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INVESTIGATION OF ANTI-INFLAMMATORY AND ANTIOXIDANTS
PROPERTIES OF PHENOLIC COMPOUNDS FROM INONOTUS
OBLIQUUS USING DIFFERENT EXTRACTION METHODS
By
Weaam Abdulwahid Abdulnabi Alhallaf
B. Sc. University of Al-Nahrain, 2005
M. Sc. University of Al-Nahrain, 2008
A DISSERTATION
Submitted in Partial Fulfillment of the
Requirements for the Degree of
Doctor of Philosophy of Science
(in Food Science and Human Nutrition/Food Chemistry)
The Graduate School
The University of Maine
May 2020
Advisory Committee:
Dr. L. Brian. Perkins, Research Assistant Professor of Food Science, Advisor
Dr. Rodney Bushway, Professor of Food Science
Dr. Angela Myracle, Assistant Professor of Human Nutrition
Dr. Karl Bishop, Associate Professor College of Science and Humanities
Dr. Balunkeswar Nayak, Associate Professor of Food Processing
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© 2020 Weaam Alhallaf
All Rights Reserved
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INVESTIGATION OF ANTI-INFLAMMATORY AND ANTIOXIDANTS
PROPERTIES OF PHENOLIC COMPOUNDS FROM INONOTUS
OBLIQUUS USING DIFFERENT EXTRACTION METHODS
By Weaam Abdulwahid Abdulnabi Alhallaf
Dissertation Advisor: Dr. L. Brian Perkins
An Abstract of the Dissertation Presented
in Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy
(in Food Science and Human Nutrition/Food Chemistry)
May 2020
Inonotus obliquus, commonly known as Chaga, is a fungal pathogen of birch
trees, known to synthesize a range of phenolic compounds with remarkable health
benefits. These presumed medicinal properties have generated increased interest in Chaga
consumption. Prior research has demonstrated the diverse chemical composition of
Chaga sourced from a variety of geographical locations. However, to our knowledge,
there is currently no available literature regarding the extraction of bioactive compounds
from Chaga grown in the United States. Additionally, the effect of the extraction method
on the antioxidant and anti-inflammation properties specifically, has yet to be validated.
Therefore, the present study was developed to examine the effects of extraction
conditions on phenolic compounds in Maine sourced Chaga and correlate these findings
to anti-inflammatory benefits.
A high-performance liquid chromatography–diode array detection (HPLC–DAD)
method was developed to determine the phenolic acids content in Chaga. The method
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demonstrated good linearity (0.994-0.999) and precision within (RSD ≤ 3) and between
(RSD ≤ 4.2) -day precisions. The procedure also produced good recovery within (≥ 90.1)
and between (≥88.5) -day precisions, as well. The majority of phenolic acids were
extracted from the base hydrolysis fraction (2794.91 µg/g).
The response surface methodology (RSM) was also applied to establish optimum
extraction conditions to obtain phenolic-rich extracts. Results indicate that an extraction
temperature of 170°C and ethanol concentration of 66% were optimal for recovering
phenolic compounds, with a total phenolic content (TPC) value of 39.32 mg GAL/g DW
and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of 76.59%. The
extractions that produced the highest yields of TPC and DPPH were then assessed for the
ability to remediate inflammation using lipopolysaccharide (LPS) activated RAW 264.7
macrophages. The results showed various Chaga extracts have significant anti-
inflammatory activity on LPS-stimulated RAW 264.7 cells. The inhibitory effect was
evident through a decrease in the production of nitric oxide (NO) and down-regulation of
tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-β (IL-1β) in RAW
264.7 macrophages. Therefore, findings confirm that Maine harvested Chaga
demonstrates anti-inflammatory properties. However, the phenolic yields (total phenolic
acids and TPC) and antioxidant activity are highly dependent upon the extraction
methodology.
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ACKNOWLEDGEMENTS
I would like to express the deepest appreciation to my committee chair, Dr. Brian
Perkins, who has supervised me throughout this research work. Without his guidance,
patience, and persistent help this dissertation would not have possible. I would also like
to extend my deepest gratitude to my committee members, Dr. Rodney Bushway, Dr.
Angela Myracle, Dr. Karl Bishop, and Dr. Balunkeswar Nayak for their suggestions and
revisions provided to my research work. I am grateful to all of those with whom I have
had the pleasure to work during this and other related projects. No one has been more
important to me in the pursuit of this project than the members of my family. I would like
to thank my parents whose love and guidance are with me in whatever I pursue. Most
importantly, I would love to thank my loving and supportive husband, Dhafer, and my
three wonderful children, Naya, Lana and Ali, who provide unending inspiration.
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TABLE OF CONTENTS
ACKNOWLEGEMENTS………………………………………………. .............................iii
LIST OF FIGURES ................................................................................................................x
LIST OF TABLES……………………………………………………. ................................xiii
INTRODUCTION……………………………………………………. ................................xv
Chapter
CHAPTER 1 LITERATURE REVIEW……………………………… ................................1
1.1 Naming Origins of Chaga………………………………………… ......................1
1.2 Distribution and Ecology…………………………………………. ......................1
1.3 Morphological Features of Chaga………………………………… ......................3
1.4 History of Medicinal Use…………………………………………. ......................4
1.5 Chemical Analysis of Chaga………………………………………….. ................5
1.5.1 Polysaccharides in Chaga ……………………………………………. ....8
1.5.2 Phenolic Compounds in Chaga ……………………………………. ........11
1.6 Extraction of Phenolic Compounds………………………………. ......................14
1.6.1 Conventional Extraction and Advanced Extraction Methods………… .....15
1.6.2 Acid- Base Hydrolysis ...............................................................................17
1.6.3 Accelerated Solvent Extraction (ASE) …………………….. ....................18
1.6.3.1 Response Surface Methodology RSM………………………... ......20
1.7 Identification and Quantification of Phenolic Compounds………. ........................21
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1.7.1 Folin-Ciocalteu Assay .................................................................................22
1.7.2 High Performance Liquid Chromatography (HPLC) Method
developme Development .......................................................................................................23
1.8 Biological Activity of Chaga……………………………………... ......................27
1.8.1 Antioxidant Activity……………………………………………. ..............27
1.8.2 Anti-Inflammatory……………………………………………… ..............29
CHAPTER 2 HPLC METHOD DEVELOPMENT TO
IDENTIFY AND QUANTIFY PHENOLIC ACIDS IN CHAGA…… ................................33
2.1 Introduction ............................................................................................................33
2.2 Materials and Methods…………………………………. .......................................36
2.2.1 Fungal Material…………………………………………………. ..............36
2.2.2 Chemicals……………………………………………………….. ..............36
2.2.3 Instrumentation…………………………………………………. ..............37
2.2.4 Chromatographic Conditions…………………………………… ...............37
2.2.5 Preparation of Standards………………………………………... ...............37
2.2.6 Methods of Extraction and Hydrolysis…………………………. ...............38
2.2.6.1 Extraction of Free Phenolic Acids……………………………. ........38
2.2.6.2 Base Hydrolysis………………………………………………. ........39
2.2.6.3 Acid Hydrolysis………………………………………………. ........39
2.2.7 Method Validation……………………………………………… ...............40
2.2.8 Statistical Analysis……………………………………………… ...............40
2.3 Results………………………………………………………… ..............................41
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2.3.1 Extraction of Phenolic Acids ......................................................................41
2.3.2. Method Validation .....................................................................................51
2.4 Discussion ..............................................................................................................54
2.5 Conclusion .............................................................................................................58
CHAPTER 3 OPTIMIZATION OF ACCELERATED SOLVENT
EXTRACTION OF PHENOLIC COMPOUNDS FROM CHAGA
USING RESPONSE SURFACEMETHODOLOGY ............................................................59
3.1 Introduction ............................................................................................................59
3.2 Materials and Methods ...........................................................................................62
3.2.1 Fungal Material ...........................................................................................62
3.2.2 Chemicals ....................................................................................................63
3.2.3 Extraction of Phenolic Compounds ............................................................63
3.2.3.1 Green extraction, Accelerated solvent extraction (ASE) ..................63
3.2.3.1.1 Experimental Design and Statistical Analyses ..........................64
3.2.3.2 Conventional Solvent Extraction (CSE) ............................................66
3.2.3.2.1 Maceration extraction (ME) .......................................................66
3.2.3.2.2 Reflux extraction (RE) ...............................................................66
3.2.3.2.3 Soxhlet Extraction (SE) .............................................................66
3.2.4 Determination of Total Phenolic Content (TPC) .........................................66
3.2.5 Determination of Antioxidant Activity (DPPH) ..........................................67
3.2.6 HPLC Analysis of the Extracts ....................................................................68
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3.3 Results ....................................................................................................................69
3.3.1 Single- Factor Experiment ............................................................................69
3.3.2 Optimization of ASE by RSM ......................................................................72
3.3.3 Comparison of ASE and Conventional Methods ..........................................78
3.3.4 High-Performance Liquid Chromatography (HPLC) Analysis
of Phenolic Acids ...........................................................................................................80
3.4 Discussion .............................................................................................................83
3.5 Conclusion ............................................................................................................93
CHAPTER 4 THE ANTI-INFLAMMATORY PROPERTIES OF
CHAGA EXTRACTS OBTAINED BY DIFFERENT EXTRACTION
METHOD AGAINST LPS INDUCED RAW 264.7 .............................................................94
4.1 Introduction ............................................................................................................94
4.2 Materials and Methods ...........................................................................................96
4.2.1 Fungal Material ...........................................................................................96
4.2.2 Reagents ......................................................................................................96
4.2.3 Preparation of Polysaccharide Extracts ......................................................97
4.2.4 Preparation of Phenolic Extracts .................................................................98
4.2.4.1 Green extraction, Accelerated solvent extraction ASE ...................98
4.2.4.2. Conventional Solvent Extraction (CSE) .........................................99
4.2.4.2.1 Maceration Extraction (ME) .............................................99
4.2.4.2.2 Reflux Extraction (RE) ....................................................99
4.2.4.2.3 Soxhlet Extraction (SE) ...................................................99
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4.2.5 Determination of total phenolic content (TPC).. ........................................99
4.2.6 Determination of Antioxidant Activity (DPPH) .........................................100
4.2.7 Determination of Total Neutral Carbohydrate Contents .............................100
4.2.7.1 Determination of Uronic Acid Content ............................................101
4.2.7.2 Determination of Protein Content ....................................................101
4.2.8 Cell Culture .................................................................................................101
4.2.8.1 Measurement of Cell Viability ..........................................................102
4.2.8.2 Measurement of NO production .......................................................103
4.2.8.3 Cytokine Measurement .....................................................................103
4.2.9 Statistical Analysis .......................................................................................103
4.3 Results ....................................................................................................................104
4.3.1 Chemical Composition .................................................................................104
4.3.2 Cell Viability ................................................................................................105
4.3.3 Inhibition of NO Production in LPS-Stimulated RAW 264.7
Macrophages .........................................................................................................107
4.3.4 Inhibition of TNF-α Production in LPS-Stimulated RAW 264.7
Macrophages .........................................................................................................110
4.3.5 Inhibition of IL-6 Production in LPS-Stimulated RAW 264.7
Macrophages ..........................................................................................................113
4.3.6 Inhibition of IL-β Production in LPS-Stimulated RAW 264.7
Macrophages .........................................................................................................116
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4.4 Discussion ...............................................................................................................119
4.5 Conclusion ...............................................................................................................123
APPENDIX Overall Conclusion ............................................................................................125
References ..............................................................................................................................127
Biography of the Auther .......................................................................................................143
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LIST OF FIGURES
Figure 1-1 Classification of the main polyphenols ........................................................12
Figure 1-2 Accelerated solvent extraction unit ..............................................................19
Figure 2-1 Effect of different extraction procedures: (A) base
hydrolysis, (B) acid hydrolysis, and (C) acidified methanol and base
and acid hydrolysis on the extractability of phenolic acids (µg/gdw)
in Chaga .........................................................................................................................43
Figure 2-2 Representative chromatograms of phenolic acids standards
recorded at (A) 280 nm and (B) 329 nm ........................................................................44
Figure 2-3 Representative chromatogram of extract obtained from
chaga using acidified methanol recorded at (A) 280 nm and (B)
329 nm ...........................................................................................................................45
Figure 2-4 Representative chromatogram of extract obtained from
Chaga using 3N NaOH hydrolysis recorded at (A) 280 nm and (B)
329 nm ...........................................................................................................................46
Figure 2-4 Representative chromatogram of extract obtained from
Chaga using 3N NaOH hydrolysis recorded at 3N NaOH hydrolysis
with 10 mM EDTA and 1% AA recorded at (C) 280 nm and (D)
329 nm ...........................................................................................................................48
Figure 2-5 Representative chromatogram of extract obtained from
Chaga using 4N HCl hydrolysis recorded at (A) 280 nm and (B)
329 nm ...........................................................................................................................49
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Figure 2-5 Representative chromatogram of extract obtained from
Chaga using 4N HCl hydrolysis with 10 mM EDTA and 1% AA
recorded at (C) 280 nm and (D) 329 nm ........................................................................50
Figure 3-1 Single factor experiment for the total phenolic yield
from Chaga with (A) accelerated solvent extraction with respect to
solvent type, (B) solvent concentration, (C) extraction temperature,
(D) number of cycles and (E) static time .......................................................................71
Figure 3-2 Counter plot analysis for the total phenolic yield from
inonotus obliquus with accelerated solvent extraction with respect to
total phenolic content (A); DPPH scavenging activity (B);
desirability response (C) ...............................................................................................76
Figure 3-3 Response surface analysis for the total phenolic yield from
inonotus obliquus with accelerated solvent extraction with respect to
total phenolic content (A); DPPH scavenging activity (B); desirability
response (C) ...................................................................................................................77
Figure 4-1 Effects of different samples on cell viability of
RAW 264.7 cells ............................................................................................................106
Figure 4-2 Effect of various samples on production of nitric oxide
(NO) in macrophage RAW 264.7 cells ..........................................................................109
Figure 4-3 Effect of various samples on tumor necrosis factor-α
(TNF-α) expression in macrophage RAW 264.7 cells ...................................................112
Figure 4-4 Effect of various samples on IL-6 expression in
macrophage RAW 264.7 cells .......................................................................................115
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Figure 4-5 Effect of various samples on IL-β expression in
macrophage RAW 264.7 cells .......................................................................................118
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LIST OF TABLES
Table 1-1 Some of the bioactive compounds extracted from Chaga using
different solvents ............................................................................................................7
Table 1-2 Structures of the important naturally occurring phenolic
acids ...............................................................................................................................13
Table 1-3 HPLC conditions to identify phenolic compounds in
Chaga .............................................................................................................................25
Table 2-1 Calibration curve, limit of detection (LOD), and limit of
quantitation (LOQ) for determination of phenolic acids in a spiked
Chaga sample .................................................................................................................51
Table 2-2 Within-day and between-day precisions and accuracy
data at three concentration levels (1, 10, and 25 µg/g) in Chaga
samples ...........................................................................................................................53
Table 3-1 Experimental design with the observed responses for
the recovery of the TPC from Chaga sclerotia samples using
ASE ................................................................................................................................68
Table 3-2 Analysis of variance (ANOVA) for the effects of
extraction temperature and ethanol concentration on TPC
of Chaga ........................................................................................................................73
Table 3-3 Comparison of predicted and experimental values for
TPC yield and DPPH from Chaga using ASE under optimized
process conditions ..........................................................................................................78
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Table 3-4 Comparison of TPC and antioxidant activity (using
DPPH radical scavenging assay) of Chaga sclerotia samples using
ASE, RE, ME and SE extraction methods .....................................................................79
Table 3-5 Quantity of individual phenolic acids (µg/ g DW) from
Chaga extracts using ASE at different extraction temperatures ....................................81
Table 3- 6 Quantity of individual phenolic acids (µg/ g DW) from
Chaga extracts using ASE optimized conditions and conventional
extraction methods .........................................................................................................82
Table 4-1 extraction conditions of Chaga using accelerated solvent
extraction ASE ..............................................................................................................98
Table 4-2 Major chemical content of the crude polysaccharide extracts
from the sclerotia of Chaga ...........................................................................................105
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INTRODUCTION
Inonotus obliquus or Chaga is an unusual polypore fungus that infects living
trunks of birch trees (Betulaceae) often resulting in white heart rot. Historically, Chaga
extracts have been utilized in medicine to prevent and treat gastrointestinal diseases as
well as certain cancers (Lee et al., 2008). More recently, researchers have scientifically
validated these practices, isolating substances from Chaga which demonstrate a range of
biological health benefits including antioxidant, anticancer, antiviral, immune
modulation, and hepatoprotective activities (Chung, Chung, Jeong, & Ham, 2010;Ham et
al., 2009;Lu, Chen, Dong, Fu, & Zhang, 2010). Chemical investigations have further
supported these findings, as I. obliquus has been shown to produce a diverse range of
bioactive compounds such as triterpenoids, polyphenols, melanin pigments, and
polysaccharides (Burmasova et al., 2019; J. H. Lee & Hyun, 2014; Chung, Chung, Jeong,
& Ham, 2010; Lu et al., 2010).
These health mediated effects have steadily increased interest in Chaga
consumption among United States consumers. As a result, there are growing research
initiatives surrounding optimization of extraction procedures defined by maximum target
compound yields. Currently, there are few investigations on United States - harvested
Chaga, or its bioactive content composition (such as phenolics) (Brydon-William, 2019).
Unsurprisingly, the extraction method(s) effect on total phenolic content, antioxidant, and
anti-inflammatory activity has yet to be confirmed. Thus, the primary objectives of this
study were to (1) develop and validate a High Performance Liquid Chromatography
(HPLC) method to assess the phenolic acids content in Chaga in both free and bound
forms, (2) apply the response surface methodology (RSM) approach to obtain the most
phenolic rich extracts from Chaga and (3) screen the anti-inflammatory activity of Chaga
extracts obtained by different extraction methods.
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CHAPTER 1
LITERATURE REVIEW
1.1 Naming Origins of Chaga
Inonotus obliquus, commonly known as Chaga, is a fungal pathogen that belongs to
the Hymenochaetaceae family (Hawksworth et al., 1995). The English name “Chaga” is
derived from the Siberian word “Czaga.” which originated from the Komi Permyak
language, spoken by the indigenous people of the Kama River Basin, in the West Uralian
region of the country (Shashkina et al., 2006). The fungus was first identified and
described by Persoon (1801), who named it Boletus obliquus, then it was later renamed
Polyporus obliquus by Fries (1830), followed by Quélet (1888) who called it Poria
obliqua (under the bark of dry Fagus). In 1927, Bourdot and Galzin termed the fungus
Xanthochrous obliquus, and its current name, Inonotus obliquus, was given by Pilàt
(1936 and 1942), who studied it thoroughly (Lee et al., 2008). The genus name, Inonotus,
directly translates to “black fiber” while the specific epithet, obliquus, refers to its
geometric shape. Specifically, the pores of the reproductive body have sloping direction
to the horizon. Inonotus obliquus is also known by other common names, such as true
tinder fungus, clinker polypore, sterile conk, cinder conk, and cancer polypore (Lee et al.,
2008).
1.2 Distribution and Ecology
Chaga is best characterized as a “circumboreal” organism, as it is found throughout
the Northern Hemisphere, mainly within dense birch forests in characteristically
temperate to subarctic climates. As a result, significant Chaga proliferation has been
reported in forests throughout Russia (Western Siberia, partial regions in the Far East,
Kamchatka peninsula), Poland, France, China (Heilongjiang province, Chinghai
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mountain area of Jilin province), Japan (Hokkaido), Korea, and Canada. In the United
States, Chaga has been harvested domestically in the Northeast (Maine through
Pennsylvania), the Great Lakes region (Michigan, Wisconsin, and Minnesota), Alaska,
and high-altitude areas of the southern Appalachian Mountains, including western North
Carolina, where there is extensive yellow birch growth (Sinclair et. al, 2005; Brydon-
Williams, 2019).
Chaga has been isolated from a variety of tree species including hardwoods such as
red alder (Alnus rubra), American and European beech (Fagus grandifolia and
sylvatica), oak (Quercus spp.), red maple (Acer rubrum), hophornbeam (Ostrya
virginiana), and poplar (Populus spp.) However, the fungus is primarily grown on the
living trunks of mature white birch (B. papyrifera, B. pendula, B. pubescens) yellow
birch (B. alleghaniensis) black birch (B. lenta), water birch (B. occidentalis), and grey
birch (B. populifolia) species. Additionally, the characteristic sterile conk has only been
isolated from birch tree varietals (Brydon-Williams, 2019; Lee et al., 2008).
A 2015 study conducted by Balandaykin and Zmitrovich, determined the preferred
growth environments of the fungal pathogen in birch stands within the Ulyanovsk region
of Russia (mainly comprised of B. pendula and B. pubescens). Particularly, the
researchers determined that Chaga prefers sprout coppiced trees to those with seedling
coppice, mature stands to a younger counterpart, and oligotrophic soils to eutrophic soils.
Furthermore, anthropogenic disturbance in the birch stands have demonstrated a positive
correlation with incidences of Chaga infestation (Balandaykin & Zmitrovich, 2015). In a
similar study conducted in seven regional forests throughout Poland between 1995-2011,
incidences of Chaga growth were higher among stands aged 60 years or older.
Additional Chaga prevalence increased in mixed birch-coniferous forest and bog forest
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compared to wet broadleaved forests, with an estimated total Chaga volume of 46 metric
tons (Szczepkowski et al., 2013). However, these studies are only applicable to European
South Boreal forests, and thus have a limited relevance to Chaga distribution in North
America (Balandaykin & Zmitrovich, 2015).
To better illustrate the relationship between tree species and Chaga fungal growth in
the United States, a recent survey conducted in the White Mountain National Forest of
New Hampshire studied Chaga growth on white birch and yellow birch trees. Among the
examined species, yellow birch was determined to be a more suitable host for the Chaga
fungus. Characteristics including comparative hardiness as well as enhanced ability to
survive through both damage and growing conditions (in particular, larger diameters and
greater heights than other birch species) contributed to these findings (Brydon-Williams,
2019).
