0747(23225):1–7. doi:10.1016/j.ijproman.2010.02.012
125. Sevag, M.G.; Lackman, D.B.;Smolens J. The isolation of the components of
streptococcal nucleoproteıns in serologıcally active form. J.Biol.Chem. 1938;124(124):425–
436.
126. Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies:
Integrating mammalian biology. Cell. 2001;104(4):487–501. doi:10.1016/S00928674(01)00237-9
127. Wolpe SD, Davatelis G, Sherry B, Beutler B, Hesse DG, Nguyen HT, Moldawer LL,
Nathan CF, Lowry SF, Cerami A. Macrophages secrete a novel heparin-binding protein with
inflammatory and neutrophil chemokinetic properties. The Journal of Experimental
Medicine. 1988;167(2):570–81. doi:10.1084/jem.167.2.570
128. Bystry RS, Aluvihare V, Welch KA, Kallikourdis M, Betz AG. B cells and professional
APCs recruit regulatory T cells via CCL4. Nature Immunology. 2001;2(12):1126–1132.
doi:10.1038/ni735
129. Wolpe SD, Sherry B, Juers D, Davatelis G, Yurt RW, Cerami A. Identification and
characterization of macrophage inflammatory protein 2. Proceedings of the National
Academy of Sciences of the United States of America. 1989;86(2):612–6.
doi:10.1073/pnas.86.2.612
130. Root RK, Dale DC. Granulocyte colony-stimulating factor and granulocyte-macrophage
colony-stimulating factor: comparisons and potential for use in the treatment of infections in
nonneutropenic patients. The Journal of Infectious Diseases. 1999;179 Suppl(Suppl 2):S342–
S352. doi:10.1086/513857
131. Angiolillo AL. Human interferon-inducible protein 10 is a potent inhibitor of
angiogenesis in vivo. Journal of Experimental Medicine. 1995;182(1):155–162.
doi:10.1084/jem.182.1.155
132. Dufour JH, Dziejman M, Liu MT, Leung JH, Lane TE, Luster AD. IFN-gammainducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell
generation and trafficking. Journal of Immunology. 2002;168(7):3195–204.
133. Carr MW, Roth SJ, Luther E, Rose SS, Springer TA. Monocyte chemoattractant protein
1 acts as a T-lymphocyte chemoattractant. Proceedings of the National Academy of Sciences.
1994;91(9):3652–3656. doi:10.1073/pnas.91.9.3652
138

134. Xu LL, Warren MK, Rose WL, Gong W, Wang JM. Human recombinant monocyte
chemotactic protein and other c-c chemokines bind and induce directional migration of
dendritic cells in vitro. Journal of Leukocyte Biology. 1996;60(3):365–371.
doi:10.1002/jlb.60.3.365
135. Tanaka T, Narazaki M, Kishimoto T. Il-6 in inflammation, immunity, and disease. Cold
Spring Harbor Perspectives in Biology. 2014;6(10):1–16. doi:10.1101/cshperspect.a016295
136. Zha S, Zhao Q, Chen J, Wang L, Zhang G, Zhang H, Zhao B. Extraction, purification
and antioxidant activities of the polysaccharides from maca (Lepidium meyenii).
Carbohydrate Polymers. 2014;111:584–587. doi:10.1016/j.carbpol.2014.05.017
137. Shi L. Bioactivities, isolation and purification methods of polysaccharides from natural
products: A review. International Journal of Biological Macromolecules. 2016;92:37–48.
doi:10.1016/j.ijbiomac.2016.06.100
138. Zhao L, Dong Y, Chen G, Hu Q. Extraction, purification, characterization and antitumor
activity of polysaccharides from Ganoderma lucidum. Carbohydrate Polymers.
2010;80(3):783–789. doi:10.1016/j.carbpol.2009.12.029
139. Ferreira SS, Passos CP, Madureira P, Vilanova M, Coimbra MA. Structure-function
relationships of immunostimulatory polysaccharides: A review. Carbohydrate Polymers.
2015;132:378–396. doi:10.1016/j.carbpol.2015.05.079
140. Soltanian S, Stuyven E, Cox E, Sorgeloos P, Bossier P. Beta-glucans as
immunostimulant in vertebrates and invertebrates. Critical Reviews in Microbiology.
2009;35(2):109–138. doi:10.1080/10408410902753746
141. Agerer R. Ectomycorrhizae of Hydnellum peckii on Norway spruce and their
chlamydospores. Mycologia. 1993;85,1(1):74–83. doi:10.2307/3760481
142. Baird RE. Type studies of North American and other related taxa of stipitate hydnums :
genera Bankera, Hydnellum, Phellodon, Sarcodon. J. Cramer; 1986.
143. Ainsworth AM, Parfitt D, Rogers HJ, Boddy L. Cryptic taxa within European species of
Hydnellum and Phellodon revealed by combined molecular and morphological analysis.
Fungal Ecology. 2010;3(2):65–80. doi:10.1016/j.funeco.2009.07.001
144. Parfitt D, Martyn Ainsworth A, Simpson D, Rogers HJ, Boddy L. Molecular and
morphological discrimination of stipitate hydnoids in the genera Hydnellum and Phellodon.
Mycological Research. 2007;111(7):761–777. doi:10.1016/j.mycres.2007.05.003
145. Hall D, Stuntz DE. Pileate Hydnaceae of the Puget Sound area III. Brown-spored genus:
Hydnellum. Mycologia. 1972;64(3):560. doi:10.2307/3757873
146. Newton AC, Watling R, Davy LM, Holden E, Ward SD. Progress towards
implementing the Biodiversity Action Plan for stipitate hydnoid fungi in Scotland. Botanical
Journal of Scotland. 2002;54(1):89–110. doi:10.1080/03746600208685031
147. Kovács D, Vetter J. Chemical composition of the mushroom Laetiporus sulphureus
(Bull.) Murill. Acta Alimentaria. 2015;44(1):104–110. doi:10.1556/AAlim.44.2015.1.10
139