1.3 Morphological Features of Chaga
Chaga is a perennial sclerotium, or resting body, which appears as a massive
black charcoal structure with a rust-colored woody texture containing internal interwoven
mycelia. Although Chaga is often referred to as a “mushroom”, scientists are skeptical of
this designation. As mentioned, it is classified as a member of the Hymenochaetaceae
family, which includes a few other dark, woody botanicals that grow on bark and
decaying trees. Unlike the sclerotium, the fruiting body occurs once during the infection
cycle. The structure of the fruiting body resembles a tumor and appears as a crust-like
layer of pores with a light-yellow inner portion surrounded by irregularly cracked
charcoal grey on the exterior. This external feature varies in size between 25-40 cm in
diameter. Chaga fungus grows wild on decaying logs and wound sites of tree stumps. The
invaded tree will utilize various defense mechanisms to resist fungal invasion; however,
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Chaga can persist for up to 80 years, producing 1–3 sclerotia on the main stem and
branches (Lee et al., 2008). It is unclear whether Chaga invasion leads to the eventual
death of the hosted tree, or if mortality of the tree is caused by infection related to other
opportunistic organisms (Niemelä et al., 1995). Various methods such as infected tree
felling, girdling, fungicide, and cutting the trunk into bolts have been tested to control
Chaga infections. None of these methods have successfully prevented the eventual
formation of Chaga conks (Sinclair et al., 2005). However, some infected trees have been
found to reject Chaga infection, ultimately resulting in the falling off of the conk, though
the decay column is left intact (Spahr, 2009).
1.4 History of Medicinal Use
Chaga has been widely recognized as a medicinal source traditionally used to treat
stomach ailments and cancer. Russian legend suggests the famous 12th century prince,
Vladimir Monomakh, was cured from a lip tumor by regularly ingesting Chaga tea. The
Russian novelist, Solzhenitsy similarly mentioned Chaga in his book “The Cancer
Ward”, in which the author describes cancer patients in rural villages, who used habitual
drinking of a hot-water Chaga as a natural remedy to cure aliments. There are also reports
of the Khanty people, an ethnic group from western Siberia, using Chaga to prevent and
treat digestive disorders, cardiac illnesses, and hepatic disorders. They also utilized the
fungus as an antiseptic agent used to clean body wounds (Saar, 1991). The Ainu people, a
tribe indigenous to northern Japan, have also utilized Chaga as a natural treatment for
stomach pain and inflammation. In addition to the perceived health effects of Chaga
consumption, the Ainu people also regarded the fungus as sacred. The act of inhaling
Chaga smoke, known as “eating the smoke” was common practice in particular religious
ceremonies. The Skolt Sami people of Northern Scandinavia, similarly, used Chaga to
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address certain illnesses including cold, flu, and stomach ailments. Additionally, the
fungus was also utilized as a recreational tea in place of other tea or coffee beverages
(Magnani Natalia, 2016). Today the fungus is still regarded for its medical properties. In
1955, following several clinical investigations, the Russian Medical Research Council
approved Chaga preparation, which is listed in the Soviet Pharmacopeia under the name
of “Befunginum”, for several forms of cancers such as genital and breast (Balandaykin
& Zmitorvich, 2015).
1.5 Chemical Analysis of Chaga
Chemical analysis of Chaga reveals that the fungus contains more than 200 bioactive
components. These compounds have been associated with a wide array of biological
activities attributed to several health-mediated effects (Diao et al., 2014; Olennikov et al.,
2012; Y. M. Park et al., 2005). The predominant bioactive constituents in Chaga include
polysaccharides (Wold et al., 2018); triterpenoids (Nakata et al., 2007); steroids (Nikitina
et al., 2016), melanin (Burmasova et al., 2019), and phenolic compounds (Nakajima et
al., 2007). Dragendorff first examined the chemical composition of Chaga in 1864. In
addition to polysaccharides, (Ludwiczak & Wrecino, 1962) first detected and identified
lanostane triterpene compounds (lanosterol-3β- hydroxyl-lanosta-8,24-diene and its
derivative inotodiol) in this mushroom as well. Kahlos and his group also isolated β-
hydroxylanosta-8,24-dien-21-oic acid (trametenolic acid), 3β-hydroxylanosta-8,24-dien-
21-al, 3β,22,25-trihydroxylanosta-8,23-diene, and d 3β,22-dihydroxylanosta-8,24-dien-7-
one (Kahlos & Hiltunen, 1983; Kahlos et al.,1984). Recently, chagabusone, a lanostane-
type triterpenoid, was also isolated following the fractionation of methanolic extracts of
this fungus (Baek et al., 2018). Careful examination of the sclerotia has led to the
isolation of additional constituents including 3 β-hydroxylanosta-8,24-diene-21,23-
!
!
!
6!
lactone, 21,24-cyclopentalanost-8-ene-3β,21,25-triol, and lanost-8-ene-3β,22,25-triol
(Shin et al., 2000; Shin et al., 2001) . In the available literature, approximately 40
triterpene compounds from the lanostane-type structure have been identified in Chaga,
including trace amounts of pentacyclic-type structure of triterpenes, such as betulin,
lupeol, and lupenon (Gao et al., 2009). Further, steroids and alkaloid-like chemical
compounds have also been extracted (Nikitina et al., 2016). Melanin; a high-molecular
weight polyphenol pigment that is synthesized by Chaga as a result of oxidative
polymerization of phenols, has similarly been isolated following physicochemical
examination (Kukulyanskaya et al., 2002). This is a rather noteworthy discovery, as
melanin extracts widely contributed to the varied medicinal properties of this fungus, due
to their high antioxidant properties (Burmasova et al., 2019).
Different solvents have been used in the extraction of bioactive compounds from
Chaga. Previous work has demonstrated that the solvent utilized for extraction may affect
the bioactivity (Kallithraka et al., 2007; Zheng et al., 2010). Chemical profiles of some
bioactive compounds of Chaga are presented in Table 1-1.
!
!
!
7!
!
Compound
Biological activities
Extraction
solvent
Reference
Melanin
Hypoglycemic;
antioxidant
Water
Burmasova et al.,
2019; Lee & Hyun,
2014).
3βHydroxylanosta8,
24diene21al
Antimutagenic and
antioxidative;
anticarcinogenic,
hypoglycemic
Ethyl acetate;
Methanol
(Chung et al., 2010;
Ham et al., 2009; Lu
et al., 2010).
Lanosta24ene3β,21
diol
Antitumor activity
Chloroform
(Taji et al., 2008).
Ergosterol peroxide
Hypoglycemic
Ethyl acetate
(Lu et al., 2010).
Lignin
Inhibits HIV-
1protease activity
Water
(Ichimura et al.,
1999).
Lignin
Antioxidant,
immunostimulants
Water
(Niu et al., 2016)
Betulinic acid
Antiproliferative
effect against human
lung adenocarcinoma
cells (A549)
Water
(Géry et al., 2018).
Ergosterol; ergosterol
peroxide, trametenolic acid
Anti-inflammatory
activity
Ethanol
(Ma et al., 2013).
Ergosterol peroxide,
trametenolic acid
Anticancer activity
Ethanol
(Ma et al., 2013).
4-hydroxy-3,5-dimethoxy
benzoic acid 2-hydroxy-1-
hydroxymethyl ethyl ester,
protocatechic acid, caffeic
acid, 3,4-
dihybenzaladehyde, 2,5-
dihydroxyterephtalic acid,
syringic acid, 3,4-
dihydroxybenzalacetone
Antioxidant
Methanol
(Nakajima et al.,
2007)
Table 1-1 some of the bioactive compounds extracted from Chaga using different solvents
!
!
!
8!
Table 1-1 continued
Compound
Biological activities
Extraction
solvent
Reference
3,4-
dihydroxybenzalacetone
Prevents hydrogen
peroxide-induced
oxidative stress in
PC12 cells
Methanol
(Nakajima et al.,
2009)
Inotodiol
Inhibits Cell
Proliferation
Chloroform
(Nomura et al., 2008)
Polysaccharides
Antioxidant;
antihyperglycaemic
effects
Water
(Liu et al., 2018; Mu
et al., 2012)
1.5.1 Polysaccharides in Chaga
Within the last decade, there has been steadily growing interest in Chaga
functionality due to its abundant polysaccharide composition. These compounds have
been associated with several therapeutic functions including antitumor, antioxidant,
hypoglycemic and nontoxigenic effects (Mu et al., 2012; Diao et al., 2014). More
specifically, Fan et al reported that the water-soluble polysaccharides within Chaga exert
in vivo antitumor activity and enhance immune defense via lymphocyte proliferation in
addition to increased tumor necrosis factor-α TNF production (Fan et al., 2012). Similarly,
Hu et al found that a purified fraction of Chaga had a therapeutic effect against chronic
pancreatitis in mice via multiple pathways including antioxidative effects (Hu et al.,
2016). Additionally, Diao et al determined that Chaga derived polysaccharides possess
antihyperglycemic effects in mice, which could be a potential therapeutic option for
diabetes (Diao et al., 2016).
!
!
!
9!
There are several previously published reports regarding Chaga polysaccharide
extraction. Some of this available literature is focused on polysaccharide extraction by
hot water, alkaline-treated hot water, or more novel methodologies. Hu et al, for example,
examined the effects of different temperatures on polysaccharide extraction efficiency.
The study determined the total polysaccharide yield was highest when an 80°C hot water
method, compared to water heated to 50°C and 70°C, was used. In addition to compound
yield, the ratio of polysaccharide to protein was also highest using this procedure (Hue et
al., 2009). This finding indicates that more substances may be extracted by water using an
elevated temperature. It has also been suggested that application of ultrasonic/microwave
will assist in extraction yield and purity of crude polysaccharides, which are advantages
over the traditional hot water methodology (Chen et al., 2010).
The monosaccharide composition of aqueous Chaga extracts consists primarily of
glucose and mannose. Kim et al previously reported that purified endo-polysaccharide
from cultivated Chaga mycelia was an α-linked fucoglucomannan, composed primarily of
mannose and glucose, with small amounts of galactose, fucose, and glucosamine as well
(Kim et al., 2006a). This is in accordance with the results of Huang et al, in which
glucose and mannose were the major monosaccharides isolated from Chaga
polysaccharides along with galactose and rhamnose, which were found in smaller ratios
(Huang et al., 2012)
Formation of polysaccharide complexed minerals has been proposed to alter both
the physiological properties and biological activity of the polysaccharides, but also
enhance the bioavailability of the minerals. Wang et al investigated the effect of an
interaction between Chaga polysaccharides and iron (III) on the antioxidant activity and
bioavailability of the final product. Data from this work suggested that the
!
!
!
10!
polysaccharide-iron (III) complex may be a suitable candidate for a new iron supplement
due to its high antioxidant capacity and increased bioavailability (Wang et al.,2015). On
the other hand, Selenizing modification polysaccharides have been successfully prepared
by a HNO3- Na2SeO3 method. Following this modification, polysaccharides were found
to have significantly increased antioxidative capacity in vivo, as well as in vitro (Hu et
al., 2017).
Polysaccharides are usually conjugated with other molecules such as proteins
exhibiting various bioactive properties. The protein content within Chaga polysaccharides
is typically determined using the Bradford method, which uses bovine serum albumin
(BSA) as a standard (Bradford M., 1976). Some studies indicate that the protein content
in polysaccharides may exist as polysaccharide-protein complexes. This hypothesis is
supported by evidence that after several deproteinization processes using the Sevage
method, a small amount of protein remained attached to the isolated polysaccharides (Mu
et al., 2012).
Uronic acid, a polysaccharide constituent, is mainly determined by the m-
hydroxydiphenyl method that utilizes galacturonic acid as a standard. The compound was
found in less than 5% Chaga polysaccharides derived from both water-soluble and alkali-
soluble samples. However, contrary to this finding, there have been several literature
reports linking high uronic acid content and protein content to increased bioactivity of
polysaccharides. Huang et al obtained five polysaccharide fractions (IOP1b, IOP2a,
IOP2c, IOP3a and IOP4) from aqueous Chaga extracts and found the higher content of
uronic acid and proteinous substances resulted in stronger antioxidant activities of
polysaccharides (Huang et al., 2012). The effects of three different drying methods
(freeze drying, hot air drying and vacuum drying) on bioactivities and chemical
!
!
!
11!
compositions of polysaccharides were also comparatively investigated. Despite its high
polysaccharide yield and nutrient content after drying application, results demonstrated
that the higher activity of the freeze-dried polysaccharides might be related to its higher
uronic acid and protein contents (Ma et al., 2013).
1.5.2 Phenolic Compounds in Chaga
Phenolic compounds are a group of secondary metabolites that are synthesized in
plants as a response to environmental stress, such as pathogens, insect attack, UV
radiation, and wounding. Chaga fungus synthesizes a range of phenolic compounds (PC),
which possess remarkable potential for free radicals scavenging (Babitskaia et al., 2000;
Nakajima et al., 2007). This property has been associated with reduced incidences of
oxidative stress-induced diseases including cancer, hypertension, neurodegenerative, and
autoimmune diseases (Zheng et al., 2010). The common structure of phenolic compounds
is characterized by the presence of aromatic ring(s) bearing one or more hydroxyl groups.
PCs are further classified into different groups based on the number of phenolic units and
other functional attributes that link these rings (Dai & Mumper, 2010). As a result,
different phenolic classes have been formed, as shown in Figure 1-2.
Different classes of phenolic comounds have been reported in Chaga from both
wild sclerotia and liquid cultures. Zheng et al determined that the production of
flavonoids from cultures of Chaga was enhanced in response to oxidative stress induced
by hydrogen peroxide H2O2 (1mM H2O 2 at a rate of 1.6 mL/h) (Zheng et al 2009b).
Similarly, a high yield of flavonoids, i.e., epicatechin-3-gallate (ECG), epigallocatechin-
3-gallate (EGCG), and naringin, was also obtained from Chaga under fermentated
conditions (Xu et al., 2016). Low molecular weight phenolic ingredients, i.e., 3,4-
dihydroxybenzalacetone (DBL), and high-molecular-weight phenolic pigments, i.e.
!
!
!
12!
melanin, have also been identified in Chaga (Nakajima et al., 2007; Olennikov et al.,
2012).
Figure 1-1 Classification of the main polyphenols (Dai & Mupper, 2010)
Among phenolic compound constituents, phenolic acids have previously been
reported in Chaga, including gallic acid, protocatechuic acid, protocatechuic aldehyde,
caffeic acid, vanillic acid, ferulic acid, and syringic acid (Ju et al., 2010; Nakajima et al.,
2007). Structurally, phenolic acids are composed of one aromatic ring, a carboxylic acid
group and one or more hydroxyl groups. Despite these structural commonalities, phenolic
acids can be further differentiated into two parent structures: hydroxycinnamic acid and
!
Phenolic Compounds!
Phenolic!
Acids!
Hydroxycinammi
c!acid
!
Hydroxybenzoic!
acid
!
Flavonoids!
Stilben
es!
Lignan
s
!
Flavonols!
Isoflavone
s!
Flavones!
Flavanols!
!
!
!
13!
hydroxybenzoic acid (Khoddami et al., 2013). The distinction within these groups is
related to the number and position of hydroxyl groups in the molecule (Pereira et al.,
2009) (Figure 2). Often, aldehyde analogues such as vanillin and protocatechuic
aldehyde are also referred to as phenolic acids (Robbins, 2003).
Table 1-2 Structures of the important naturally occurring phenolic acids
!
Hydroxybenzoic Acids Hydroxycinnamic Acids
Phenolic acids
Hydroxybenzoic Acids
Hydroxycinnamic Acids
R1
R2
R3
R4
R1
R2
R3
R4
Protocatechuic
acid
H
OH
OH
H
Gallic acid
H
OH
OH
OH
Vanillic acid
H
OCH3
OH
H
Syringic acid
H
OCH3
OH
OCH3
Caffeic acid
H
OH
OH
H
Cinnamic acid
H
H
H
H
!
!
!
14!
Table 1-2 continued
Phenolic acids
Hydroxybenzoic Acids
Hydroxycinnamic Acids
R1
R2
R3
R4
R1
R2
R3
R4
Ferulic acid
H
OCH3
OH
H
Sinapic acid
H
OCH3
OH
OCH3
1.6 Extraction of Phenolic Compounds
In general, the extraction of phenolic compounds from natural sources is
challenging due to several factors including chemical diversity of the compounds, uneven
distribution within the cell matrix, and interference from other components within the
plant. However, determining optimal extraction conditions is key in the recovery of
phenolic compounds of interest (Dai & Mumper, 2010). In Chaga, the structural diversity
of its components and the presence of interfering substances such as terpenes and
carbohydrates, results in large variability of the chemical properties including different
biomolecules polarity and hydrogen bonding, which can greatly influence the extraction
yields and biological activities of the phenolic extracts. Prior work has indicated that the
efficiency of an extraction method and resulting bioactivity of phenolic compounds is
strongly dependent on specific extraction conditions such as methodology, solvent type,
and extraction temperature. Published data has shown varying levels of total phenolic
content from Chaga depending on the extraction method and conditions as well. For
example, Zheng et al. investigated solvent effects on phenolic metabolites and
antioxidant activities of Chaga extracts using a heated reflux system. The study showed
that polar solvents (ethyl acetate, acetone, ethanol, and water) had a higher extraction rate
!
!
!
15!
for PC compared to extractions using non‐polar solvents. The authors also reported
enhanced antioxidant activities following the increase in solvent polarity. For instance,
the scavenging for superoxide anion, DPPH, and hydroxyl radicals reached 71.48, 357.73
and 52.18%/mg, respectively, via ethyl acetate extraction (Zheng et al., 2011a). However,
under ultrasonic extraction conditions, an additional report deemed aqueous acetone
(70% v/v) as the most efficient solvent for extracting total phenolic compounds in Chaga
(Zhao et al., 2013). In addition to solvent type, temperature of the extraction protocol
needs to be considered when extracting phenolic compounds. Relatively high
temperatures are reported to increase extraction efficiency by decreasing viscosity of the
solvent. This leads to better diffusion of the solvent through the cell walls which
ultimately increases phenolic compounds solubility in the solvent (Ju et al., 2010; Seo &
Lee, 2010). However, there are upper limits to this observation, with extremely high
temperatures causing degradation of phenolic compounds (Lindquist & Yang, 2011).
1.6.1 Conventional Extraction and Advanced Extraction Methods
Different extraction methods have been used to recover phenolic compounds
from Chaga. Conventionally, a large amount of organic solvent such as methanol,
ethanol, acetone, ethyl acetate, or their mixture with water, either at room temperature, or
at the boiling point of the chosen solvent(s) is used. However, the major concern with
these methods are long extraction times, the large amount of solvent required to execute
the procedure, potential negative environmental impacts, as well as decomposition of
thermo-labile compounds. Therefore, advanced “green” extraction methods have been
designed to reduce energy consumption and allow the use of alternative solvents and/or
reduce organic solvent consumption, while producing safe and high-quality extracts
(Axelsson et al., 2012; Chemat et al., 2012; Cvjetko et al., 2018). Some of these
!
!
!
16!
technologies such as supercritical fluid extraction (SWE), microwave-assisted extraction
(MAE), ultrasound extraction (UAE) and accelerated solvent extraction (ASE) have
previously been applied to the extraction of phenolic compounds from a variety of natural
sources (Andrade et al., 2012;Şahin & Şamli, 2013; Tripodo et al., 2018; Švarc-Gajić et
al., 2013). Zhao et al has applied aqueous two-phase extraction (ATPE) associated with
ultrasonic extraction to extract these compounds from Chaga, specifically (Zhao et al.,
2013). ATPE technique depends on the selective separation of the compounds of interest
from the sample matrix into two separate phases. Ultrasonic extraction on the other hand,
breaks the cell wall structure, which allows for a better diffusion rate, improving the
solubility of the analyte in the extraction media. Applying the combined methods to
Chaga derived phenolic compound extraction, previous literature has reported increased
yields in phenolics (37.8 mg gallic /g dw compared to 29.0 mg gallic/ g) obtained by
traditional ultrasonic extraction. Additionally, the anti-oxidative activities of the extract
obtained by the combined methods have been shown to enhance scavenging superoxide
anions SOD, hydroxyl radicals, and DPPH radical isolation. Microwave assisted
extraction (MAE) compared to the traditional aqueous Chaga extraction (maceration)
methods, also increased melanin output and extracted antioxidative properties. It was
found that the melanin extraction efficiency and antioxidative properties were equal to or
higher than those of the melanin obtained by the classic extraction method. Further, the
MAE reduced the extraction time by 2–3.5 times that of the traditional method (Nitriles
1975; Parfenov et al., 2019). Seo & Lee highlighted that the use of a high temperature
during Subcritical Water Extraction (SWE) of Chaga-derived phenolic compounds
resulted in 30 times more phenolic content than that obtained using a lower extraction
temperature. The researchers found the highest total phenolic content (TPC)
(10.72 mg/mL) was obtained in SWE extracts produced at 250°C for 30 minutes,
!
!
!
17!
compared to TPC (0.61 mg/mL) produced at 50°C for 10 minutes. The authors also
suggested utilizing SWE as a tool to increase the antioxidant activities of Chaga extracts
(Seo & Lee 2010). As suggested, phenolic levels within these fungal extracts are largely
dependent on the extraction methods.
1.6.2 Acid- Base Hydrolysis
In general, phenolic acids occur as free, soluble conjugates (e.g., glycosides and
esters of fatty acids) and insoluble-bound forms (Naczk & Shahidi, 2004). These different
forms of phenolic acids require specific extraction conditions considering the chemical
nature of these acids in terms of stability and susceptibility to degradation (Mattila &
Kumpulainen, 2002). Aqueous organic solvents such as methanol, acetone, and/or water,
sometimes with a small portion of acetic acid, have been used to extract soluble phenolic
acids (free, soluble esters, and soluble glycosides) (Escarpa & Gonzlez, 2001; Mattila &
Kumpulainen, 2002). However, organic solvents cannot extract the bound forms of
phenolic acids. These phenolics are coupled to the cell wall components through ester,
ether, and glycosidic bonds (Escarpa & Gonzlez, 2001; Luthria, 2012; Mattila &
Kumpulainen, 2002; Nardini & Ghiselli, 2004). Base and/or acid hydrolysis are typically
utilized to cleave the ester linkage to the cell walls and release the bound phenolic acids
(Mattila & Kumpulainen, 2002). Enzymatic treatments (mainly pectinases, amylases, and
cellulases) have been reported as being a less prevalent technique to release phenolic
acids from their corresponding conjugates (Robbins & Bean, 2004) Generally, acid
hydrolysis involves treating the sample with HCl ranging from 1 to 4N, either at room
temperature or at high extraction temperatures; while in base hydrolysis procedures,
samples are treated with NaOH ranging from 2 to 10N for a few hours or overnight at
room temperature (Nardini & Ghiselli, 2004). Some studies have adopted a sequential
!
!
!
18!
extraction regime to systematically release the phenolic acids from their respective forms.
In this extraction system, the sample is first subjected to organic solvent or acidified
organic solvent to extract soluble phenolic acids. The base hydrolysis is then utilized to
release the bound phenolic acids, followed by acid hydrolysis to liberate the bound
phenolics that have not been hydrolyzed during the base hydrolysis process (Li et al.,
2020; Ross et al., 2009).
1.6.3 Accelerated Solvent Extraction (ASE)
Many modern techniques such as accelerated solvent extraction (ASE) have been
developed to overcome the previously discussed phenolic extraction challenges. The
most notable benefits of ASE include increases in extraction efficiency, extract
selectivity, and procedure reproducibility. Specifically, using elevated pressure and
temperatures for a relatively short duration, results in successful degradation of the cell
membranes, which considerably reduces the extraction time and increases product yields.
The application of high pressure (>1000 psi) allows solvents to be heated to temperatures
higher than their boiling point, which disrupts the cell wall structure, decreases liquid
solvent viscosity, and accelerates diffusion through membranes; thus, allowing better
penetration into the matrix and further improving the extraction procedure (Ameer et al.,
2017; Mustafa & Turner, 2011).
!
!
!
19!
!
Figure 1-2 Accelerated solvent extraction unit
!In the literature, accelerated solvent extraction ASE has been described to
improve the extraction of phenolic compounds in cereals and grains. The phenolic
compounds within these mediums are typically bound to the cell wall components,
presenting considerable challenges in their extraction. Barros et al evaluated the
extraction efficiency of phenolic compounds from sorghum bran and found that using
ASE compared to conventional methods, significantly improved extraction of phenolic
compounds from the product (Barros et al., 2013). Gomes et al optimized these phenolic
compound extraction conditions (including temperature, solvents, extraction time, and
number of extractions) to primarily isolate flavonoids, from species of Passiflora. The
!
!
!
20!
researchers’ methodology employed ASE with a Box-Behnken design. Compared to
traditional extraction approaches, ASE results in substantial decreases in solvent
consumption and extraction time Gomes et al. (2017. This is because the technique can
be automated in an inert atmosphere, which results in minimal phytochemical
degradation due to limited extraction times. Further, ASE allows the operator to better
control the extraction parameters including temperature, static extraction time, and the
number of extraction cycles, further enhancing the method efficiency (Ameer et al., 2017;
Mustafa & Turner, 2011).
ASE relies on the use of elevated pressure (500-2000 psi) and temperature (40-
200 °C) for a relatively short time to accelerate the rate of extraction. Pressure allows the
extraction cell to be filled faster, forcing liquid into the pores and maintaining the liquid
state of extraction solvents, even at high temperatures. This elevated temperature results
in an increase in the solvation ability of the solvent due to a decrease in both its’ viscosity
and surface tension. The result is an accelerated diffusion rate and mass transfer of the
analyte into the solvent; thereby improving the recovery of compounds of interest (Ameer
et al., 2017; Carabias-Martínez et al., 2005; Wang & Weller, 2006).
1.6.3.1 Response Surface Methodology RSM
The response surface methodology (RSM) has been at the forefront of recent
extraction optimization research. RSM has been used to develop and improve process
parameters among various food, biology, and chemistry applications (Ferreira et al.,
2007). RSM is a combined mathematical and statistical tool that facilitates a multifaceted
evaluation of the impact of several parameters and their interactions relative to the
desired response. This technique simultaneously optimizes experimental conditions,
while reducing the required number of experimental trials (Bezerra et al., 2008; Gunst,
!
!
!
21!
Myers, & Montgomery, 1996). This methodology is widely used to overcome the
limitations of a single factor approach, in which only one variable can be analyzed with
respect to all other factors remaining constant. As suggested, the single factor approach is
time-consuming, labor-intensive, and lacks critical data on factor interaction outcomes.
Thus, employment of RSM can aid in ensuring optimal processing or extraction
conditions (Bezerra et al., 2008; Liyana-Pathirana & Shahidi, 2005).
1.7 Identification and Quantification of Phenolic Compounds
Several techniques have been used for the identification and quantification of
Chaga-derived phenolic compounds. The Folin-Ciocalteu (FC) assay and high
performance liquid chromatography (HPLC) combined with various detectors are the
most frequently applied methods (Table 3 and Table 4). Other chromatographic
approaches such as gas chromatography (GC) and ultra-fast liquid chromatography
(UFLC) have also been reported. Ju et al employed GC in combination with a mass
spectrometer detector (MS) to identify and quantify 2,5-dihydroxyterephthalic acid, a
low-level phenolic compound, previously reported to be present in Chaga (Ju et al.,
2010). This proposed procedure boasts higher sensitivity and selectivity to identify
compounds present in Chaga at low concentrations. Similarly, Hwang et al utilized the
same GC-MS method to quantify minor levels of vanillic acid and 2,5-
dihydroxyterephthalic acid in Chaga under various extraction conditions (Hwang et al.,
2019). In addition to these compounds, hispin analogs and melanins have also been
detected in the diverse range of phenolic metabolites that are present in the complex
Chaga matrix (Zheng et al., 2009b). Such compounds are difficult to identify using
chromatographic approaches; establishing suitable extraction conditions and/or
subsequent metabolite fractionation is required prior to chromatographic analysis.
!
!
!
22!
Further, the need of pure reference standards, many of which are unstable and/or not
available, is essential for accurate quantification of these compounds (Olmo-Cunillera et
al., 2020).
Unlike the previously described procedures, nuclear magnetic resonance (NMR)
spectroscopy is a potent tool used for both qualitative and quantitative analysis of
phenolic compounds in Chaga without extraction or derivatization. NMR spectroscopy is
known to be non-destructive, non-selective, and capable of acquiring a comprehensive
range of organic metabolite profiles. Previous studies have examined the NMR profiles
of phenolic compounds from cultured mycelia of Chaga grown under different light
conditions (Olmo-Cunillera et al., 2020; Zheng et al., 2009a; Zheng et al., 2009b),
conducted NMR- spectroscopy analysis to compare the phenolic profiles of mycelia
exposed to fungal elicitor from cultures grown under normal physiological conditions.
1.7.1 Folin-Ciocalteu Assay
Folin–Ciocalteu assay is widely used to quantify the total phenolic content from
different naturally derived extracts. The FC is a calorimetric method based on the
oxidation/reduction reaction. In this assay, the phenolic group becomes oxidized while
the metal ion is reduced. When conducted in an alkaline medium, electrons from the
phenolic compounds are transferred to the FC reagent, forming a blue chromophore that
can be read spectrophotometrically. This newly formed blue coloration absorbs UV-vis
radiation in wavelength range of 700- 760 nm(Cicco et al., 2009). Generally, gallic acid
is used as the reference standard compound and results are expressed as gallic acid
equivalents (mg/mL). For many years, the FC method was commonly used to measure
the phenolic content in natural products because it is simple, standardized, and the
reagent is commercially available(Magalhães et al., 2010;Ramirez-Sanchez et al., 2010).
!
!
!
23!
Nevertheless, there are limitations to this assay, including lack of specificity. This is
because other products of oxidation can interfere, causing over-estimation of the
polyphenol content within the sample. Substances such as sugars, aromatic amines,
organic acids and bases can react with the FC reagent (Ramirez-Sanchez et al., 2010).
1.7.2 High Performance Liquid Chromatography (HPLC) Method Development
A combination of water and organic solvents such as methanol and acetonitrile
are typically used for the reverse phase HPLC separation of phenolic compounds. For
satisfactory chromatographic resolution and peak shapes of phenolics, the pH of the
mobile phase should be maintained at the range (2-4) to avoid the ionization of phenolic
compounds (Michalkiewicz et al., 2008). Formic acid, acetic acid, and phosphoric acid
have been utilized most often for MP acidification. Generally, acetonitrile and acidified
water are the dominant mobile phases utilized in HPLC quantification of phenolic
compounds extracted from Chaga. Previous studies have shown that the increase in the
pH of the mobile phase dramatically reduces the retention of phenolic acids. This is
because phenolic acids are weak acids and exist primarily in the protonated form at a pH
below their pKa, so in order to increase the retention capacity of phenolic acids, the pH of
the eluent should be lower than their pKa (Michalkiewicz et al.,2008 ;Joseph & Palasota,
2001). The choice of a stationary phase is key for phenolic compound separation.
Typically, C18 or reversed phase (RP-C18) column 1.8-4.6mm ID and 2.0- 5 µm particle
size has been used for phenolics separation in Chaga. It has been reported that most
HPLC assays of phenolic compounds are carried out at ambient column temperature and
recently, however, higher temperatures have also been reported (Khoddami et al., 2013).
HPLC elution time to detect phenolic compounds in Chaga have ranged from 35 to 75
min. phenolic compounds in Chaga are detected using UV-VIS, PDA coupled with
!
!
!
24!
fluorescence, Mass spectrometric (MS), and electrospray ionization mass spectrometry
(ESI-MS). Table 1- 3 presents some of the HPLC procedures that have been used to
identify and quantify phenolic compounds in Chaga.
!
!
!
25!
Chaga
Phenolic Compounds
Column/Detector
Solvent/flow
rate
Temperature
C/Time min
Reference
Wild
gallic acid,
protocatechuic acid,
protocatechuic
aldehyde, caffeic acid,
syringic acid
Zorbax Eclipse
Plus C18 (250 ×
4.6mm × 5 µm)
column/DAD
A: water
containing
0.1% formic
acid; B:
acetonitrile
containing
0.1% formic
acid; Elution
profile: 0–6
min, 8–12%
B; 6–15 min,
12–17.4% B;
15–27 min,
17.4–27% B;
27–27.1 min,
27-8% B; and
27.1–35 min,
8% B/Flow
rate:1
mL/min:
Injection
volume: 20µL
NM/35
(Hwang et
al., 2019)
Wild
2,5-
Dihydroxyterephthalic
and vanillic acids
U-VDSpher PUR
C18-E
(100 × 2.0 mm
× 1.8 µm)
column/ ESI-MS
A: water
containing
0.1% formic
acid; B:
acetonitrile
containing
0.1% formic
acid; Elution
profile: 0–6
min, 8–12%
B; 6–15 min,
12–17.4% B;
15–27 min,
17.4–27% B;
27–27.1 min,
27-8% B; and
27.1–35 min,
8% B/Flow
rate:1
mL/min:
Injection
volume: 20µL
NM/35
(Hwang et
al., 2019)
!!
Table 1-3 HPLC conditions to identify phenolic compounds in Chaga
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!
!
26!
Table 1-3 continued
Chaga
Phenolic Compounds
Column/Detector
Solvent/flow
rate
Temperature
C/Time min
Reference
Wild
Protocatechuic acid,
vanillic acid, syringic
acid
Synergi Hydro-
RP
(250 × 4.60 mm ×
4 µm)
column/PDA
A: water
containing
0.1% formic
acid; B:
acetonitrile;
Elution
profile: 0-20
min,5-15%B,
20-50 min,
15-30%B, 50-
65 min, 30-
50%B, 65-75
min, 50-95%
B; Flow rate:
1.0 mL/min;
Injection
volume:10 µL
NM/75
(Ju et al.,
2010)
Wild
2,5-
dihydroxyterephthalic
acid, Protocatechuic
acid, vanillic acid,
syringic acid
Synergi Hydro-
RP
(250 × 4.60 mm ×
4 µm)
column/ESI-MS
A: water
containing
0.1% formic
acid; B:
acetonitrile;
Elution
profile: 0-20
min,5-15%B,
20-50 min,
15-30%B, 50-
65 min, 30-
50%B, 65-75
min, 50-95%
B; Flow rate:
1.0 mL/min;
Injection
volume:10 µL
NM/75
(Ju et al.,
2010)
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!
!
!
!
27!
Table 1-3 continued
Chaga
Phenolic Compounds
Column/Detector
Solvent/flow
rate
Temperature
C/Time min
Reference
Fermented
ferulic acid, gallic
acid, epicatechin-3-
gallate (ECG),
epigallocatechin-3-
gallate (EGCG),
naringin.
NM/DAD–ESI–
MS/MS
A: water
containing
0.1% formic
acid; B:
acetonitrile;
Elution
profile: 0-20
min, 95-
90%A, 20-40
min, 90-5%A
NM/40
(Xu, Zhao,
& Shen,
2016b).
Fermented
gallic acid, ferulic
acid, epicatechin-3-
gallate (ECG),
epigallocatechin-3-
gallate (EGCG),
phelligridin G,
davallialactone,
inoscavin B
Synergi Hydro-RP
(250 × 4.60 mm ×
4 µm) column/
DAD–ESI–
MS/MS
A: water
containing
0.1% formic
acid; B:
acetonitrile;
Elution
profile: 0-20
min, 5-10% B,
20-45 min, 10-
95%B
NM/45
(Zhu & Xu,
2013)
Wild
gallic acid,
protocatechuic
acid, p-
hydroxybenzoic
acids, cinnamic acid
NM/PDA
NM
NM
(Glamočlija
et al., 2015)
!
1.8 Biological Activity of Chaga
1.8.1 Antioxidant Activity
The DPPH assay is based on the reduction of the radical DPPH• by receiving a
hydrogen atom from the antioxidant species. The DPPH• is a stable radical, which has an
unpaired valence electron at one bridge nitrogen atom.
As an antioxidant source, Chaga has been previously reported to have the
strongest antioxidant activity among other fungi in both superoxide and hydroxyl radical
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28!
scavenging activities (Nakajima et al., 2007). Chaga contains an abundance of
polyphenols and natural black pigments known as melanin, which are responsible for this
antioxidant action. Babitskaia et al investigated the melanin complex production of
Inonotus obliquus in fermented conditions. The researchers found that copper ions
(0.008%), pyrocatechol (1.0 mM), and tyrosine (20.0) stimulated the formation of this
complex. As a result of its formation, melanin in Inonotus obliquus demonstrates both
high antioxidant and genoprotective activities for the fungus (Babitskaia et al., 2000). Hu
et al similarly tested the antioxidant activity of ethanol crude and hot water (50°C, 70 °C,
and 80 °C) extracts of Inonotus obliquus. The total antioxidant capacity was measured by
superoxide anion scavenging activity (SOD) and DPPH assays. The results from this
work showed that the ethanol extract exhibited the maximum SOD-like activity, whereas
the hot water extract (70 °C) exhibited the maximum DPPH radical-scavenging activity
(Hu et al., 2009). Cui et al demonstrated that the polyphenolic extract, although dose-
dependent, was most effective in scavenging superoxide radicals, followed by the
extracts containing triterpenoids and steroids (Cui et al., 2005). The polysaccharide
extracts on the other hand were entirely inactive. Further, the data from this study suggest
that there are several direct human health benefits attributed to Chaga extract
consumption. More specifically, the polyphenolic extract efficiently may protect human
keratinocytes from oxidative stress. Yun et al also showed that the antioxidant properties
of Chaga could have a tremendous benefit on the human aging process. Particularly,
conditions such as enhanced apoptosis and elevated levels of Reactive Oxygen Species
(ROS), which are caused by direct cell exposure (several types) to various oxidants (i.e.
hydrogen peroxide or ultraviolet (UV) radiation) play a significant role in oxidative stress
and the aging process. This study determined that a pretreatment with Chaga extract
scavenged intracellular ROS and prevented lipid peroxidation in H2O2-treated human
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29!
fibroblast. An additional finding was that MMP-1 and MMP-9 activities in the fibroblasts
were inhibited, leading to increases in collagen synthesis. A third benefit that the
researchers observed was that the deleterious effects of UV exposure, such as skin
thickening, and wrinkle formation was impeded on a hairless mice model. The oxidative
stress in PC12 cells after H2O2 toxicity was suppressed by Chaga extract as well (Yun et
al., 2011).
1.8.2 Anti-Inflammatory
Inflammation is a complex biological response of body tissues elicited by harmful
physical, chemical, and biological stimuli such as chemical toxins and pathogens (Bak et
al., 2013). The inflammatory response is regulated by a cascade of inflammatory
mediators and growth factors that are produced by activated macrophages. Macrophages
are important cells that play a key role in the immune system and are associated with
inflammatory diseases. Activated macrophages produce various cytokines such as tumor
necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), as well as other
inflammatory mediators including the nuclear factor kappa-B, nitric oxide (NO), and
prostaglandin which primarily serves to protect the host. However, excessive
uncontrolled production of these defense molecules can lead to severe inflammation and
tissue damage (Dai et al., 2019; Robbins et al., 2016).
The production of pro-inflammatory cytokines such as the nuclear factor kappa-B
NF- κB is an indication of the inflammation reaction. NF- κB is a protein complex that is
implicated in the inflammation process of both macrophages and lymphocytes. It also
regulates the expression of genes involved in cellular proliferation and adhesion. The
activation of NF-κB has been reported to play a key role in the induction of the
transcriptions of multiple pro-inflammatory mediators, including tumor necrosis factor α
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30!
(TNF-α) and interleukin-1β (IL-1β). During normal physiological conditions, NF-KB is
sequestered in the cytosol of the unstimulated cell as an inactive complex bound to
inhibitor kappa B (I κB). Exposure to one of several possible inflammatory agents
activates the cell and initiates the inflammation reaction. These stimuli agents can be
exogenous (such as lipopolysaccharides, LPS) or endogenous (TFN- α or IFN-) (Mendis
et al., 2008). The stimulation of this reaction then causes the enzyme IjB-kinase (IjK) to
phosphorylate the inactive complex, and release NF-KB molecules to move freely into
the nucleus. This movement then leads to the induction of multiple inflammatory
mediators, such as iNOS, COX-2, TNF-α, interleukins (IL)-6 and -8, and others (Cheung
et al., 2013).
Nitric oxide (NO) is an important inter and intracellular messenger participating
in cellular signaling and a wide range of physiological and pathological functions within
many cells and tissues, including the brain, the cardiovascular system, and certain host
immune cells (Paige & Jaffrey, 2006). NO is synthesized enzymatically, through the
conversion of L-arginine to L-citrulline by nitric oxide synthase (NOS) in the presence of
molecular oxygen and NADPH (Alderton et al., 2001). Nitric oxide synthase enzymes are
present in three main isoforms which exist in various cell types. nNOS is the first type of
NOS that localizes in the central nervous system (neurons). Those which localize in the
vascular endothelial cells are termed eNOS, and NOS that are induced in hepatocytes and
macrophages after infection are called iNOS. All three isoforms have similar molecular
structures; however, each show tissue specific variations in their nitric oxide expression
and pathway regulations (Choudhury & Saha, 2012). Both nNOS and eNOS are
constitutive (cNOS), and capable of producing suitable amounts of NO that plays a key
role in various biological functions in the human body. In contrast, the inducible isoform
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31!
iNOS is expressed in response to pro-inflammatory cytokines and bacterial LPS. It has
previously been reported that the overproduction of NO due the action of iNOS is a major
contributor in inflammatory processes is indicative of the degree of the inflammation
(Alderton et al., 2001).
The anti-inflammatory effects of various Chaga extracts which are characterized
by high phenolic content have been reported in both LPS induced macrophages and
dextran sulfate sodium_(DSS)_induced colitis mice models. Methanol–based Chaga
extracts have also been shown to significantly inhibit the productions of NO, PGE2, and
TNF-. This suppresses the expressions of both inducible nitric oxide (NO) synthase
(iNOS) and cyclooxygenase-2 (COX-2) via the down-regulation in the binding activity of
nuclear factor B (NF-B) in LPS induced macrophage cell systems 2005. Kim et al
investigated the effect of the addition of different portions of water to organic solvent on
both antioxidant and anti-inflammatory activities of the extract (Kim et al., 2007).
Seventy percent aqueous ethanol extracts of chaga performed better than the 100%
ethanol extract alternative for both antioxidant and anti-inflammatory activity in
macrophage cells. This finding could be attributed to the addition of water to polar
solvent, leading to an increase in the efficiency of phenolic compound extraction by
increasing their solubility. Van et al reported that all four fractions (triterpenoids, water-
soluble polysaccharides, polyphenolic, and low molecular weight polysaccharides)
separated from methanol extract were found to significantly reduce NO production in the
murine macrophage cell system as well as reduce the level of other inflammatory
cytokines depending on dosage (Van et al., 2009). In addition, water Chaga extracts have
demonstrated significant anti-inflammatory activity when used against a DSS- induced
colitis mice model, due to down-regulation of the expression of inflammatory mediators
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32!
(Mishra et al., 2012). Debnath et al similarly demonstrated the in vivo anti-inflammatory
activity of ethanol extracts from Chaga grown on germinated brown rice against DSS
-induced colitis mice model (Debnath et al., 2012).
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CHAPTER 2
HPLC METHOD DEVELOPMENT TO IDENTIFY AND QUANTIFY
PHENOLIC ACIDS IN CHAGA
2.1 INTRODUCTION
Epidemiological studies indicate that the consumption of chaga extracts is related
to reduction in oxidative DNA damage in human lymphocytes, as well as to diabetes and
cancer incidence rates (Geng et al., 2013; Kim et al., 2006; Park et al., 2004). These
health benefits have been partially attributed to a wide variety of potential
chemopreventive compounds present in Chaga, called phytochemicals, and include
antioxidants such as phenolic compounds (Cui et al., 2005; Nakajima et al., 2007). There
are large number of scientific investigations in the literature based on the extraction and
analysis of different classes of phenolic compounds including phenolic acids (Dai &
Mumper, 2010; Luthria & Mukhopadhyay, 2006; Luthria & Pastor-Corrales, 2006;
Mattila & Kumpulainen, 2002).