148. Sinanoglou VJ, Zoumpoulakis P, Heropoulos G, Proestos C, Ćirić A, Petrovic J,
Glamoclija J, Sokovic M. Lipid and fatty acid profile of the edible fungus Laetiporus
sulphurous. Antifungal and antibacterial properties. Journal of Food Science and
Technology. 2015;52(6):3264–3272. doi:10.1007/s13197-014-1377-8
149. Liu X, Wang L, Zhang C, Wang H, Zhang X, Li Y. Structure characterization and
antitumor activity of a polysaccharide from the alkaline extract of king oyster mushroom.
Carbohydrate Polymers. 2015;118:101–106. doi:10.1016/j.carbpol.2014.10.058
150. Smiderle FR, Alquini G, Tadra-Sfeir MZ, Iacomini M, Wichers HJ, Van Griensven
LJLD. Agaricus bisporus and Agaricus brasiliensis (1 → 6)-β-d-glucans show
immunostimulatory activity on human THP-1 derived macrophages. Carbohydrate Polymers.
2013;94(1):91–99. doi:10.1016/j.carbpol.2012.12.073
151. Barad A, Mackedenski S, Li WM, Li XJ, Lim BCC, Rashid F, Tackaberry LE,
Massicotte HB, Egger KN, Reimer K, Cheung PCK, Lee CH. Anti-proliferative activity of a
purified polysaccharide isolated from the basidiomycete fungus Paxillus involutus.
Carbohydrate Polymers. 2018;181(August):923–930. doi:10.1016/j.carbpol.2017.11.058
152. GlycoProfileTM Beta-Elimination Kit | Sigma-Aldrich. [accessed 2018 May 16].
https://www.sigmaaldrich.com/life-science/proteomics/post-translationalanalysis/glycosylation/beta-elimination-kit.html
153. N-Glycosidase F PNGase F of Flavobacterium meningosepticum, recombinant from E.
coli | Sigma-Aldrich. [accessed 2018 May 16].
https://www.sigmaaldrich.com/catalog/product/roche/nglyfro?lang=en&region=CA
154. Zhu F, Du B, Xu B. A critical review on production and industrial applications of betaglucans. Food Hydrocolloids. 2016;52:275–288. doi:10.1016/j.foodhyd.2015.07.003
155. Singh RS, Bhari R, Kaur HP. Mushroom lectins: current status and future perspectives.
Critical reviews in biotechnology. 2010;30(2):99–126. doi:10.3109/07388550903365048
156. Synytsya A, Novák M. Structural diversity of fungal glucans. Carbohydrate Polymers.
2013;92(1):792–809. doi:10.1016/j.carbpol.2012.09.077
157. Synytsya A, Novak M. Structural analysis of glucans. Annals of Translational Medicine.
2014;2(2):17. doi:10.21037/3397
158. Ruthes AC, Smiderle FR, Iacomini M. D-Glucans from edible mushrooms: A review on
the extraction, purification and chemical characterization approaches. Carbohydrate
Polymers. 2015;117:753–761. doi:10.1016/j.carbpol.2014.10.051
159. Zhu F, Du B, Bian Z, Xu B. Beta-glucans from edible and medicinal mushrooms:
Characteristics, physicochemical and biological activities. Journal of Food Composition and
Analysis. 2015;41:165–173. doi:10.1016/j.jfca.2015.01.019
160. Chan G, Chan W, Sze D. The effects of β-glucan on human immune and cancer cells.
Journal of Hematology & Oncology. 2009;2(1):25. doi:10.1186/1756-8722-2-25