In general, phenolic acids occur as free, soluble conjugates (e.g., glycosides and
esters of fatty acids) and insoluble-bound forms (Naczk & Shahidi, 2004). These different
forms of phenolic acids require specific extraction conditions considering the chemical
nature of these acids in terms of stability and susceptibility to degradation (Mattila &
Kumpulainen, 2002). Aqueous organic solvents such as methanol, acetone, and/or water,
sometimes with a small portion of acetic acid, have been used to extract soluble phenolic
acids (free, soluble esters, and soluble glycosides) (Escarpa & Gonzlez, 2001; Mattila &
Kumpulainen, 2002). However, organic solvents cannot extract the bound forms of
phenolic acids. These phenolics are coupled to the cell wall components through ester,
ether, and glycosidic bonds (Luthria, 2012; Escarpa & Gonzlez, 2001; Mattila &
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34!
Kumpulainen, 2002; Nardini & Ghiselli, 2004). Base and/or acid hydrolysis are typically
utilized to cleave the ester linkage to the cell walls and release the bound phenolic acids
(Mattila & Kumpulainen, 2002). Enzymatic treatments (mainly pectinases, amylases, and
cellulases) have been reported as being a less prevalent technique to release phenolic
acids from their corresponding conjugates (Robbins & Bean, 2004). Generally, acid
hydrolysis involves treating the sample with HCl ranging from 1 to 4N, either at room
temperature or at high extraction temperatures, while in base hydrolysis procedures,
samples are treated with NaOH ranging from 2 to 10N for a few hours or overnight at
room temperature (Nardini et al., 2002). Some studies have adopted a sequential
extraction regime to systematically release the phenolic acids from their respective forms.
In this extraction system, the sample is first subjected to organic solvent or acidified
organic solvent to extract soluble phenolic acids. The base hydrolysis is then utilized to
release the bound phenolic acids, followed by acid hydrolysis to liberate the bound
phenolics that have not been hydrolyzed during the base hydrolysis process (Li et al.,
2020; Ross et al., 2009).
Reported extraction methods of phenolic acids from Chaga are still incomplete.
One reason for this may be the lack of information regarding the total content of phenolic
acids in Chaga. Most of the extraction procedures described in the literature describe the
content of the free forms of phenolic acids of these extracts. Specific knowledge
pertaining to the content of bound forms is scarce or missing. Common extraction
solvents used to separate free phenolic acids from Chaga include methanol, ethanol,
acetone, ethyl acetate and/or water (Nakajima et al., 2007; Van et al., 2009). Previous
work has employed a high temperature during the extraction of phenolic compounds from
Chaga as an attempt to liberate these compounds from the cell wall components (Seo &
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Lee, 2010). Phenolic extracts produced by utilizing thermal processes such as steam
treatment or high pressure and temperature have been found to contain a higher content
of phenolic acids than those extracts produced without such treatments (Ju et al., 2010;
Hawng et al, 2019). From what is stated above, it can be hypothesized that some phenolic
acids in Chaga are bound to the cell wall structure, and thus, a hydrolysis procedure is
required in order to liberate these bound acids from their respective forms.
A major problem associated with the hydrolysis procedures is the loss of several
phenolic acids, particularly dihydroxy-cinnamic acids such as caffeic acid due to the
oxidation of phenolic acids (Krygier et al., 1982). However, previous work of Nardini et
al has introduced the addition of ascorbic acid AA, a powerful antioxidant, and
ethylenediaminetetraacetic acid (EDTA), a metal chelator, to prevent the degradation of
phenolic acids during the hydrolysis procedures (Nardini et al.,2002). No work has
examined the addition of AA and EDTA during the extraction of phenolic acids from
Chaga.
Several methods have been developed for the quantification of phenolic acids
from different sources, including the Folin–Ciocalteu assay and chromatographic
approaches (HPLC-UV, GC-MS, LC-MS) (Acosta-Estrada et al., 2014; Robbins, 2003;
Shahidi and Yeo, 2016). The Folin–Ciocalteu assay, which provides an estimate of total
phenolic content by quantifying the antioxidant capacity, is not specific to phenolic
groups and suffers from interference by ascorbic acid and reducing sugars (Stalikas,
2007). Chromatographic methods occupy a leading position in the separation and
quantification of individual phenolic acids (Stalikas, 2007). Therefore, there is a need for
development of a chromatographic HPLC method that can separate and simultaneously
measure the prominent phenolic acids in Chaga in a single run. The aims of this study
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36!
were thus (1) to examine the effect of acid and base hydrolysis on the extractability of
total phenolic acids from Chaga and (2) to develop a suitable HPLC method for
simultaneous identification and quantification of five phenolic acids in Chaga.
2.2 MATERIALS AND METHODS
2.2.1 Fungal Material
Chaga sclerotia were collected from a yellow birch (Betula alleghaniensis) from a
forest in Maine, USA. Samples were freeze-dried (Model 7754511, Labconco
Corporation, Kansas City, Missouri, USA), then ground using an electrical grinder
(Nutribullet, model-NBR-1201M, Los Angeles, USA). The ground powder was then
passed through a 20-mesh (0.84 mm) sieve, and only particles with a diameter smaller
than 0.84 mm (20-mesh) were collected and pooled. All material was stored in a -20°C
freezer until subsequent extraction preparation.
2.2.2 Chemicals
Phenolic acid standards—3,4-dihydroxybenzoic acid (PA), caffeic acid (CA),
syringic acid (SA), and 3,4-dihydroxybenzaldehyde (PCA)—were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Methanol, ethyl acetate, petroleum ether,
ethylenediaminetetraacetic acid (EDTA), ascorbic acid (AA), ethyl acetate, sodium
carbonate, vanillic acid (VA), hydrochloric acid, acetic acid, and formic acid were
provided by Fisher Chemicals (Fair Lawn, NJ, USA). Ultrapure water was obtained from
a Millipore water system (EMD Millipore, Billerica, MA, USA). All reagents and organic
solvents were HPLC or analytical grade.
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2.2.3 Instrumentation
Chromatography of phenolic acids was carried out on an HPLC system (Hewlett
Packard model 1100) consisting of a pump, a vacuum degasser and Diode-Array Detector
(Agilent Technologies, Paolo Alto, CA, USA). Chromatograms were recorded and
evaluated by the software Agilent 3D Chemstation, version B.04.01 (Agilent
Technologies).
2.2.4 Chromatographic Conditions
The chromatographic separation of the examined analytes was carried out on a
reverse-phase Hypersil GOLD aQ C18 column (150 mm × 4.6 mm i.d., 3 µm particle
size) (Thermo Scientific, USA) preceded by a Hypersil GOLD guard column (Thermo
4 × 3.0 mm). The column and guard column were held at 30°C, and the flow rate was set
at 1 mL/min. The mobile phase of the HPLC system consisted of (A) methanol and (B) a
0.1% V/V formic acid/ultrapure-water, pH = 2.8. The gradient was linear at a flow rate of
1 mL/min The solvent gradient in volume ratios was as follows: 95% to 50% B for
16 min; the solvent gradient was reduced to 95% B at 18 min, and maintained at 95% for
2 min; the latter was followed by washing with methanol and re-equilibration of the
column for 10 min. Total acquisition time was 30 min. The wavelength used for the
quantification of the phenolic acids with the diode array detector DAD was 280 nm for
all the phenolic acids except for caffeic acid, which was monitored at 329 nm.
Identification of the phenolic acids was achieved by comparing retention times and UV
spectra of the unknowns with the standards.
2.2.5 Preparation of Standards
All the identified phenolic acids were quantified with external standards.
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38!
Stock solutions of the phenolic acids were initially prepared at 1 mg/mL in an appropriate
volume of methanol/water (75:25, v/v). Working standard solutions were obtained by
appropriate dilution of the stock solutions with methanol/water (75:25, v/v) in the
concentration range of 0.1–100 µg/mL. All solutions were kept at -20°C for further
analysis. The standard solutions were stable for at least three months (Nnane & Damani,
2002).
2.2.6 Methods of extraction and hydrolysis
Following the general procedure of Ross et al. (2009), three different methods of
extractions combined with hydrolysis regimes were tested.Method 1 employed acidified
methanol to extract free phenolic acids; Method 2 used a pure base hydrolysis with
NaOH at different concentrations with and without AA (1%) and EDTA (10 mM); and
Method 3 was accomplished with a pure acid hydrolysis, with HCl at different
concentrations - with and without the presence of AA (1%) and EDTA (10 mM).
Experimental steps after hydrolysis were performed as prescribed by Luthria & Pastor-
Corrales (2006) and Mattila & Kumpulainen (2002).
2.2.6.1 Extraction of free phenolic acids
Extraction was achieved by sonication of 0.5 g of ground Chaga sample in 10 mL
methanol containing 10% acetic acid for 15 min. After centrifugation at 6,700 rpm (2,000
x g) for 10 min, the supernatant was removed, and extraction was repeated once more in a
similar way. The combined extracts were evaporated to dryness under a gentle flow of
nitrogen, and the residues were re-dissolved in 200 µL of methanol: water (75:25, v/v).
The samples were filtered through a 0.45 µm filter (PTFE) before being analyzed by
HPLC.
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39!
2.2.6.2 Base hydrolysis
The ground samples of Chaga 0.5 g were hydrolyzed by adding 10 mL of NaOH
at each of the following concentrations: 2, 3, 6, and 10N (with or without 10 mM EDTA
and 1% AA). The mixture was flushed with nitrogen and allowed to hydrolyze for 30 min
at 40–45°C. After 30 min, the sample allowed to cool and 1.4 mL of 6N HCl was added
to acidify the reaction mixture. The liberated phenolic acids were extracted with 20 mL
of DE/EA (1:1, v/v). The mixture was vortexed for 45 s and centrifuged (eppendrof 5430,
Hauppauge, NY) for 10 min at 6,700 g. Centrifuging caused the DE/EA organic phase to
separate from the aqueous phase. The DE/EA organic phase containing the phenolic acids
liberated from the base hydrolysis was collected by removing the upper organic
(supernatant) phase from the bottom aqueous residue phase using a pipette. The DE/EA
organic supernatants were combined, and the combined DE/EA was evaporated to
dryness under a rotary vacuum. The residue was resuspended in 5 mL methanol: water
(75:25, v/v). The samples were then filtered through a 0.45µm filter (PTFE), and were
analyzed with HPLC.
2.2.6.3 Acid hydrolysis
The phenolics were extracted as previously described (Krygier et al., 1982), with
some modifications. The ground samples of Chaga 0.5 g were hydrolyzed by adding 10
mL of HCl at one of the following concentrations: 1, 2, 3, and 4N (with or without
10 mM EDTA and 1% ascorbic acid). The mixture was incubated for 30 min at room
temperature. The experimental steps after hydrolysis were performed as described in
section 2.6.1.
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2.2.7 Method validation
The method was validated in terms of linearity, accuracy, within-day and
between-day precision, and sensitivity based on the limit of detection (LOD) and limit of
quantification (LOQ). Linearity was evaluated using linear regression analysis of six-
point calibration curves, which were obtained by plotting the peak area versus the
concentration of each standard. Linear regression analysis was used to calculate the slope,
intercept, and correlation coefficient of each calibration line. The within-day and
between-day precisions were evaluated by analyzing Chaga samples fortified at three
concentration levels (1, 10, and 25 µg/g). Each solution was measured three times in the
same day for within-day precision and three times over four consecutive days for
between-day precision. Both precision estimates are expressed as relative standard
deviations (RSD), i.e. standard deviation of repeated measurements as a percentage of the
mean value. Recovery was used to evaluate the accuracy. There were three different
concentrations of standards added to the sample extract method (3N NaOH with AA and
EDTA protection) at three concentration levels (1, 10, and 25 µg/g) for each compound
examined. The recovery was calculated as follows:
𝑅𝑒𝑐𝑜𝑣𝑒𝑟𝑦 =100% ×
𝑎𝑚𝑜𝑢𝑛𝑡 𝑓𝑜𝑢𝑛𝑑 −𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑎𝑚𝑜𝑢𝑛𝑡
𝑎𝑚𝑜𝑢𝑛𝑡 𝑠𝑝𝑖𝑘𝑒𝑑
The calculations for the (LOD) were based on the standard deviation of y-intercepts of
the regression lines (σ) and the slope (S), using the following equation LOD = 3.3 σ/S.
(LOQ) were calculated using the equation LOQ = 10 σ/S.
2.2.8 Statistical analysis
All experiments were repeated three times. Data are expressed as mean of the
replicates ± standard deviation (SD). The software IBM SPSS Statistics (version 25) was
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41!
used to perform statistical analysis of experimental results. A one-way analysis of
variance (ANOVA) test was applied to compare means followed by Tukey’s test. P
values <0.05 were considered statistically significant.
2.3 RESULTS
2.3.1 Extraction of Phenolic Acids
Three different methods of extraction combined with hydrolysis regimes were
employed to extract phenolic acids from Chaga. The free phenolic acids were extracted
using acidified methanol, while bound phenolic acids were liberated by base hydrolysis
and acid hydrolysis, respectively. Figure 2-1 represents the effect of base hydrolysis with
NaOH at 1, 2, 3, and 4N with and without AA and EDTA on the extractability of total
phenolic acids from chaga. Up to 47.42% higher extractability of total phenolic acids was
observed after incubation of chaga with 3N NaOH compared to incubation with 1N
NaOH. However, degradation of CA initiated at 2N NaOH and resulted in about 65% and
81% lower CA at 2N and 3N of NaOH, respectively. The results also showed a protective
effect for AA and EDTA; higher extractability of total phenolics was achieved after
extraction using NaOH at all concentrations with protection (AA and EDTA), in
comparison to extracts obtained without protection. For example, the base hydrolysis (3N
NaOH with AA and EDTA) resulted in 37% higher total phenolic acids content
compared to 3N NaOH without AA and EDTA, and the same trend was observed at the
other examined NaOH concentrations (Figure 2-1A).
For the acid hydrolysis extraction, it was observed that increasing the
concentration of HCl up to 4N resulted in an increase of total phenolic acids by 31%, but
the stability of CA was negatively affected, and extraction resulted in 65% lower CA.
The results also showed a protective effect of AA and EDTA when added to HCl
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!
!
42!
hydrolysis, at all concentrations. The highest extractability of total phenolic acids was
achieved after acid hydrolysis (4N HCl with protection) and resulted in 38% higher total
phenolic acids content compared to the same concentration without protection (Figure 2-
1 B).
The results in Figure 2-1C indicate that the majority of phenolic acids in chaga
were extracted from the base hydrolysis (3N NaOH with EDTA and AA) followed by the
acid hydrolysis (4N HCl with AA and EDTA), whereas the acidified methanol resulted in
extracting the lowest total phenolic acids content. Base hydrolysis liberated nearly 3.5
and 10 times the amount of phenolics as acid hydrolysis and acidified methanol,
respectively.
Qualitative and quantitative analyses of individual phenolic compounds of Chaga
extracts at different extraction conditions were executed using high-performance liquid
chromatography HPLC. Figure 2-2A and Figure.2-2B depict the HPLC chromatograms
of the five phenolic acids (PA, PCA, CA, VA, CA, and SA with elution times of 6.2, 7.7,
11.5, 12.7, and 13.7 min, respectively.
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Figure 2-1 Effect of different extraction procedures: (A) base hydrolysis, (B) acid hydrolysis,
and (C) acidified methanol and base and acid hydrolysis on the extractability of phenolic
acids (µg/gdw) in Chaga. Columns represent means of duplicate samples (n = 3). Different
superscripts represent significant differences (P <0.05).
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44!
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Figure2-3A and Figure2-3B show reprehensive HPLC chromatograms of chaga
treated with 0.1 acetic acid 70% (methanol: water) at 280 and 329 nm, respectively. All
the examined phenolic compounds were detected, and the UV spectra of these peaks
matched the UV spectra of their respective authentic standards. For this condition, all the
examined compounds were detected, and the UV spectra of these peaks matched the UV
spectra of their respective authentic standards.
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Figure 2-2 Representative chromatograms of phenolic acid standards recorded at (A)
280 nm and (B) 329 nm. 1 = protocateuchuic acid (6.2 min); 2 = protocatechuic
aldehyde (7.7 min); 3 = caffeic acid (11.5 min); 4 = vanillic acid (12.7 min), and
5 = syringic acid (13.7 min).
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Figure 2-4A and Figure 2- 4B show representative chromatograms of the Chaga
sclerotia hydrolyzed with 3N NaOH without protectors monitored at 280 and 329 nm,
respectively. For this condition, all the examined compounds were detected, except for
CA, which was not detected at 329 nm. The UV spectra of these peaks matched the UV
spectra of their respective authentic standards. Figure 2-4C and Figure 2-4D show
representative chromatograms of the chaga sclerotia hydrolyzed with 3N NaOH with
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Figure 2-3 Representative chromatogram of extract obtained from Chaga using acidified
methanol recorded at (A) 280 nm and (B) 329 nm. 1 = protocatechuic acid (6.2 min);
2 = protocatechuic aldehyde (7.7 min); 3 = caffeic acid (11.5 min); 4 = vanillic acid
(12.7 min); and 5 = syringic acid (13.7 min).
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13.4 mM EDTA and 2% AA monitored at 280 and 329 nm, respectively. For this
condition, all the examined phenolic compounds were detected, and the UV spectra of
these peaks matched the UV spectra of their respective authentic standards.
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Figure 2-4 Representative chromatogram of extract obtained from Chaga using 3N NaOH
hydrolysis recorded at (A) 280 nm and (B) 329 nm. . 1 = protocatuchuic acid (6.2 min);
2 = protocatechuic aldehyde (7.7 min); 3 = caffeic acid (11.5 min); 4 = vanillic acid
(12.7 min), and 5 = syringic acid (13.7 min).
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Figure 2-5A and Figure 2-5B show representative chromatograms of the Chaga
sclerotia hydrolyzed with 6N HCl without protectors monitored at 280 and 329 nm,
respectively. For this condition, all the examined compounds and the UV spectra of these
peaks matched the UV spectra of their respective authentic standards. Figure2-5C and
Figure2-5D representative chromatogram of the Chaga sclerotia hydrolyzed with 3N HCl
with 13.4 mM EDTA and 2% AA monitored at 280 and 329 nm, respectively. For this
condition, all the examined phenolic compounds were detected, and the UV spectra of
these peaks matched the UV spectra of their respective authentic standards.
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Figure 2-4 Representative chromatogram of extract obtained from Chaga using 3N NaOH
hydrolysis recorded at 3N NaOH hydrolysis with 10 mM EDTA and 1% AA recorded at
(C) 280 nm and (D) 329 nm. 1 = protocatuchuic acid (6.2 min); 2 = protocatechuic
aldehyde (7.7 min); 3 = caffeic acid (11.5 min); 4 = vanillic acid (12.7 min), and
5 = syringic acid (13.7 min).
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Figure 2-5 Representative chromatogram of extract obtained from Chaga using 4N HCl
hydrolysis recorded at (A) 280 nm and (B) 329 nm. 1 = protocatuchuic acid (6.2 min);
2 = protocatechuic aldehyde (7.7 min); 3 = caffeic acid (11.5 min); 4 = vanillic acid
(12.7 min), and 5 = syringic acid (13.7 min).
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Figure 2.5 Representative chromatogram of extract obtained from Chaga using 4N HCl
hydrolysis with 10 mM EDTA and 1% AA recorded at (C) 280 nm and (D) 329 nm.
1 = protocatuchuic acid (6.2 min); 2 = protocatechuic aldehyde (7.7 min); 3 = caffeic
acid (11.5 min); 4 = vanillic acid (12.7 min), and 5 = syringic acid (13.7 min).
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2.3.2. Method Validation
The method was validated in terms of linearity, accuracy, within-day and between-day
precision, and sensitivity based on LOD and LOQ according to the International
Conference on Harmonization (ICH,1996/2005) and the Commission Decision
(2002/657/EC) guidelines. The calibration curves were constructed with six
concentrations, in triplicate, for each phenolic compound, using the external standard
method. The results in Table 2-1 show good linear correlation and high sensitivity under
these conditions as confirmed by correlation coefficients (R2, 0.994–0.999). The LODs
and LOQs determined in the chaga sample ranged between 0.13–0.17 µg/g and 0.39–0.52
µg/g, respectively. Detailed information of these compounds regarding calibration curves,
linear ranges, LODs, and LOQs is summarized in Table 2-1.
Table 2-1 Calibration curve, limit of detection (LOD), and limit of quantitation (LOQ)
for determination of phenolic acids in a spiked Chaga sample.
Compound
Regression equation
R2
LOD (µg/g)
LOQ (µg/g)
PA
y = 32,149x - 5.9164
0.994
0.17
0.51
PCA
y = 48,482x - 19.389
0996
0.16
0.48
CA
y = 50,114x - 21.822
0.999
0.13
0.39
VA
y = 32,505x - 16.607
0.999
0.15
0.45
SA
y = 28,432x - 10.508
0.999
0.17
0.52
PA, protocatechuic acid; PCA, protocatechuic aldehyde; CA, caffeic acid; VA, vanillic; and SA, syringic
acid.
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Precision of the method was assessed by performing within-day precision and
between-day precision experiments. As shown in Table 2, the results showed satisfactory
within- and between-day precision for the analytical method, with RSD lower than 4.2%
for all compounds tested. Accuracy, expressed as % recovery of the five standards,
ranged with the base hydrolysis method (3N NaOH with AA and EDTA protection) from
89.1% to 103.3% for within-day analysis and from 85.5% to 103.5% for between-day
analysis.
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Table 2-2 Within-day and between-day precisions and accuracy data at three
concentration levels (1, 10, and 25 µg/g) in Chaga samples.
Compound
Within-day precision (n=3)
Between-day precision (n=3 replicates ×4
days)
1µg/g
10µg/g
25µg/g
1µg/g
10µg/g
25µg/g
RSD
R
RSD
R
RSD
R
RSD
R
RSD
R
RSD
R
PA
2.6
98.1
2.1
97.7
0.9
98.2
3.8
98.3
3.5
97.2
4.1
96.5
PCA
2.4
99.2
2.5
98.4
1.5
99.2
2.3
97.3
3.2
98.1
3.0
99.1
CA
1.2
89.1
1.3
91.4
0.5
91.1
2.7
91.7
4.0
85.5
3.7
93.1
VA
1.5
95.8
2.9
98.8
3.0
99.8
4.2
99.2
0.8
97.3
4.1
101.3
SA
2.1
101.9
2.3
99.2
2.3
103.3
0.9
103.5
3.3
98.5
4.2
98.5
PA, protocatechuic acid; PCA, protocatechuic aldehyde; CA, caffeic acid; VA, vanillic; and SA,
syringic acid. RSD: relative standard deviation, expressed as%, R: recovery rate, expressed as%.
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2.4 DISCUSSION
The first aim of this study was to examine the effect of extraction method and
hydrolysis on the extractability of total phenolic acids from Chaga, including free, bound,
and conjugated forms. Many studies have established that phenolic acids in nature are
rarely present in soluble free form and that they most often occur as esters, glycosides,
and complexes bound to the cell wall. Organic solvents have been described to separate
the soluble phenolic acids, while base and acid hydrolysis is known to liberate the
insoluble phenolic acids through breaking their linkages to the cell wall components. Our
results show that base hydrolysis (3NaOH with AA and EDTA) is the most effective
system for the extraction of phenolic acids from the Chaga matrix followed by acid
hydrolysis (4N HCl with AA and EDTA), while acidified aqueous methanol is much less
effective. Previous investigations have revealed that base hydrolysis primarily breaks the
ester bonds, while acid hydrolysis often breaks the glycosidic bonds (Acosta-Estrada et
al., 2014; Ross et al., 2009; Nardini et al., 2002). This suggests that most phenolic acids
in Chaga may be present in the bound form, mainly ester-bonded and to a lesser extent
glycosidic-bonded to the cell wall of Chaga. It is difficult to compare the results obtained
in this study to those of previous studies because of the lack of investigations concerning
the forms of phenolic acids in chaga. However, one study has suggested a heat
pretreatment as an attempt to liberate bound phenolic acids from Chaga (Ju et al., 2010).
The authors revealed that the levels of the identified phenolic acids were significantly
increased as a result of the thermal treatment. Other reports employed high temperature
and pressure during the extraction of phenolic compounds from Chaga to release these
phenolics from the cell wall components (Hawng et al, 2019; Seo & Lee, 2010). The
results from our study and the previous studies support the idea that hydrolysis
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procedures are required during the extraction of phenolic acids from Chaga to release
these acids from their respective conjugates.
The highest total phenolic content was achieved after base hydrolysis (3N NaOH
with AA and EDTA). At this concentration, PCA was the dominant phenolic acid in the
acid hydrolysates and made up 68.36% of the total identifiable phenolic pool. An
increase in the base concentration beyond 3N resulted in lower total phenolic acids
content. For example, raising the concentration to 4N NaOH with AA and EDTA resulted
in 13.22% lower extractability of total phenolic acids with 83.91% less CA compared to
3N NaOH with AA and EDTA. Ross et al reported higher extractability of phenolic acids
after extraction using 10N NaOH compared to 2N NaOH (Ross et al., 2009). However, it
should be noted that, in their study, the 10N NaOH was diluted with water before
extraction, and therefore, the final concentration was 3.3N NaOH, which is similar to the
concentration used in this study.
Although acid hydrolysis was found to increase the total content of phenolic
acids, the concentration of CA decreased at a high level of HCl. For example, raising the
concentration of HCl up to 4N, even with the presence of AA and EDTA, resulted in the
extraction of less CA. This is consistent with previous studies that have reported lower
stability of CA under strong acidic conditions (Krygier et al., 1982; Verma et al., 2009).
Conversely, acidic conditions favored the extraction of SA, supporting Verma et al who
suggested that acidic hydrolysis is the optimum method to liberate some phenolic acids,
particularly SA, from wheat grains (Verma et al.,2009). At 4N HCl (with AA and
EDTA), PCA was the dominant phenolic acid in the acid hydrolysates and made up
51.26% of the total identifiable phenolic pool. PCA is the predominant phenolic acid in
both acid and base hydrolysis conditions.
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In terms of the stability of phenolic acids, our study showed that the addition of
AA and EDTA during acid and base hydrolysis improves the stability of phenolic acids.
For example, CA, which was found in all concentrations of the base hydrolysis, with AA
and EDTA protection, was not detected at a high level of NaOH without AA and EDTA
protection, supporting the findings of Krygier et al who reported low stability of CA
when hydrolyzed under alkaline conditions (Krygier et al.,1982). In contrast, Nardini &
Ghiselli (2004) reported good stability of phenolic compounds during alkaline hydrolysis
in the presence of AA and EDTA. Similarly, SA was detected in higher levels in the acid
hydrolysis fractions with AA and EDTA protection than those without protection,
indicating a protective effect of EDTA and AA when added to the acid hydrolysis
methods. This is consistent with a previous study (Ross et al., 2009) where, in
investigating the use of HCl with and without EDTA and AA protection, the authors
reported higher total phenolic acids content with the use of AA as EDTA from dry beans.
The effectiveness of the HPLC method was tested using standard solutions of five
phenolic acids. Generally, a combination of water and organic solvents such as methanol
and acetonitrile are utilized for the separation of phenolic acids. For satisfactory
chromatographic resolution and peak shapes of these acids, the use of acidified mobile
phases is required (Michalkiewicz et al., 2008). Generally, formic acid, acetic acid, and
phosphoric acid have been used. In this study, formic acid was selected. It has a
sufficiently low pH to ensure better phenolic acids separation; methanol was selected as
an organic modifier. Previous studies have shown that the increase in the pH of the
mobile phase dramatically reduces the retention of phenolic acids. This is because
phenolic acids are weak acids and exist primarily in the protonated form at a pH below
their pKa, so in order to increase the retention capacity of phenolic acids, the pH of the
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eluent should be lower than their pKa (Seema et al., 2001; Michalkiewicz et al., 2008).
Therefore, following preliminary trials, a pH of 2.8 with 1% v/v formic acid (acidified
water) was chosen as the optimum pH of the elution solvent. The analysis was completed
in about 20 min, but elution of the rest of the material in the column took almost 30 min.
Using this HPLC method, four phenolic acids and one phenolic acid aldehyde were
identified in each extract and compared to the retention times and calibration curve of
external standards. The elution times of PA, PCA, VA, and SA were 6.2, 7.7, 11.5, and
13.7 min, respectively, at 280 nm. CA was eluted at 12.7 min at a wavelength of 329 nm.
In all HPLC chromatograms of Chaga extract, under all conditions, there was a peak
eluting around 16.5 min at a wavelength of 329 nm, which was too early to be ferulic
acid. Also, the UV spectra for this compound did not match the UV spectra of the ferulic
acid standard. Therefore, the peaks were not identified as ferulic acid. Identification of
these peaks was not pursued.
The method performance was validated by the determination of linearity,
precision, and detection limits. The calibration curves, obtained by triplicate injections,
were constructed from peak areas of the reference compounds versus their
concentrations. The calibration curves showed good linearity with regression coefficients
>0.994 (Table 2-1). The detection limit is the lowest amount of analyte in a sample that
can be detected but not necessarily quantified. The LOD and LOQ were estimated based
on the standard deviation of y-intercepts of the regression lines (σ) and the slope (S). The
LOD for all the compounds was in the range of 0.13–0.17 µg/g (Table 2-2). The LOQ is
defined as the lowest concentration that can be determined with acceptable accuracy and
precision. The LOQ for all the compounds was in the range of 0.39–0.52 µg/g (Table 2-
2). In this study, the limit of linearity in analytical ranges was studied, and the results
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agreed with the required criteria (Irakli et al., 2012). The within- and between-day
precisions of the analytical method were determined in terms of percent relative standard
deviation (%RSD). The %RSD values for evaluated concentrations (1, 10, and 25) were
0.5–3.0% and 0.8–4.2% for the within- and between-day precisions, respectively.
Recoveries of the experiment were performed in order to study the accuracy of the
method. There were three different concentrations of standards (1, 10, and 25 µg/g) added
to the sample extract (3N NaOH with AA and EDTA protection) for determination of
percentage recovery of the five phenolics in Chaga extract. Recoveries ranged from
90.1% to 103.3% for within-day analysis and from 88.5% to 103.5% for between-day
analysis.
2.5 CONCLUSION
A simple, sensitive, precise, and accurate HPLC method for the simultaneous
quantification of five phenolic compounds from Chaga was developed and validated.
Different hydrolysis conditions, with and without AA and EDTA protection, were
explored in order to liberate phenolic acid from chaga. The majority of phenolic acids
were extracted from the base hydrolysis (3N NaOH with AA and EDTA). This work
shows that both base and acid hydrolysis procedures have a substantial effect on the
amount of phenolic acids present in Chaga. For the first time, addition of AA and EDTA
has shown a protective effect on degradation of phenolic acids in Chaga during the
hydrolysis conditions.
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CHAPTER 3 OPTIMIZATION OF ACCELERATED SOLVENT EXTRACTION
OF PHENOLIC COMPOUNDS FROM CHAGA USING RESPONSE SURFACE
METHODOLOGY
3.1 INTRODUCTION
An effective extraction method is required to isolate phenolic compounds from the
cellular matrix of various food substrates. The efficiency of the extraction process is
determined by the composition of the natural source of phenolic compounds, the
chemical structure of the compounds themselves, and the extraction condition and
methodology that is utilized. In general, due to chemical diversity and uneven distribution
of the individual phenolic compounds within the cell matrices, no universal approach is
ideal for efficient extraction of all kinds of phenolic compounds. Moreover, the presence
of other molecules in the cellular matrix such as sugars, pigments, and terpenes may
complicate the extraction process of the compounds of interest due to interfering effects
(Dai & Mumper, 2010; Luthria & Mukhopadhyay, 2006). Several extraction parameters
such as solvent polarity, time and temperature, pH, solid-liquid proportion and particle
size, also contribute to the efficiency of the extraction process (Dai & Mumper, 2010;
Khoddami et al., 2013). Hence, it is of critical importance to optimize the extraction
conditions and select an appropriate extraction technique for each phenolic source in
order to achieve good extraction efficiency.
Response surface methodology (RSM) has been at the forefront of recent extraction
optimization research. It has been used to develop, improve and optimize process
parameters among various food, biology, and chemistry applications (Ferreira et al.,
2007). RSM is a combined mathematical and statistical tool that facilitates a multifaceted
evaluation of the impact of several parameters and their interactions, relative to the
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desired response. This technology simultaneously optimizes experimental conditions
while reducing the required number of experimental trials (Gunst et al., 1996). This
methodology is widely used to overcome the limitations of a single factor approach, in
which only one variable can be analyzed with respect to all other factors remaining
constant. Traditional single factor methods are time-consuming, labor-intensive, and
often lack data on factor interaction outcomes. Thus, establishing true optimum
conditions may be inadvertently jeopardized (Liyana-Pathirana & Shahidi, 2005).
Conventional and modern extraction techniques have been reported in phenolic
compound recovery among several different natural sources, including Chaga (Dai &
Mumper, 2010; (Khoddami et al., 2013). Typically, conventional methods such as
maceration, soxhlet, and reflux are used by employing large volumes of organic solvents
such as methanol, ethanol, acetone, ethyl acetate, or their mixture with water, either at
room temperature, or at the boiling point of the solvent (Espinoet al., 2018). However,
these methods have many demonstrated drawbacks such as low selectivity, labor intensity
and prolonged extraction time. This ultimately results in high-energy consumption in
addition to targeted compound degradation, due to both internal and external factors such
as light, air, high temperatures and enzymatic reactions (Palma et al., 2001). Moreover,
environmental constraints associated with excessive organic solvent usage during the
extraction process evoke serious concern (Khoddami et al., 2013; Liazid et al., 2007). To
overcome these difficulties, “green” extraction techniques have been introduced with
special emphasis on environmental and economic aspects. Green extraction technologies
have been reviewed in several comprehensive studies. Briefly, the main goal of these
green strategies is to design extraction processes that reduces energy consumption and
allow the use of alternative solvents or reduces organic solvents consumption while
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producing safe and high-quality extract (Chemat et al., 2012). Some of these technologies
such as supercritical fluid extraction (SFE), microwave-assisted extraction (MAE),
ultrasound extraction (UAE) and accelerated solvent extraction (ASE) have been applied
to phenolic compound extraction method development from different plant materials
(Andrade et al., 2012; Şahin & Şamli, 2013; Švarc-Gajić et al., 2013;Tripodo et al.,
2018).
Among these extraction methods, accelerated solvent extraction (ASE) is a good
alternative to conventional procedures. Compared with traditional approaches, there is a
substantial decrease in the solvent consumption and extraction time required for ASE.
This is because ASE can be automated in an inert atmosphere, which results in minimal
phytochemical degradation due to long extraction time. Further, ASE allows better
control over several extraction conditions such as temperature, static extraction time, and
the number of extraction cycles, further enhancing method efficiency (Ameer et al., 2017;
Mustafa & Turner, 2011).
ASE relies on the use of elevated pressure (500-2000 psi) and temperature (40-
200 °C) for a relatively short time to accelerate the rate of extraction. Pressure allows the
extraction cell to be filled faster, helps to force liquid into the sample pores and maintains
the liquid state of extraction solvents, even at high temperatures. This elevated
temperature results in an increase in the solvation ability of the solvent due to a decrease
in both its’ viscosity and surface tension. The result is an accelerated diffusion rate and
mass transfer of the analyte into the solvent; thereby improving the recovery of
compounds of interest (Ameer et al., 2017).
Although there are several reports that assess the different biological activities
of inonotus obliquus and conventional methods used for phenolic content recovery, very
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little attention has been given to green technology impacts on Chaga target compound
extraction efficiency. In addition, a thorough investigation of several experimental factors
that can significantly affect the efficiency of the extraction and the quality of the phenolic
compounds is lacking. Therefore, this study aims to (i) apply the response surface
methodology (RSM) approach to ASE extraction parameters (extraction temperature and
ethanol concentration) in order to optimize maximum total phenolic and antioxidant
activity of inonotus obliquus. This study also aims to (ii) evaluate the efficacy of
accelerated solvent extraction (ASE) as an environmentally friendly (green) technique for
phenolic compounds extraction in Chaga. The differences among the total phenolic
content and the antioxidant activity of the extracts are discussed and compared with those
obtained by conventional extraction methods. Finally, High-Performance Liquid
Chromatography HPLC analysis was developed (see Chapter 2) to assess the effect of
ASE extraction temperatures on individual phenolic compounds
3.2 MATERIALS AND METHODS
3.2.1 Fungal Material
Chaga sclerotia were collected from a yellow birch (Betula alleghaniensis) from
forest in Maine, USA. Samples were freeze dried (Model 7754511, Labconco
Corporation, Kansas City, Missouri, USA), then ground using an electrical grinder
(Nutribullet, model-NBR-1201M, Los Angels, USA). The ground powder was then
passed through a 20-mesh (0.84 mm) sieve and only particles with a diameter smaller
than 0.84 mm (20-mesh) were collected and pooled. All material was stored in a -20 °C
freezer until subsequent extraction preparation.
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3.2.2 Chemicals
Folin–Ciocalteu (FC) reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 3,4-
dihydroxybenzoic acid (PA), caffeic acid (CA), syringic acid (CA), and 3,4-
dihydroxybenzaldehyde (PCA) were purchased from Sigma–Aldrich (St. Louis, MO,
USA). Methanol, absolute ethanol, acetone, and ethyl acetate, sodium carbonate, vanillic
acid (VA), diatomaceous earth, and Ottawa sand were provided by Fisher Chemicals
(Fair Lawn, NJ, USA). Ultrapure water was obtained from a Millipore water system (EMD
Millipore, Billerica, MA, USA). All reagents and solvents were HPLC or analytical grade.
3.2.3. Extraction of Phenolic Compounds
3.2.3.1. Green extraction, Accelerated solvent extraction (ASE)
Accelerated solvent extraction of chaga derived phenolic compounds was
executed using a Dionex ASE 200 (Dionex Corp., Sunnyvale, CA) system. The dried
samples (1 g) were mixed with diatomaceous earth (0.25 g), and the mixture was placed
into a stainless-steel cell (11 mL). The cells were equipped with a stainless-steel frit and a
cellulose filter at the bottom to avoid accumulation of suspended particles in the
collection vial. The extraction cells were loaded in the cell tray and were extracted using
single-factor and RSM guided-experimental design conditions.
More specifically, for all sample extractions, the cell containing the ground sclerotia
sample was preheated for 2 minutes, filled with extraction solvent (methanol, ethanol,
acetone, and ethyl acetate) or a varied concentration (40-100% v/v ethanol: water),
depending on the desired solvent, up 1500 psi of pressure. Samples were then heated for
a specific time depending at the desired extraction temperature. The ASE device
automatically controlled the heating duration. The automatic settings for this equipment
requires a 5 min heating period with extraction temperature set to 60, 80, 100 °C, 6 min
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at 120 °C, 7 min at 140 °C, 8 min at 160 °C, and 9 min at 180 and 200 °C, respectively.
This step was followed by a specific static period (1-20 min). These operations were
repeated during (1-5 cycles). At the conclusion of the final static period, the cell was
rinsed with fresh extraction solvent (100% of the extraction cell volume) and purged with
nitrogen for 90 sec. Once the extraction was completed, the suspension obtained was
centrifuged (10 min, 2000 x g) and the solvent was removed using a rotary evaporator
(Rotavapor R3000, Buchi, Switzerland). The resulting residue was then dissolved in 10
mL of 75% methanol and filtered through a 0.45µm membrane filter for further analysis.
3.2.3.1.1. Experimental Design and Statistical Analyses
This analysis was performed in two stages. A set of preliminary single-factor
testing was first developed to investigate the effect of extraction variables (such as
solvent type, solvent concentration, extraction temperature, number of cycles and static
time) in order to assess the effects of these conditions on the total phenolic content
(TPC). The experimental results were analyzed using SPSS statistical software (Version
25.0). All data were expressed as means ± standard deviations, in triplicate
measurements. One-way analysis of variance (ANOVA) with Tukey’s test was used to
determine significant differences (P < 0.05). Based on the results from the single-factor
experiments, major variables influencing the extraction process were selected and
optimal ranges for each were determined.
In the second stage, a response surface methodology was then employed to
optimize phenolic compound extraction based on the dependent variables (Y1, total
phenolic content TPC (mg GAL/ g DW) and Y2, DPPH), respectively. A Face Central
Composite Design (FCD) was also used to optimize Chaga phenolic compound
extraction. The two independent variables chosen for this study were extraction
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temperature (X1, °C) and ethanol concentration (X2, %). Both factors had three equally
spaced levels in the design, coded as follows: -1, 0, +1, corresponding to the low, middle
and high level of Xi, respectively. The variables were coded according to the equation:
𝑥!=
𝑋!−𝑋!
𝛥𝑋 (1)
Where xi the (dimensionless) coded value of the variable is Xi, X0 represents X at the
center point, and ΔX denotes the step change. This design required thirteen experiments,
which were performed in a random order to avoid systematic errors. Experimental data
from FCD were fitted to the following second-order polynomial model and regression
coefficients were obtained.
Y= β!+β!X!+
!
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β!!X!+ β!" X!X! (2)
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Where Y is the predicted extraction yield of TPC; B0 is a constant; βi, βii, and βij are the
coefficients of the linear, quadratic and interactive terms, of the model. Xi and Xj are the
code values of extraction temperature and ethanol concentration. In order to determine
optimum conditions and assess the relationship between the responses and experimental
levels of each factor, the regression coefficients were used to generate 3D surface plots
from the fitted polynomial equation. The Design Expert (Version 7.0.12, Stat-Ease Inc.,
Minneapolis) statistical software was used to design the FCD and to analyze the
experimental data in RSM. The goodness of fit of the model was evaluated by the
coefficient of determination (R2) and the lack of fit obtained from the analysis of variance
(ANOVA). Coefficients with a p-value lower than 0.05 were defined significant. To
verify the adequacy of the model, additional experiments were performed at optimal
conditions predicted with the RSM, and the experimental data were then compared to
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values predicted by the model. ASE method at optimized condition was compared with
ME; SE and RE based on the TPC, DPPH scavenging activity, and HPLC results.
3.2.3.2. Conventional Solvent Extraction (CSE)
3.2.3.2.1. Maceration extraction (ME)
Chaga powder (1g) was macerated with 25 mL of aqueous ethanol (70%) for 48 h at
room temperature. After filtration through Whatman no. 1 filter paper, the solvent was
removed using a rotary evaporator (Rotavapor R3000, Buchi, Switzerland). The resulting
residue was then dissolved and filtered in accordance with the procedure defined in
section 3.2.3.1. Extraction was carried out in triplicate.
3.2.3.2.2. Reflux extraction (RE)
Chaga powder (1 g) was mixed with 25 mL of aqueous ethanol (70%) in a round-
bottom flask. The extraction mixture was then refluxed in a water bath at 70°C for 3 h.
The resulting residue was dissolved and filtered in accordance with the procedure defined
in section 3.2.3.1. Extraction was carried out in triplicate.
3.2.3.2.3. Soxhlet Extraction (SE)
One gram of Chaga powder was continuously extracted with 500 mL of aqueous
ethanol (70%) for 48 h at 70 °C in a Soxhlet apparatus. The resulting residue was
dissolved and filtered in accordance with the procedure defined in section 2.3.1.
Extraction was carried out in triplicate.
3.2.4. Determination of Total Phenolic Content (TPC)
The total phenolic content (TPC) of each extract was determined by the Folin–
Ciocalteu method described by (Jaramillo-Flores et al., 2003). Briefly, 20 µL of
supernatant was mixed with 90 µL of a 10-fold diluted Folin–Ciocalteu reagent in a 96-
!
!
!
67!
well microplate. After standing for 5 min at room temperature, 90 µL of 6% sodium
carbonate (Na2CO3) solution was added and the mixture was incubated at room
temperature for 90 min. The absorbance of the reaction mixtures was measured at 750 nm
in a spectrophotometric microplate reader (Bio-Tek ELx808, Vermont, USA). The
absorbance of the extract was compared with a gallic acid standard curve for estimating
the concentration of TPC in the sample. The TPC was expressed as milligrams of gallic
acid equivalent per gram of dry weight Chaga (mg GAL/g DW).