140

Appendix
A1. DNA Consensus Sequences
*Consensus sequences were produced by Dr Keith Egger
Hericium coralloides
GAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCCCCTTGG
TATTCCGAGGGGCACGCCTGTTCGAGTGTCGTGAAATTCTCAACTCARTCCTCTT
GTTATGAGAGGGCTGGGCTTGGACTTGGAGGTCTTGCCGGTGGTTCCTTCGGGAC
CGTCGGCTCCTCTTGAATGCATGAGTGGATCCCTTTTTGTAGGGTTTGCCCTTGGT
GTGATAATTATCTAYGCCGCGGGTAGCCTTGCGCGCTGGTCTGCTTCTAACCGTC
CTTCGGGACATGTTTTTCATCTCAACTTGACCTCGAATCAGGCGGGACTACCCGC
TGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACTAACAAGGATTCCCCTA
GTAACTGCGAGTGAAGCGGGAAAAGCTCAAATTTAAAATCTGGCGGTCTTCGGC
CGTCCGAGTTGTAGTCTGGAGAAGTGCTTTCCGCGCTGGACCGTGTACAAGTCTC
CTGGAATGGAGC
Hydnellum diabolus
GAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAaTCTTTGAA
CGCACCTTGCGCTCCTTGGTATTccgaggagcaTGCCTGTTTGAGTGTCATGAAATTCT
CAACTGCTTTGGCATTTTTTGTTCAAAGTGAAGTTGGACTTTGGAGGGTTTGCTG
GCGTGTTCGGCTCCTCCCAAAAGCATAAAGCCTCTTTTCTTGGTAGATCTTGGCG
GAAAAGTATCTTCGACGTGATAATCATCTACGTTGTGGAGAAAGCCTTGAGCAA
AGAGCCGTTCTTTGTCTTTGACAAGGAAAGGCTTCTTCTACTGGGGTGGCTCCAC
GAGGGTTACACCTCACTTGACCAATTCGACCTCAAATCAGGCAGGACTACCCGC
TGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACTAACAAGGATTCCCCTA
GTAACTGCGAGTGAAGCGGGAAAAGCTCAAATTTGAAATCTGGCGTGCCCTTGG
CCGTCCGAGTTGTAGTCTGGAGAAGCGTCTTCCGTGCTGGACCATGTACAAGTCC
CTTGGAACAGGGC
Phaeolepiota aurea
GAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGA
ACGCACCTTGCGCTCCTTGGTATTCCGAGGAGCATGCCTGTTTGAGTGTCATTAA
ATTCTCAACCTTTCCAAGTTTTATTACCTGGTCAGGCTTGGATTTGGGGGTTGCAG
GTTTCTTTCATGAAGTCAGCTCCTCTTAAATACATTAGCGGAACCTTTTTGTGGAC
TGTCAAATGGTGTGATAATTATCTACACTATTGTACGCTGCAATCTTATAGGGCT
TCAGCTTCTAACTGTCCATTGACTTGGACAATTCTTGACCATTTGACCTCAAATCA
GGTAGGACTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACTA
ACAAGGATTCCCCTAGTAACTGCGAGTGAAGCGGGAAAAGCTCAAATTTAAAAT
CTGGCGGTCTTTGGCTGTCCGAGTTGTAATCTAGAGAAGTGTTATCCGCGCTGGA
CCGTGTACAAGTCTCCTGGAATGGAGC
141

Laetiporus sp.
*Sample Contaminated*
Letharia vulpina
GAaAtGcgATaagTAATGTgAATtGCAGAAtTCAGTgAATCATcGAATcTTTGAACGCA
CAtTGCgCCCcTcgGTATTCCGGGGGGCAtgCcTGTTcGAGCGTCATTGCACCCCTCA
AGcGTAGctTGGTATTGGGTCTTcGCCCCCGCGgcGtGCCcGAAAatCAgtggcggTccGGT
GtGActTtAagcgTAGTAAATTtTcGTccgcTTTGAAAGTTcGcgCcGTgcCcGgCCAGaCaAC
CCCccctYTATTTCAATAaTTGaCCTcGGaTCagGtagggAtACCCGctGAAcTTAAGCATA
TCAATAAgCGGAGGAAAAgAAACCaWcaGGGaTTGCCTCAgtAACGGCgAgtGAagcG
GCATCAgCTCAAATTTGAAATCTGGCCCTTTCggGGtCCgAGTTGtAATTTgtAgAgAG
TGTttCggGGGAgAccaCggtCTaagtCCattGgAACATGGc
Vulpicida canadensis
GAAATGCGATAACTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGA
ACGCACATTGCGCCCCTCGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTAT
ACCCTTCAAGCGTAGCTTGGTATTGGGCCTCGCCCCCACGGCGTGCCCGAAAAG
CAGTGGCGGTCCGGGGCGACTTTGAGCGTAGTAAAATCATCCCGCTTTGAAAGC
TCGCCTCGCGGCCGGCCAGACAACCCCATTCATTCTATACATTGACCTCGGATCA
GGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACC
AACAGGGATTGCCTCAGTAACGGCGAGTGAAGCGGCAACAGCTCAAATTTGAAA
TCTGGCCCCGTCGGGGTCCGAGTTGTAATTTGTAGAGAGTGCTTCGGGTGAGACC
GCGGTCTAAGTCCATTGGAACATGGC

142