3.2.5 Determination of Antioxidant Activity (DPPH)
The antioxidant activity of the extract was measured with the DPPH method
according to the procedure reported by (Brand-Williams et al., 1995) with some
modifications. Briefly, 150 µL of DPPH• solution prepared in methanol (0.2 mM) was
mixed with 150 µL of chaga extract and the mixture was incubated at 37 °C. The tests
were performed on a micro-plate reader (Bio-Tek ELx808, Vermont, USA). Absorbance
readings of the mixture were taken at 517 nm over a period of 20 min. The percentage
inhibition of radicals was calculated using the following formula:
%𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 =(𝐴!"#$%"&! 𝐴!"#$%& )
𝐴!"#$%"&
×100
where A control is the absorbance of DPPH solution without extract; and A sample is the
absorbance of the sample with DPPH solution. The half-maximal inhibitory
concentration (IC50) was reported as the amount of antioxidant required to decrease the
initial DPPH concentration by 50%. At minimum, all tests were performed in triplicate,
and graphs were plotted using the average of three determinations.
!
!
!
68!
Table 3-1 Experimental design with the observed responses for the recovery of the TPC
from Chaga sclerotia samples using ASE. The codes (-1, 0, 1) and values used for X1
(130, 150, 170) and X2 (50,70, 90)
Run
Independent variables
Responses
Extraction temperature
(°C)
Ethanol
concentration (%)
TPC (mg GAL/ g DW)
DPPH%
1
0 (150)
0 (70)
29
69
2
1 (170)
1 (90)
30
70
3
0 (150)
0 (70)
28
69
4
0 (150)
-1 (50)
19
64
5
0 (150)
1 (90)
20
61
6
-1 (130)
-1 (50)
7
55
7
0 (150)
0 (70)
27
68
8
1 (170)
0 (70)
39
77
9
0 (150)
0 (70)
28
70
10
-1 (130)
1 (90)
13
51
11
1 (170)
-1 (50)
30
70
12
0 (150)
0 (70)
29
68
13
-1 (130)
0 (70)
18
59
3.2.6. HPLC Analysis of the Extracts
The chromatographic separation of the examined analytes was carried out on a
reversed-phase Hypersil GOLD aQ C18 column (150 mm × 4.6 mm i.d., 3 µm particle
size) (thermo scientific, USA) preceded by a Hypersil GOLD guard column (thermo
4 × 3.0 mm). The column and guard column were held at 30°C, and the flow rate was set
at 1 ml/min. The mobile phase of the HPLC system consisted of (A) methanol and (B) a
0.1% V/V formic acid/ultrapure-water, pH = 2.8. The gradient was linear at a flow rate of
1 mL/min The solvent gradient in volume ratios was as follows: 95% to 50% B for
16 min; the solvent gradient was reduced to 95% B at 18 min, and it was maintained at
!
!
!
69!
95% for 2 min; the latter was followed by washing with methanol and re-equilibration of
the column for 10 min. Total acquisition time was 30 min. The wavelength used for the
quantification of the phenolic acids with the diode array detector (DAD) was 280 nm for
all the phenolic acids except for caffeic acid, which was monitored at 329 nm.
Identification of the phenolic acids was achieved by comparing retention times and UV
spectra of the unknowns with the standards.
3.3 RESULTS
3.3.1 Single- Factor Experiment
The results in Figure 3-1 represent the effect of different extraction parameters on
the total content of phenolic compounds of Chaga extract. The TPC increased as the
polarity of the extracting solvents also increased. Thus, the highest phenolic content was
obtained in methanol (2.24 ± 0.04 mg GAL/g DW), followed by ethanol (1.05 ± 0.05 mg
GAL/g DW), acetone (0.76 ± 0.01 mg GAL/g DW), with the least recovery in ethyl
acetate (0.44 ± 0.01 mg GAL/g DW) (Figure 3-1A).
The effect of various concentrations of ethanol ranging from 40% to 100% (v/v)
as an extraction solvent on the recovery of Chaga phenolic compounds was investigated.
Data showed that TPC yield increased with increasing ethanol concentration, reaching a
maximum (7.4 7.25 ± 0.14 mg GAL/g DW) at 70%. Beyond 70%, the amount of isolated
TPC gradually decreased (Figure 3-1B).
In this study, the impact of temperature (ranging between 40 °C to 200 °C) on the
TPC extraction level was investigated using 70% ethanol. The results showed that the
extraction temperature significantly affected the total phenol value of the extracts. TPC
!
!
!
70!
increased as the extraction temperature increased, reaching a maximum of (40 ± 0.1 mg
GAL/ g DW) at 200 °C (Figure 3-1C).
The static cycle of ASE was determined by performing consecutive accelerated
solvent extractions on the same samples (1-5) times at 130 °C for 1 min. The TPC value
of the extract was found to increase significantly after two cycles, reaching a value of
(16.53 ± 0.06 GAL/ g DW). Beyond two cycle repetitions, there was no significant
increase in the TPC recovery (Figure 3- 1D)
The impact of the extraction duration of chaga phenolics was analyzed at six
static periods (1, 5, 7, 10, 15, and 20 min) with 70% ethanol and an extraction
temperature of 130 °C with 2 extraction cycles. The results showed that the extraction
yields of the phenolic compounds significantly increased (16.53 ± 0.06 to 18.52 ± 0.42
mg GAL/g DW) as the time of extraction increased from 1 to 7 min. Maximum yield was
obtained at 7 min. Further, an increase to 20 min resulted in a slight decrease in the
extraction yield of phenolic!compounds!(Figure!3-!1E).!
!
!
!
71!
!
!
Figure 3-1 Single factor experiment for the total phenolic yield from Chaga with (A)
accelerated solvent extraction with respect to solvent type, (B) solvent concentration, (C)
extraction temperature, (D) number of cycles and (E) static time
!
!
!
!
72!
3.3.2 Optimization of ASE by RSM
The results of the second-order response surface model fitting in the form of
ANOVA are listed in Table 2. It can be seen that the two models were statistically
significant and good predictors of the influence of the independent variables on their
corresponding responses, as evidenced from the high values of F-test (270.57 and 245.1
for Y1 and Y2, respectively) and from the low p-values (p < 0.0001) for both (Y1 and Y2)
(Yang et al., 2009).
The values of (R2) were 0.9949 and 0.9943 for Y1 and Y2, respectively, which
indicates that more than 0.99 of the variability of the responses were accounted for. Also,
the values of R2 and the R2adj (0.9912 and 0.9903 for Y1 and Y2, respectively) imply high
degree of correlation between the observed and the predicted data from the regression
models. ANOVA results also indicated non-significant lack of fit for both response
surface models at a 95% confidence level (0.5522 and 0.8050 for Y1 and Y2,
respectively), confirming the suitability of the models to accurately predict the variation.
Additionally, the relatively low values of coefficients of variation (CV) 3.29 and 1.08 %
for Y1 and Y2 respectively, suggest a high degree of precision and good reliability of the
experimental values (Table 3-2).
!
!
!
73!
Table 3-2 Analysis of variance (ANOVA) for the effects of extraction temperature and
ethanol concentration on TPC of Chaga.
Parameter
TPC
DPPH
Estimated
coefficient
std error
F-value
p-value
Estimated
coefficient
std error
F-value
p-value
Model
270.57
< 0.0001
245.1
< 0.0001
Intercept
-174.6221
0.3327
-94.62787
0.2934
Linear
X1
0.4734
0.3271
965.94
< 0.0001
0.9307
0.2884
902.82
< 0.0001
X2
3.632
0.3271
53.25
< 0.0091
1.8054
0.2884
16.63
0.0049
Quadratic
X21
0.0009
0.4821
0.6765
0.4379
0.0022
0.4251
4.45
0.0729
X22
-0.0215
0.4821
318.42
< 0.0001
-0.0159
0.4251
226.38
< 0.0001
Interaction
X1X2
-0.0037
0.4006
14.02
0.0072
0.0025
0.3533
8.01
0.0254
Lack of fit
0.8068 0.5522
0.3306 0.805
R2
0.9949
0.9943
R2 Adj
0.9912
0.9903
CV %
3.29
1.08
TPC: total phenolic content; X1: Extraction temperature (°C); X2: Ethanol concentration.
!
!
!
74!
The effect of extraction temperature and solvent concentration on TPC was
significant in first-order linear effects (X1) at P < 0.001 and (X2) at P < 0.01, second-
order quadratic effect (X22) at P < 0.001, and an interactive effect (X1X2) at p < 0.01. The
predicted model obtained for Y1 is given below:
𝑌
!=−174.6221 +0.4734𝑋!+3.6320𝑋!−0.0037𝑋!𝑋!+0.0009𝑋!
!−0.0215𝑋!
!
The results also show that the effect of extraction temperature and solvent
concentration on DPPH was significant in first-order linear effects (X1) at P < 0.001 and
(X2) at P < 0.01, second-order quadratic effect (X22) at P < 0.001, and an interactive
effect (X1X2) at p < 0.01. The predicted model obtained for Y2 is listed below:
𝑌
!=−94.6278 +0.9307𝑋!+1.8054 𝑋!+0.0025𝑋!𝑋!−0.00221𝑋!
!−0.0159 𝑋!
!
The effects of the independent variables and their mutual interactions on the TPC
and DPPH values of Chaga extract can be visualized on the three-dimensional response
surface plots and two dimensions contour plots shown in figure 3-2 and figure 3-3,
respectively. The effect of extraction temperature and ethanol concentration on the
recovery of TPC is presented in (figure 3-2A and figure 3-3A). The extraction
temperature displayed a linear effect on the response TPC, while ethanol concentration
demonstrated both linear and quadratic effects. TPC increased with a simultaneous
increase in temperature (130-170 °C) and ethanol concentration up to 70%. The TPC then
remained constant or slightly decreased along with the increase of ethanol concentration
at a fixed extraction temperature.
(Figure 3-2B and figure 3-3B) presented similar linear and quadratic effect of
extraction temperature and ethanol concentration on the response DPPH; raising the
extraction temperature up to the highest level allow high recovery of TPC, while
!
!
!
75!
increasing the concentration of ethanol to a moderate level show the highest DPPH
scavenging activity at constant extraction temperature.
!
!
!
!
!
!
!
!
!
!
!
76!
!
Figure 3-2 Counter plot analysis for the total phenolic yield from inonotus obliquus with
accelerated solvent extraction with respect to total phenolic content (A); DPPH
scavenging activity (B); desirability response (C).
!
!
!
!
77!
!
Figure 3-3 Response surface analysis for the total phenolic yield from inonotus obliquus
with accelerated solvent extraction with respect to total phenolic content (A); DPPH
scavenging activity (B); desirability response (C).
!
!
!
!
78!
A comparison between the predicted value by the model and experimental values
for TPC yield and DPPH from Chaga using ASE under optimized process conditions is
presented in Table 3-3. The results indicate that there was no significant difference
between the experimental values and the predicted values at (p > 0.05).
Table 3-3 Comparison of predicted and experimental values for TPC yield and DPPH
from Chaga using ASE under optimized process conditions.
Optimum conditions
Responses
Extraction
Temperature
(° C)
ETOH
%
TPC (mg GAL/g DW)
DPPH%
Experimental
value
Predicted
value
Experimental
value
Predicted
value
170
66
39.32
38.45
76.59
76.37
!
3.3.3 Comparison of ASE and Conventional Methods
The results of comparing ASE to conventional methods of extraction in terms of total
phenolic content, antioxidant activity, extraction time and solvent consumption are
presented in Table 3-4. Data show that extract obtained using ASE at optimum conditions
had significantly higher phenolic content and antioxidant activity than!those! obtained!
via conventional methods (p < 0.05) (Table 3-4). The data also show that the time of
!
!
!
79!
extraction was reduced from 48 hours using SE to 30 minutes using ASE with solvent
consumption being reduced from 500 mL to 25 mL (Table 3-4).
Table 3-4 Comparison of TPC and antioxidant activity (using DPPH radical scavenging
assay) of Chaga sclerotia samples using ASE, RE, ME and SE extraction methods.
Results are expressed as means ± standard deviation.
Extraction
Method
TPC (mg GAL/ g
DW)
Antioxidant
(IC50 mg/mL)
Extraction
time (hour)
Solvent
Consumption
(mL)
ASE
38.5 ± 0.87a
0.18± 0.01a
0.35
25
RE
8.61 ± 0.19b
0.64± 0.05b
3
30
SE
6.59 ± 0.58c
0.78± 0.12b
48
500
ME
3.11 ± 0.2d
1.36± 0.32c
48
30
!
Same letters in the same column denote non statistically different means according to ANOVA and
Tukey’s test; TPC (total phenolic content); GAL (gallic acid equivalents); DW (dry weight); ASE
(accelerated solvent extraction); RE (reflux extraction); ME (maceration extraction); SE (soxhlet
extraction).
!
!
!
!
80!
3.3.4 High-Performance Liquid Chromatography (HPLC) Analysis of Phenolic
Acids
The effect of different extraction temperatures using ASE on the extractability of
phenolic acids is shown in Table 3-5. As the extraction temperature increased from (40-
180 ◦C), a significant increase in the extractability of phenolic acids in the extracts is
observed. For example, the total yield of phenolic acids was increased from (376.31 to
2407.87 µg/g). However, a further increase in the temperature (up to 200 °C) resulted in a
reduction in the total yield to 1843.16 µg/g. Another example, the lowest quantity of SA
(71.99± 0.74 µg/g) was observed when ASE temperature was 40 ◦C, whereas an
extraction temperature of 180 ◦C provided the highest yield (585.03 ± 0.35 µg/g). Raising
the temperature to 200 °C led to a significant reduction in the yield of SA (489.98 ± 0.08
µg/g). A similar trend was observed for all compounds except for CA, which undergoes
degradation at a lower temperature.
!
!
!
!
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!
!
!
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81!
Table 3-5 Quantity of individual phenolic acids (µg/ g DW) from Chaga extracts using
ASE at different extraction temperatures
ASE
temperature
(◦C)
PA
PCA
VA
CA
SA
Total
40
66.84± 0.83
120.79±0.74
44.36 ± 0.45
72.33 ± 0.68
71.99 ± 0.74
376.31h
60
65.75± 0.62
191.29 ± 0.96
74.09 ± 0.9
75.90 ± 0.58
73.45 ± 0.34
480.48g
80
85.92 ± 0.75
250.00 ± 0.27
75.48 ± 2.28
91.04 ± 0.54
126.00 ± 0.52
628.44f
100
97.36 ± 0.97
303.69 ± 0.65
102.44 ± 1.9
117.77 ± 0.4
181.04 ± 0.53
802.30e
120
118.58 ±1.68
470.90 ± 0.36
132.74 ± 1.21
193.69 ± 0.57
202.05 ± 1.01
1117.96d
140
153.52 ±1.03
623.53±1.95
154.19 ± 0.29
224.36 ±1.2
360.69 ± 0.28
1516.29c
160
218.99 ±1.61
798.90 ± 0.52
184.46 ± 0.1
199.87 ± 0.22
506.94 ± 1.5
1909.16b
180
256.07 ± 0.72
1097.86 ± 1.76
284.66 ± 1.17
184.25 ± 1.21
585.03 ± 0.35
2407.87a
200
189.61 ± 0.99
907.53 ± 1.78
191.08 ± 1.01
64. 96 ± 0.83
489.98 ± 0.08
1843.16b
PA, protocatechuic acid; PCA, protocatechuic aldehyde; CA, caffeic acid; VA, vanillic; and SA,
syringic acid. Different letters in the same column denote statistically different means (p <0.05)
according to ANOVA and Tukey’s test
!
The effect of different extraction methods on the extractability of phenolic acids
from Chaga is presented in Table 3- 6. The yield of the phenolic acids obtained from
extract made with ASE at optimized conditions contained higher concentrations of
individual phenolic acids than the extracts produced from conventional extraction
!
!
!
82!
methods. For example, the content of SA varied from 541.84 ± 0.4 µg/g in ASE at
optimized conditions, to 115.66 ± 0.57 µg/g in RE, 200.55 ± 0.96 µg/g in SE and 47.43 ±
0.4 µg/g in ME, respectively. The results show that the content of SA ‘extracted by ASE
was over 3, 5, and 11 times higher than those extracted by RE, SE, and ME, respectively.
Table 3- 6 Quantity of individual phenolic acids (µg/ g DW) from Chaga extracts using
ASE optimized conditions and conventional extraction methods
Extraction
method
PA
PCA
VA
CA
SA
Total
ASE
Optimized
250.39 ± 0.57
1121.58 ±0.94
276.25 ± 0.39
206 ± 0.83a
541.84 ± 0.4
2396.06a
RE
83.49 ± 0.66
230.19 ± 1.53
29.68 ± 0.54
82.67 ± 0.55
115.66 ± 0.57
514.69b
SE
80.31 ± 0.65
248.56 ± 3.02
39.7 ± 0.28
81.38 ± 0.8
200.55 ± 0.96
650.50b
ME
45.29 ± 0.95
90.65 ± 1.12
Nd
23.76 ± 0.98
47.43±0.4 0.4
216.87c
PA, protocatechuic acid; PCA, protocatechuic aldehyde; CA, caffeic acid; VA, vanillic; and SA,
syringic acid. Different letters in the same column denote statistically different means (p <0.05)
according to ANOVA and Tukey’s test. nd, not detected.
!
!
!
83!
3.4 DISCUSSION
The first part of this research was to optimize the extraction of phenolic
compounds from Chaga. Many studies have established that extraction of phenolic
compounds from natural sources is governed by several parameters including extraction
conditions such as solvent type, extraction temperature, and the applied extraction
method (Dai & Mumper, 2010; Khoddami et al., 2013). Selection of extraction solvents
plays a key role in the recovery of phenolic compounds from their sources. The polarity
of solvent used as an extraction medium as well as the solubility of the phenolic
compounds in the solvent, strongly influences the quality and the quantity of the phenolic
compounds that are extracted. In this study, the effectiveness of four common solvents
(Luthria & Mukhopadhyay, 2006) in extraction efficiency of Chaga sclerotia-derived
phenolic compounds was evaluated. Methanol, ethanol, acetone, and ethyl acetate were
used in this analysis, and the degree of effectiveness for each was determined by the total
phenolic content (TPC) measured by Folin–Ciocalteu assay. As shown in Fig.3-1A, the
highest yield of TPC obtained in methanol which could be attributed to better solvation of
the phenolic compounds present in Chaga due to the high polarity of methanol. Although
ethanol has similar polarity to methanol, the solvation efficiency of phenolic extracts was
less. This could be due to the presence of the ethyl group in ethanol, which is longer than
the methyl group in methanol. The elongated ethyl group may result in a lower solvation
degree due to the larger hydrophobic characteristics of this structure (Boeing et al., 2014).
Generally, methanol is more efficient for the extraction of low molecular weight
polyphenols, as aqueous acetone presents a better yield for high molecular weight
polyphenols. However, ethanol is a preferable solvent with respect to safety and
!
!
!
84!
environmental considerations (Dai & Mumper, 2010). For these reasons, ethanol was
selected as the extracting solvent for subsequent experiments.
The results in Figure 3-1B show TPC yield increased with increasing ethanol
concentration to a certain level and then gradually decreased. The results from this
experiment agree with previous reports, suggesting that the addition of water to organic
solvents help to improve the relative polarity of the solvent. The addition of water results
in swelling of the raw matrix, allowing the solvent surfaces to contact more phenolic
compounds, leading to enhanced solubility of the phenolic compounds in the solvent
(Luthria, 2012). Additionally, the high dielectric constant of water, leads to increased
polarity indices of ethanol when in a water-based solution (Spigno & De Faveri, 2009).
The structure of phenolic compounds themselves, also influences their solubility. More
specifically, the presence of several hydroxyl groups makes these compounds
hydrophilic, increasing solubility in hydroalcoholic mixtures compared to a mono-
alcoholic solvent. However, a high concentration of organic solvent may lead to the
dissolution of phenolic compounds as a result of a decline in the solvent polarity; thus,
decreasing the solubility of phenolic compounds and the extracting rate (Yang et al.,
2009). Based on these results, the concentration range 50–90% ethanol: water was
selected for the RSM trials and 70% was fixed for the next single-factor experiments.
As a general rule, a relatively high extraction temperature of extraction assists in
reducing the solvent viscosity, allowing better diffusion of the solvent into the solid
matrix. This in turn, increases the solubility of the phenolic compounds, making the
extraction process more efficient. Yet, there are upper limits to this applied temperature,
as an excessive amount of heat can decrease the extraction efficiency due to the thermal
degradation of phenolic compounds. Therefore, selecting an appropriate extraction
!
!
!
85!
temperature is a necessary step in procedure optimization for phenolic compounds
isolation. As it can be discerned from Figure 3-1C, raising the extraction temperature to
the highest level resulted in increase the yield of TPC, However, severe degradation of
phenolic compounds have been previously reported at 200 °C (Lindquist & Yang, 2011),
Therefore, to prevent changes in the natural phenolic profile of the extracts, 130 °C was
selected for subsequent single-factor trials and 130, 150 and 170 °C, were selected for the
RSM study.
The number of extraction cycles represents the number of times the static heating
and flushing steps are repeated. The number of extraction cycles can be manipulated to
avoid prolonged heat exposure of the samples during the extraction process. For example,
instead of conducting one extended cycle to extract specific compounds from their
matrices, the extraction could be completed in multiple shorter cycles (Sarker, 2012). The
results in Figure 3- 1D showed no significant increase in the TPC recovery after two
cycle repetitions. Therefore, a total of two extraction cycles was selected as the optimum
cycle number for phenolic extraction.
Extraction time is an additional factor which influences TPC recovery and
extraction efficiency and was therefore further explored with a static cycle (Sarker 2012).
A maximum yield in TPC was obtained at 7 min followed by reduction, which may be
due to induced thermal degradation with the longer extraction time. Therefore, 7 min was
fixed for RSM optimization (Figure3-1E).
In this study, the influence of extraction temperature (X1), and ethanol
concentration (X2) on total phenolic content (Y1) and the corresponding antioxidant
activity (Y2), was investigated. The ranges of these variables were determined as
extraction temperature (X1: 130, 150, and 170°C), and ethanol concentration (X2: 50%,
!
!
!
86!
70%, and 90%), based on the preliminary single-factor experiment determined described
above.
The determination coefficient (R2), adjusted determination coefficient (R2adj),
lack of fit, and coefficient of variation (CV) were estimated to check the models'
adequacy (Erbay & Icier, 2009). R2 measures the proportion of the total variation of the
dependent variable attributed to each independent variable. This value falls between 0
and 1. Hence, a model with a higher R2 value is a better model. However, R2 is not
always a representative indicator of model adequacy as this value either increases or
never decreases, when more independent variables are introduced in the model. This may
lead to an inflated R2 value that does not explain the relationship between the
independent variables and the response. Therefore, the R2adj is used to compensate for
the addition of more variables that do not improve the model, by considering both the
number of independent variables and the sample size. With regard to this, the R2adj will
only increase if the added variables correlate with the dependent variables (Bonate,
2006). Therefore, a good statistical model, assesses the gap between R and the R2adj,
with these values reasonably close to 1 (R2 value > 0.7 and R2adj ≥ 7) representing an
appropriate model.
The three-dimensional (D) response surface plots and two dimensions (D) contour
plots explain the effects of the independent variables and their mutual interactions on the
TPC and DPPH values of Chaga extracts. Each 3D plot represents the number of
combinations of the two-test variables. 3D response surface and 2D contour plots are the
graphical representations of the regression equation and are useful for assessing the
relationship between independent and dependent variables. Different shapes of the
contour plots indicate whether the mutual interactions between the variables are
!
!
!
87!
significant. The circular contour plot indicates negligible interactions between the
corresponding variables, while elliptical contour suggests significant interactions between
the corresponding variables (Liu et al., 2013).
The results of the response surface plots confirmed the data of single factor
experiments; raising the extraction temperature to allow linear increase of the yield of
TPC, while raising ethanol concentration resulted in quadratic effect (Figure3- 2A and
figure 3-3A). A rise in extraction temperature can disrupt the phenolic matrix bonds and
weaken the cell wall structure, thus allowing an increase in phenolic compound release
into the solvent. Higher temperatures can also improve the extractability of phenolic
compounds through reducing both solvent viscosity and surface tension while increasing
solute solubility and the diffusion coefficient, resulting in a higher extraction rate
(Richter et al., 1996). These findings are in accordance with several other studies that
reported a significant contribution of high extraction temperature towards phenolics
extraction. Luthria (2012), recovered higher yields of total phenolic acids from vegetable
waste when extractions were carried out between 100 and 160 °C, obtaining the optimum
value at 160 °C. Tripodo et al. (2018) reported goji berry phenolic compound extraction
was positively influenced by temperature. The researchers found that for all employed
solvents, TPC increased with increasing extraction temperature, reaching the optimum
value at 180 °C.
A similar quadratic effect of solvent concentration on the recovery of phenolic
compounds was observed among different sources including blackcurrant (Cacace &
Mazza, 2003) and rosemary extract (Hossain et al., 2011). This effect might be due to the
change in solvent polarity with a change in ethanol concentration. It was reported that
changes in ethanol concentration could enhance phenolic compound solubility by altering
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the physical properties of the solvent such as density, dynamic viscosity, and dielectric
constant (Cacace & Mazza, 2003). However, further increase of ethanol concentration
might lead to dissolution of phenolic compounds due to a decline in the solvent polarity
and the decrease solubility (Yang et al., 2009).
In parallel with phenolic content, extraction temperature and ethanol
concentration displayed similar linear and quadratic effects on the antioxidant activity,
reaching a peak at the highest temperature and a moderate ethanol concentration level
(Figure 3-2B and figure 3-3B). These results are consistent with the significant increase
in TPC, which was observed at the same extraction conditions. Our results agree with
prior studies, which have reported the significant contribution of extraction temperature
and ethanol concentration on the antioxidant activities from different sources in linear
and quadratic effects, respectively (Karacabey & Mazza, 2010; Liyana-Pathirana &
Shahidi, 2005).
Since both response variables are equally important, verification experiments
were performed at optimal conditions derived for maximizing the desirability of the two
responses (Figure3- 2C and figure 3-3C). Under these optimal conditions, the
experimental values were found to be not significantly different from the predicted values
at (p > 0.05), further confirming the validity and the adequacy of the predicted models
(Table 3-3).
In previous work, a linear relationship was reported between the DPPH
scavenging activity and the total phenolic content of Chaga extract (Debnath et al., 2013).
Our results show a similar trend where the antioxidant activity of the extracts tends to
increase with increasing phenolic content. A significant positive correlation of R = 0.98
(p < 0.001) between the phenolic content and the antioxidant activity, as determined by
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the DPPH scavenging assay, was observed. Therefore, it is suggested that the phenolic
compounds extracted from Chaga contribute strongly to the antioxidant capacity of the
examined extracts. However, it should also be mentioned that the extracts were crude and
may contain other components such as terpenoids and polysaccharides, which are also
known radical scavengers (Handa et al., 2010). Significant correlations between the
phenolic concentration in plants and the antioxidant activity displayed by the plants have
been previously reported in literature. Piluzza & Bullitta investigated the relationship
between phenolic content and the antioxidant capacity of several Mediterranean plant
species, and obtained a good linear correlation using DPPH and ABST assays. They also
suggested that phenolic content could be used as an indicator of the antioxidant properties
of the examined plant species (Piluzza & Bullitta, 2011). Similarly, the high correlation
coefficient between the antioxidant activity and the total phenolic content of grape cane
extracts has been reported (Karacabey & Mazza, 2010).
In this study ASE were compared to conventional extraction methods in terms of
total phenolic content, antioxidant activity, extraction time and solvent consumption.
Data showed that extract obtained using ASE at optimum conditions had significantly
higher phenolic content and antioxidant activity than those obtained via conventional
methods (p < 0.05) (Table 3-4). The higher efficiency of the ASE technique could be
explained by the combined application of high temperature and pressure leading to
increased solubility and mass transfer rate between the phenolic analytes and the solvent.
It also causes disruption in solute- matrix bonds (dipole-dipole, van der Waals, and H2-
bonding) and weakens the cell wall structure. These combined effects allow the diffusion
of targeted phenolic compounds to the outer surfaces of Chaga matrices, improving the
phenolic recovery rate (Mustafa & Turner, 2011). In addition, ASE may have prevented
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the degradation of phenolic compounds due to limited extract exposure to light and
oxygen during processing. Moreover, the short extraction times possible (about 35 min)
may have reduced the adverse effect of enzyme activity that could be produced during the
extraction process (Khoddami et al., 2013; Palma et al., 2001).
The results from the present study agree with other reports, which indicated that
ASE is an effective technique for extracting phenolics from different sources as
compared to other extraction methods (Ameer et al., 2017; Ju & Howard, 2003; Tripodo
et al., 2018). However, other studies have reported that high temperature in ASE resulted
in reductions of both phenolic content and antioxidant activity of the extract (Nayak et
al., 2015). Differences in the extraction efficiency of phenolic compounds using ASE
may be related to the vast structure diversity of extracted phenolic compounds and their
differences in reactivity stability at higher extraction temperatures. Ju & Howard reported
that the highest recovery of anthocyanins, obtained from dried red grape skin using
acidified methanol, occurred at 60 °C, whereas the highest recovery of total phenolics
from the same source occurred at 120 °C. The researchers attributed the increased yield
of total phenolics at higher temperatures to the improved extraction of more heat-stable
procyanidins and phenolic acids (Ju & Howard 2003). Similarly, higher extraction
temperatures lead to lower catechin and epicatechin recovery, whereas no decrease was
observed in the recovery of the other assayed phenolics and their acid and aldehyde
derivatives under the same conditions (Palma et al., 2001). Consequently, it is necessary
to adjust the extraction method depending on the nature of the target compounds that are
to be extracted.
Data has also shown that extracts obtained by RE and SE methods had higher
TPC and DPPH values compared to extracts obtained by ME (Table3-4). Kähkönen et al
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reported that the reflux extraction found superior to room temperature for phenolic
extraction from apple, cowberry, and bilberry (Kähkönen et al., 2003). The above results
support previous work which suggested that the absence of heat and long extraction time
in maceration (few days) contribute to the low efficiency of the extraction (Brglez et al.,
2016).
With consideration of environmental and practical aspects, our data shows
prevalence in ASE over conventional methods with respect to a reduction in both
extraction time and solvent consumption (Table 3- 4). Green extraction methods such as
ASE, provides high-quality extract with less impact to the environment as a result of
limited energy usage and solvent consumption (Chemat et al., 2012). The present study,
therefore, proposes ASE as an efficient green technology to extract phenolic compounds
from Chaga.
Qualitative and quantitative analyses of individual phenolic compounds of Chaga
extracts at ASE and conventional conditions were executed using HPLC-DAD method.
Many studies have reported different behavior of individual phenolic acids affected by
extraction temperature and the applied extraction method. However, to our knowledge,
no studies have analyzed phenolic acid profiles in Chaga. Firstly, to define the
relationship between ASE extraction temperature and the phenolic acids profile, we
evaluated extraction temperatures (40-200 °C) on the quantity of individual phenolic
acids isolated from Chaga. The results showed that raising the extraction temperature
resulted in higher phenolic acids extracted from Chaga. However, a decrease in the
quantity of all phenolic acids is observed at 200°C. The results also showed that CA was
more affected by raising temperature and less quantity of CA is obtained at 160 °C. The
earlier decline in CA level at 160 °C could be attributed to its chemical structure. CA has
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two hydroxyl groups in its aromatic ring which translates into reduced stability under
MAE conditions (Liazid et al., 2007). Other studies have also reported the stability of
phenolic acids at different elevated temperatures using ASE and MAE. These studies are
in agreement with the result in the present study in which that the extraction temperature
has a significant effect on the content of the individual phenolic compounds. Elevated
extraction temperatures generate greater yields of those compounds, however,
degradation at a certain temperature will result, depending on the chemical structures of
the assayed compounds (Luthria, 2012; Palma et al., 2001).
Secondly, the yield of the selected individual phenolic compounds obtained using
different extraction methods was compared. The results demonstrated that more phenolic
acids are extracted using ASE at optimized conditions in comparison to the extracts
produced from conventional extraction methods Table 3- 6. A similar trend was observed
in the other assayed phenolic acids. The experimental results of phenolic acids agreed
with a previous study, which presented ASE as the most efficient method in the
extraction of chlorogenic acid from black eggplant compared to other conventional
techniques (Luthria & Mukhopadhyay, 2006). The greater yield of individual phenolic
acids obtained under ASE at optimized conditions may be linked to the presence of
phenolic compounds (e.g. phenolic acids are rarely found in free form and are primarily
conjugated to other components in the matrix (Luthria & Pastor-Corrales, 2006).
Therefore, a pretreatment may be required to release them from their strong conjugate.
The previous report showed that steam treatment of Chaga samples before extraction
facilitated the release of more phenolic acids from Chaga matrices due to the disruption
of the cell wall, as a result of thermal processing (Ju et al., 2010). Considering that the
most important effect of ASE is related to the use of higher extraction temperatures, it
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seems that the phenolic acids in Chaga are strongly bonded to the matrix and therefore
higher temperatures are required to break these bonds and release the phenolic analytes.
Based on the results obtained from this study, ASE is an efficient method to extract
phenolic compounds from Chaga.
3.5 CONCLUSION
Isolation of bioactive compounds is only possible through determining an
efficient extraction method. There has been growing interest in establishing
environmentally conscious and green extraction methods, while also evaluating the
economic feasibility of these procedures. In the present study, an ASE based green
extraction method was developed for the first time to obtain phenolic compounds from
Chaga. An experimental design was applied to establish optimum extraction conditions
yielding both maximum total phenolic content as well as antioxidant activity within the
extract. The optimal ASE conditions were achieved at 170°C and 66% ethanol in water as
the solvent and it was found that ASE not only provided higher recovery of TPC, but also
high quality phenolic compounds, with high levels of antioxidant capacity. The results of
this study contribute to enhancement and utilization of ASE Chaga extract in food,
pharmaceutical, and cosmetic industries while providing an environmentally method of
isolating these healthful compounds.
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CHAPTER 4 THE ANTI-INFLAMMATORY PROPERTIES OF CHAGA
EXTRACTS OBTAINED BY DIFFERENT EXTRACTION METHOD AGAINST
LPS INDUCED RAW 264.7
4.1 INTRODUCTION
Inflammation is a physiological immune response of the body to injury,
characterized by fever, swelling, and pain. Inflammation is usually implicated in the
pathogenesis of a variety of diseases including, asthma, heart disease, cancer, and
diabetes. (Taofiq et al., 2016). During the inflammatory process, large amounts of pro-
inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), tumor
necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) are generated,
mostly for the primary protection of the host (Garlanda et al., 2007; Jeong & Jeong,
2010). However, excess uncontrolled production of these inflammatory products can lead
to oxidative stress (Dai et al., 2019; Garlanda et al.,2007). Recently, there has been
considerable interest in finding anti-inflammatory agents from natural sources without or
with low toxic effect (Taofiq et al., 2016). Several compounds have shown anti-
inflammatory activity; among them, phenolic compounds have attracted great attention
due to their wide variety of biological activities.
Previous studies have indicated that Chaga contains various bioactive components
with different chemical characteristics and polarities (Duru et al., 2019; Shashkina et al.,
2006). Accordingly, extraction of bioactive compounds from Chaga using various
solvents leads to separate extracts with different profiles. For instance, petroleum ether
and chloroform were used to extract lanostane-type triterpenoids from Chaga; whereas
water and aqueous alcohol were suitable solvents to separate polysaccharides, melanin
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pigments, and phenolic compounds from Chaga (Mazurkiewicz, 2006; Zhang, et al.,
2011). Aqueous preparations of Chaga have been used since the 12th century in Eastern
Europe to treat a variety of ailments without toxic effect. The traditional way to prepare
Chaga is that pieces of Chaga are either macerated or boiled in water for a few hours or a
few days. The resultant extracts from these processes are readily consumed or saved at a
suitable temperature until consumed (Géry et al., 2018). Currently, quick brewing is a
more common way to consume Chaga as Chaga tea; Chaga in powder form or in tea bags
are steeped in hot water for a short period of time, strained, and then consumed as a tea.
Investigations into the chemical composition and biological properties of aqueous
extracts from Chaga have revealed that aqueous extract of Chaga had therapeutic effects
against diabetes via multiple pathways—including antioxidative effects (Diao et al.,
2014). Research has found that orally administered aqueous extract of Chaga could
ameliorate acute inflammation (Mishra et al., 2012). However, no investigation has been
carried out to examine chemical composition of Chaga tea in powder and tea bag form.
Alcoholic extracts from Chaga are characterized by high phenolic content. It has
been reported that phenolics are the main chemical compounds involved in Chaga
biological effects—including antioxidant, anti-cancerous, antimicrobial, and anti-
inflammatory activities (Glamočlija et al., 2015; Nakajima et al., 2007; Nakajima et al.,
2009a; Park et al., 2004; Park et al., 2005;Van et al., 2009). Different conventional and
advanced extraction methods have been used to separate phenolic compounds from
Chaga (Hwang et al., 2019; Seo & Lee, 2010). Data from the second chapter showed that
accelerated solvent extraction (ASE) increased the total phenolic content and enhanced
the DPPH scavenging activity of Chaga extracts—compared to conventional extraction
methods. In addition, the concentrations of various individual phenolic acids significantly
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increased in relation to their concentrations in other extracts. Previous studies have
demonstrated that alcoholic extracts from Chaga possess significant anti-inflammation
effects in vivo and in vitro. However, no studies have investigated the effect of the
extraction method on the anti-inflammatory properties of Chaga extracts.
This study investigated the effect of Chaga extracts obtained by different
extraction methods on anti-inflammation in LPS-stimulated 264.7 cells. Our work is the
first experimental approach on Chaga collected in Maine, USA. The study also aimed to
investigate the chemical features of Chaga tea steeping in bag and powder form.
4.2 MATERIALS AND METHODS
4.2.1 Fungal Material
Chaga sclerotia were collected from Maine birch forests. Samples were
lyophilized (Model 7754511, Labconco Corporation, Kansas City, Missouri, USA), then
ground using an electrical grinder (Nutribullet, model-NBR-1201M, Los Angeles, USA).
The ground powder was passed through a 20-mesh (0.84 mm) sieve and only particles
with a diameter smaller than 0.84 mm (20-mesh) were collected. All particles were stored
in a -20 °C freezer until subsequent extraction preparation.
4.2.2 Reagents
Folin–Ciocalteu (FC) reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 3,4-
dihydroxybenzoic acid, caffeic acid, syringic acid, 3,4-dihydroxybenzaldehyde, bovine
serum albumin (BSA), galacturonic acid, Griess reagent, 3-(4,5- Dimethylthiazol-2-yl)-
2,5-diphenyl-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Escherichia coli
LPS, and were purchased from Sigma–Aldrich (St. Louis, MO, USA). Ethanol, sodium
carbonate, vanillic acid, diatomaceous earth, and Ottawa sand were purchased from
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Fisher Scientific (Fair Lawn, NJ, USA). Ultrapure water was obtained from a Millipore
water system (EMD Millipore, Billerica, MA, USA). The murine macrophage (RAW 264.7)
cell line, Dulbecco’s modified media (DMEM), heat inactivated fetal bovine serum
(FBS), and penicillin-streptomycin were obtained from Gibco Life Technologies. For the
enzyme-linked immunosorbent assay (ELISA) the TNF-α, IL-6, and IL-1β ELISA kits
were obtained from e-Bioscience, Inc. (Cincinnati, OH, USA). Cytokine ELISA kits were
obtained from R&D Systems (Minneapolis, MN, USA). All reagents and solvents were
HPLC or analytical grade.
4.2.3 Preparation of Polysaccharide Extracts
Chaga sample (1.5g) in the bagged form (B) or the powder form (P) was infused in 200
mL of boiled distilled water at 100 °C for (3, 6, and10 min). The infusions were filtered
through sterilized gauze. Four volumes of cold 95% ethanol were added to the aqueous
extract after concentrating to 30% of the original volume with rotary evaporator under
reduced pressure at 60°C. The extracts were kept at 4°C overnight to isolate the crude
polysaccharides. The precipitate was recovered by centrifugation at (20 min, 2000 x g)
(Rotavapor R3000, Buchi, Switzerland), washed with absolute acetone to remove
adherent sugar residue and other small molecules and dialyzed for two days with distilled
water (cut-off Mw 8000 Da). The retained portion was concentrated; deproteinated with
Sevag reagent (CHCl3: BuOH = 4:1, v/v) for 30 min under the magnetic force stirring
and the procedure was repeated two times. Finally, the extracts were centrifuged to
remove insoluble material and the supernatant was lyophilized in the freeze–dry
apparatus for 48 h to give the crude polysaccharide extracts from the powder form P3,
P6, and P10 and from the bagged form B3, B6, and B10, depending on the brewing time.
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4.2.4 Preparation of Phenolic Extracts
4.2.4.1 Green extraction, Accelerated solvent extraction ASE
ASE was performed with a Dionex (Sunnyvale, CA, USA) ASE 200 instrument with
solvent controller. According to our previously optimized method, briefly, dried ground
sample of Chaga (1 g) was placed in a stainless-steel extraction cell, preheated for 2 min,
and extracted with 70% aqueous ethanol or 66% aqueous ethanol. The extractions were
performed at three temperature ranges (130 °C, 150 °C, and 170 °C) for 30 min (two
cycles for every sample) at a pressure of 1500 psi. Once the extraction was complete, the
suspension obtained was centrifuged (10 min, 2000 x g) and the solvent was removed
using a rotary evaporator (Rotavapor R3000, Buchi, Switzerland). The resulting powders
were stored at −20 °C for further experiments.
Table 4-1 extraction conditions of Chaga using accelerated solvent extraction ASE
Extraction conditions
Extract
Temperature °C
ETOH %
170
66
ASE1
150
70
ASE2
130
70
ASE3
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4.2.4.2. Conventional Solvent Extraction (CSE)
4.2.4.2.1 Maceration Extraction (ME)
Chaga powder (1 g) was macerated with 25 mL of aqueous ethanol (70%) for 48 h at
room temperature. After filtration through a Whatman no. 1 filter paper, the solvent was
removed using a rotary evaporator (Rotavapor R3000, Buchi, Switzerland). The resulting
residue was then dissolved and filtered in accordance with the procedure defined in
section 4.2.4.1. Extraction was carried out in triplicate.
4.2.4.2.2 Reflux Extraction (RE)
Chaga powder (1 g) was mixed with 25 mL of aqueous ethanol (70%) in a round-bottom
flask. The extraction mixture was then refluxed in a water bath at 70°C for 3 h. The
resulting residue was dissolved and filtered in accordance with the procedure defined in
section 2.3.1. Extraction was carried out in triplicate.
4.2.4.2.3 Soxhlet Extraction (SE)
Chaga (1 g) was continuously extracted with 500 mL of aqueous ethanol (70%) for 48 h
at 70 °C in a Soxhlet apparatus. The resulting residue was then dissolved and filtered in
accordance with the procedure defined in section 2.3.1. Extraction was carried out in
triplicate.
4.2.5 Determination of total phenolic content (TPC)
The total phenolic content (TPC) of the extracts was determined by the Folin–Ciocalteu
method described by (Jaramillo-Flores et al., 2003). Briefly, 20 µL of supernatant was
mixed with 90 µL of a 10-fold diluted Folin–Ciocalteu reagent in a 96-well microplate.
After standing for 5 min at room temperature, 90 µL of 6% sodium carbonate (Na2CO3)
solution was added and the mixture was incubated at room temperature for 90 min. The
absorbance was measured at 750nm in a spectrophotometric microplate reader (Bio-Tek
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ELx808, Vermont, USA). The absorbance of the extract was compared with a gallic acid
standard curve for estimating the concentration of TPC in the sample. The TPC was
expressed as milligrams of gallic acid equivalent per gram of dry weight Chaga (mg
GAE/g DW).
4.2.6 Determination of Antioxidant Activity (DPPH)
The antioxidant activity of the extract was measured with the 1,1-diphenyl-2-
picrylhydrazyl DPPH method according to the procedure reported by (Nakajima et al.,
2007). A solution of DPPH was freshly prepared by dissolving 11 mg DPPH in 100 mL
methanol (about 0.28 mM). The extract (150 µL) with varying concentrations (60-220
mg/mL) and DPPH solution (150 µL) was mixed in a 96-well microplate and incubated
for 30 min at room temperature. The decrease in absorbance was measured at 517 nm
using a spectrophotometric microplate reader (Bio-Tek ELx808, Vermont, USA). The
percentage inhibition of radicals was calculated using the following formula:
%𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 =(𝐴!"#$%"&! 𝐴!"#$%& )
𝐴!"#$%"&
×100
where A control is the absorbance of DPPH solution without extract; and A sample is the
absorbance of the sample with DPPH solution. The half-maximal inhibitory
concentration (IC50) was reported as the amount of antioxidant required to decrease the
initial DPPH concentration by 50%. At minimum, all tests were performed in triplicate,
and graphs were plotted using the average of three determinations.
4.2.7 Determination of Total Neutral Carbohydrate Contents
The carbohydrate content of the polysaccharide extracts was determined with a
slightly modified phenol-sulphuric acid method (Masuko et al., 2005). Briefly, 1 mL of
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sample solution, 0.05 mL of 80% phenol, and 5mL of concentrated sulphuric acid were
mixed and shaken. After the mixture was kept at room temperature for 10 min, the
absorbance was measured at 490 nm. The total carbohydrate content was calculated with
D-glucose as standard.
4.2.7.1 Determination of Uronic Acid Content
The uronic acid contents of the polysaccharide extracts were measured according
to the method of Blumenkrantz (Allen & Brock, 2000) using D-galacturonic acid as a
standard. Briefly, 0.2 mL of sample solution and 1.2 mL of sulphuric acid\ tetraborate
solution were mixed and shaken. The mixture was kept at 100 °C for 5 min, 20 mL of m-
hydroxydiphenyl reagent was added, the absorbance was measured within 5 minutes at
520 nm.
4.2.7.2 Determination of Protein Content
The total protein content of the polysaccharide extracts was measured by the
method of Bradford (Emami Bistgani et al., 2017) with bovine serum albumin as a
standard. Briefly, 10 µL of sample solution and 200 µL of Bradford reagent were mixed.
After the mixture allowed to stand at room temperature for 5 minutes, the absorbance was
read at 595 nm.
4.2.8 Cell Culture
The RAW264.7 cell line derived from murine macrophages was purchased from
the American Type Culture Collection (Rockville, MD, USA). The cells were maintained
at 37 °C in a humidified atmosphere of 5% (v/v) CO2 in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with glutamine (1 mM), 10% fetal bovine serum (FBS
FBS; ATCC; Manassas, VA, USA), penicillin (50 U/mL), and streptomycin (50 µg/m).
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Medium was changed every two days. In all experiments, cells were grown to 70-80%
confluence and subjected to no more than 20 cell passages.
4.2.8.1 Measurement of Cell Viability
Cell viability was assessed by the MTT 3-(4,5-dimethylthiazolyl-2)-2,5-
diphenyltetrazolium bromide) assay. The assay is based on the ability of mitochondria in
viable cells to reduce the yellow tetrazolium salt MTT to purple formazan crystals. The
method was performed according to the manufacturer’s procedure (Mosmann, 1983) with
some modifications. The cells were cultured in 96-well plates at a density of
1 × 104 cells/well for 24 h then the cells were treated with the samples at different
concentrations (50, 100, and 150 µg/mL (Chaga extract) or 25, 50, and 100 µM (vanillic
acid, caffeic acid, and syringic acid) or 5, 10, and 20 µM (protocatechuic acid and
protocatechuic aldehyde) for 24 h in a humidified 5% CO2 atmosphere at 37 °C. After the
incubation period, the media was removed and 100 µL of fresh medium and 10 µL of
MTT solution were added to each well, and the plate was incubated for 2h at 37 °C.
Finally, the cell culture medium was discarded, and the formazan blue formed in the cell
were resuspended in 200µL solubilization solution. The quantity of formazan (an
indicator of cell viability) is measured by recording changes in absorbance at 540 nm
using a spectrophotometric microplate reader (Bio-Tek ELx808, Vermont, USA). Of
note, extracts and standards were dissolved in 0.05% DMSO. Cells treated with 0.05%
DMSO were used as control and cells were treated with 2µM gallic acid was used as
positive control. All experiments were performed in triplicate. % Cell viability was
calculated using:
% 𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 =
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑒𝑥𝑡𝑟𝑎𝑐𝑡
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑚𝑒𝑑𝑖𝑎 × 100
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4.2.8.2 Measurement of NO production
Inhibitory effects of Chaga extracts and the pure phenolic acid standards on the
production of NO in RAW 264.7 cells were evaluated using a method modified from the
previously reported (Sun et al., 2003). RAW 264.7 cells in 10% FBS-DMEM (without
phenol red) were seeded at (1 × 105 cells/well) in 12 well plates. Cells were incubated for
24h at 37 °C. Cells were then treated with varying concentrations of samples (50, 100,
and 150 µg/mL (Chaga extract) or 25, 50, and 100 µM (vanillic acid, caffeic acid, and
syringic acid) or 5, 10, and 20 µM (protocatechuic acid and protocatechuic aldehyde) for
2h. The cells were then treated with LPS (1 µg/mL; Sigma–Aldrich,) for 24 h at 37 °C.
After 24 h, 100 𝜇L of cell culture medium was mixed with 100 𝜇L of Griess reagent,
incubated at room temperature for 15 min and the absorbance was measured at 540 nm in
an ELISA microplate reader (Bio-Tek ELx808, Vermont, USA). The values were
compared with a sodium nitrite standard curve (5-100µM).
4.2.8.3 Cytokine Measurement
To assess the anti-inflammatory effect of Chaga extracts and the pure phenolic acid
standards on the expression of TNF-α, IL-6, and IL-1β were quantified using ELISA kits
(e-Bioscience, Inc., Cincinnati, OH, USA). The assays were performed according to
instructions provided by the manufacturer.
4.2.9 Statistical Analysis
Statistical analyses were performed with SPSS v25 (SPSS, Chicago, IL, USA). All results
were expressed as the mean ± the standard error of triplicate analysis. Statistical
significance was determined using one-way analysis of variance for independent means,
followed by Tuckey’s test. Differences were considered significant at P < 0.05.
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4.3 RESULTS
4.3.1 Chemical Composition
The yield and the bioactive content of the crude extracts from the powdered form
of Chaga were significantly higher (p < 0.05) than those of the bagged form at all
extraction temperatures (Table 4- 1). For example, P6 yielded 30.21 ± 0.01% crude
polysaccharide that contains 17.56 ± 0.01% carbohydrate, 10.23 ± 1.02% protein, 4.12 ±
0.44% uronic acid, and 17.61 ± 0.05% phenolic content, while B6 gave 17.33 ± 0.03%
crude polysaccharide containing 11.56 ± 0.01% carbohydrate, 7.11 ± 0.5% protein, 2.57
± 0.71% uronic acid, and 8.33 ± 0.05% phenolic content. The results also indicated that
there was no significant effect of brewing time on the yield and the chemical content of
the crude extracts of both bagged and powdered form. For example, B3 extract resulted in
a carbohydrate content of 10.26 ± 0.08%, which was comparable to the carbohydrate
content of (11.56 ± 0.01% and 11.81 ± 0.04%) obtained from B6 and B10 extracts,
respectively. Similarly, P3, P6, and P10 extracts gave carbohydrate content of 17.02 ±
0.01%, 17.56 ± 0.01%, and 18.31 ± 0.05%, respectively. The same trends were observed
for the yield, protein, uronic acid, and total phenolic content of crude polysaccharide
extracts of Chaga tea obtained from different brewing times.
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Table 4-2 Major chemical content of the crude polysaccharide extracts from the sclerotia
of Chaga. The data represent the mean ± SD of triplicate experiments.
Sample
Yield%
Carbohydrate %
Protein%
Uronic acid%
TPC%
P3
30.66±0.05a
17.02 ± 0.01a
10.53±1.66a
4.11±0.47a
16.77±0.13ab
P6
30.21±0.01a
17.56 ± 0.01a
10.23±1.02a
4.12±0.44a
17.61±0.05a
P10
31.33±0.04a
18.31 ± 0.05a
11.02±1.28a
4.11±0.26a
17.04±0.03a
B3
15.53±0.01c
10.26 ± 0.08b
7.14± 1.83b
3.01±0.53b
6.49 ±0.04d
B6
17.33±0.03b
11.56 ± 0.01b
7.11 ± 0.5b
2.57±0.71b
8.33 ±0.05c
B10
17.33±0.02b
11.81 ± 0.04b
7.23± 2.01b
2.84±1.22b
8.33 ±0.06c
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4.3.2 Cell Viability
RAW 264.7 cells were treated with various Chaga extracts or pure phenolic
standards: (50, 100, and 150 µg/mL Chaga extracts) or (25, 50, and 100 µM vanillic acid
(VA) or caffeic acid (CA) or syringic acid SA) or (5, 10, and 20 µM protocatechuic acid
PA or protocatechuic aldehyde PCA) to assess their effects on cell viability using the
MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay. This assay
measures the activity of mitochondrial succinate-tetrazolium reductase of living cells and
their ability to cleave the tetrazolium salts to formazan crystals resulting in a color change
that can be monitored spectrophotometrically (Mosmann, 1983). The data were expressed
as percent cell viability compared to control (0.05% DMSO). The results showed that
Chaga extracts and the pure standards did not cause any cytotoxicity at the examined
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concentrations in RAW 264.7 cells (Figure 4-1). Therefore, subsequent experiments were
performed at these concentrations.
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Figure 4-1 Effects of different samples on cell viability of RAW264.7 cells. Extracts
were obtained by (A) ASE conditions, (B) pure phenolic acid standards, (C) crude
polysaccharide extracts, and (D) different extraction methods. Cells were stimulated with
1 µg/mL of LPS plus varying concentrations of samples (0 = media, GA = 2 µM; 1 = 5
µM; 2 = 10 µM; 3 = 20 µM; 4 = 50 µg/mL; 5 = 100 µg/mL; 6 = 150 µg/mL; 7= 25 µM; 8
= 50 µM; and 9 = 100 µM).
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4.3.3 Inhibition of NO Production in LPS-Stimulated RAW 264.7 Macrophages
We examined the inhibitory effects of various Chaga extracts and pure phenolic
acid standards on NO production on LPS-induced RAW 264.7 cells. Stimulation of the
cells with 1 µg/mL LPS increased the NO levels to 44.53 ± 0.23 µM compared to 4.8 ±
0.12 µM in the negative control group (Figure 4-2). All ASE extracts significantly (p <
0.05 and p < 0.01) reduced the level of NO production at all tested concentrations (Figure
4-2A). For example, at 150 µg/mL, ASE1, ASE2, and ASE3 reduced the concentration of
NO released from RAW 264.7 cells by 66.82%, 61.61%, and 43.34%, respectively,
compared with the LPS group. We further investigated the inhibitory effect of the main
phenolic acids found in Chaga extracts on the inhibition of NO production. Results
showed that treating the LPS induced cells with various concentrations of PA, PCA, and
CA significantly (p < 0.05 and p <0.01) reduced the production of NO, while treatments
with SA and VA did not exhibit any effect on the induced cells (Figure 4-2B). For
example, at the highest examined concentrations, the levels of NO released from the
induced cells decreased by 54.35%, 58.66%, and 41.60%, after treatments with PA, PCA,
and CA, respectively. However, the VA and SA treatments did alter the level of NO
productions at any concentration.
All crude polysaccharide extracts separated from Chaga tea in the powder form
showed significant inhibitory effect (p < 0.05 and p < 0.01) on NO production Figure 4-
2C. However, crude polysaccharide extracts separated from the bag form of Chaga tea
showed significant inhibitory effect (p <0.05), on NO production only at the highest
examined concentration of the extracts compared to the LPS group. For example, at 150
µg/mL, P6 and B6 extracts reduced the nitrile concentration in the supernatants by
67.76% and 37.31%, respectively, compared to the LPS group
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To investigate the effect of the conventional extraction methods on the inhibition
of NO production, RAW264.7 cells were incubated with extracts obtained by different
extraction methods in the presence of 1 µg/mL of LPS. There was no significant effect of
the ME extract on the inhibition of NO production. However, both RE and SE extracts
showed significant inhibition (p < 0.05 and p <0.01) on the production of NO, compared
to the LPS group (Figure 4-2D). For example, at 150 µg/mL, RE and SE extracts reduced
the nitrile concentration in the supernatants by 51.42% and 52.61%, respectively,
compared to the LPS group.
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Figure 4-2 Effect of various samples on production of nitric oxide (NO) in macrophage RAW 264.7
cells (A) extracts obtained by ASE (B) pure phenolic standards (C) crude polysaccharide extracts
and (D) different extraction methods. Cells were cultured in the absence or presence of LPS (1 µg
/mL) with various concentrations of different samples for 24h (0 = media; GA=2 µM; 1= 5 µM;
2=10 µM; 3= 20 µM; 4 = 50 µg/mL; 5=100 µg/mL; and 6= 150 µg/mL; 7= 25 µM; 8 = 50 µM; and
9 = 100 µM). NO production was measured by the Griess reagent and was represented as mean ±
standard error (SE) in the bars. Significant different values at P < 0.05 and p <0.01.
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4.3.4 Inhibition of TNF-α Production in LPS-Stimulated RAW 264.7 Macrophages
To evaluate the anti-inflammatory effect of Chaga, we investigated the inhibitory
effects of various Chaga extracts and pure phenolic standards on the expression of pro-
inflammatory cytokines TNF-α, 1L-6, and IL-1β, in LPS-stimulated RAW 264.7 cells.
We found an increase in the expression of TNF-α in the LPS stimulation group compared
to the control group (Figure 4-3). Twenty-four hours of incubation with ASE extracts
significantly inhibited the level of TNF-α (P < 0.05) in the LPS-induced cells, compared
with the LPS group. For example, at 150 µg /mL, ASE1, ASE2, and ASE3 extracts reduced the
level of TNF-α, released from RAW 264.7 cells by 42.90%, 35.94%, and 31.15%, respectively,
compared with the LPS group (Figure 4-3A). The effect of the pure phenolic standards on
the expression of TNF-α is presented in Figure 4-3B. All phenolic acids except VA and
SA significantly (p <0.05) decreased the secretion of TNF-α at all examined
concentrations, compared to the LPS treatment. For example, at the highest concentrations,
PA, PCA, and CA reduced the expression of TNF-α by 44.3%, 46.7%, and 32.5%, respectively,
compared to the LPS treatment. The results from the crude polysaccharide extracts showed
that extracts separated from the powder form of Chaga tea had significant inhibitory
effects (P < 0.05) on the level of TNF-α, compared with the LPS group. For example, at a
concentration of 150 µg/mL, P3 and P6 extracts reduced the level of TNF-α in the supernatants
by 37.2% and 37.5%, respectively (Figure 4- 3C).
The level of NO was inhibited by Chaga extracts depended on the extraction
method. We further examined the effect of the extraction method on the ability of the
extracts to attenuate the level of TNF-α. At a concentration of 150 µg/mL, extracts made
by RE and SE had significant effects (p < 0.05) on the level of TNF-α, compared to LPS
treatment. For example, TNF-α expression was inhibited by 22.3%, and 22.1%, after treatment
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with 150 µg/mL of RE, and SE, respectively. The results also showed that ME extract had no
inhibitory effect on the level of TNF-α at any concentration (Figure 4- 3D).!
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Figure 4-3 Effect of various samples on tumor necrosis factor-α (TNF-α) expression in
macrophage RAW 264.7 cells (A) extracts obtained by ASE (B) pure phenolic standards
(C) crude polysaccharide extracts and (D) different extraction methods. Cells were
cultured in the absence or presence of LPS (1 µg /mL) with various concentrations of
different samples for 24h (0 = media; GA=2 µM; 1= 5 µM; 2=10 µM; 3= 20 µM; 4 = 50
µg/mL; 5=100 µg/mL; and 6= 150 µg/mL; 7= 25 µM; 8 = 50 µM; and 9 = 100 µM).
TNF-α production was determined through an ELISA. The data represent the mean ± SE
of triplicate experiments. Significant different values at P < 0.05
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4.3.5 Inhibition of IL-6 Production in LPS-Stimulated RAW 264.7 Macrophages
The expression of IL-6 cytokine increased to (755 ± 0.42 pg/mL) in macrophage cells
after stimulation with LPS. However, when various Chaga extracts obtained by ASE
were added at 50, 100, and 150 µg/mL, these increases were significantly (p < 0.05)
reduced. For example, at 150 µg/mL, ASE1, ASE2, and ASE3 extracts reduced the level
of IL-6 released from RAW 264.7 cells by 57.3%, 49.4%, and 50.4%, respectively,
compared with the LPS group (Figure 4-4A). The inhibition activity of phenolic acid
standards on the expression of IL-6 in LPS-induced 264.7 RAW cells is displayed in
(Figure 4-4B). We observed the same trend of TNF-α expression inhibition for the level
of IL-6; all assayed phenolic acids except VA and SA significantly (p < 0.05)
suppressedthe level of IL-6 at all examined concentration, compared to the LPS
treatment. For example, at the highest concentrations, PA, PCA, and CA reduced the
expression of IL-6 by 53.7%, 56.6%, and 39.5%, respectively, compared to the LPS
treatment. The effect of the polysaccharide extracts on the expression of IL-6 was similar
to their effect on TNF-α; the expression of IL-6 was significantly reduced (p < 0.05) after
treating the induced cells with P3 and P6 extracts at different concentrations. For
example, the IL-6 level was reduced by 56.8% and 57.1% after the LPS-induced cells
were treated with 150 µg/mL of P3 and concentration (Figure 4-4C). The results in
(Figure 4-4D) showed that extracts obtained by SE and RE have significant inhibitory
effect (P < 0.05) on the expression of IL-6 cytokine, compared with the LPS group. The
results in (Figure 4-4D) showed that extracts obtained by SE and RE have significant
inhibitory effect (P < 0.05) on the expression of IL-6 cytokine, compared with the LPS
group. For example, at a concentration of 150 µg/mL, RE and SE inhibited the cytokine
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level by 26.6% and 26.9%, respectively, compared with the LPS group. No inhibitory
effect of ME extract on the level of IL-6 was observed at any concentration.
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Figure 4-4 Effect of various samples on IL-6 expression in macrophage RAW 264.7 cells (A)
extracts obtained by ASE (B) pure phenolic acid standards (C) crude polysaccharide extracts and
(D) different extraction methods. Cells were cultured in the absence or presence of LPS (1 µg /mL)
with various concentrations of different samples for 24h (0 = media; GA=2 µM; 1= 5 µM; 2=10
µM; 3= 20 µM; 4 = 50 µg/mL; 5=100 µg/mL; and 6= 150 µg/mL; 7= 25 µM; 8 = 50 µM; and 9 =
100 µM). IL-6 production was determined through an ELISA. The data represent the mean ± SD of
triplicate experiments. Significant different values at P < 0.05.
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4.3.6 Inhibition of IL-β Production in LPS-Stimulated RAW 264.7 Macrophages
Our data indicated an increase in the expression of IL-β in the LPS-stimulation
group compared to the control group (Figure 4-5). At 150 µg/mL, all Chaga extract
obtained by ASE displayed significant inhibitory effect (p<0.05) on the level of IL-1β as
compared to the LPS group (Figure 4-5A). For example, ASE1 reduced the level of IL-1β
by 22.6% compared to the LPS group. Similarly, the level of IL-1β decreased
significantly (p <0.05) after treating the LPS-induced cells with the highest
concentrations of PA and PCA, respectively (Figure 4-5B). For example, the
concentration of IL-1β in the supernatants was decreased by 22.6% and 21.5%, after
incubated the LPS-induced cells with highest concentration of PA, PCA, respectively.
The effect of the polysaccharide extracts on the expression of IL-β is presented in (Figure
4-5C). Polysaccharide extracts from the Chaga obtained by the tea bag form had no
ability to attenuate the level of inflammatory cytokine. However, at 150 µg/mL, P6
significantly (p <0.05) reduce the level of IL-1β by 21.5% , compared to the LPS
treatment. (Figure 4-5D) presents the effect of the extraction method on the inhibition
activity of the extracts against IL-1β in the LPS-induced macrophages. None of the
extracts obtained by the conventional methods affected the expression of the IL-1β at any
concentration, compared to the LPS group.
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Figure 4-5 Effect of various samples on IL-β expression in macrophage RAW 264.7 cells (A)
extracts obtained by ASE (B) pure phenolic acid standards (C) crude polysaccharide extracts and
(D) different extraction methods. . Cells were cultured in the absence or presence of LPS (1 µg
/mL) with various concentrations of different samples for 24h (0 = media; GA=2 µM; 1= 5 µM;
2=10 µM; 3= 20 µM; 4 = 50 µg/mL; 5=100 µg/mL; and 6= 150 µg/mL; 7= 25 µM; 8 = 50 µM; and
9 = 100 µM). IL-β production was determined through an ELISA. The data represent the mean ±
SD of triplicate experiments. Significant different values at P < 0.05.
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4.4 DISCUSSION
Inflammation is a physiological immune response of body tissues against
physical, chemical, and biological stimuli such as tissue injury, chemical toxins, or
pathogens (Jeong & Jeong, 2010). Lipopolysaccharide is an endotoxin, an integral outer
membrane component of gram-negative bacteria, and the most potent trigger for
microbial initiators of inflammatory response (Dobrovolskaia & Vogel, 2002).
Macrophages are essential for the initiation and resolution of pathogen- or tissue damage-
induced inflammation. Macrophages are cells that play a vital role in the immune system
and are associated with inflammatory diseases. Macrophages activated by LPS treatment
produce wide variety of inflammatory markers mostly for the primary protection of the
host including NO TNF-α, IL-6, and IL-1β. However, excess and uncontrolled production
of these inflammatory product lead to excessive inflammatory response and oxidative
stress (Dai et al., 2019; Han et al., 2002; Park et al., 2007). Anti-inflammatory agents
produce anti-inflammatory effect through regulating cytokines and these inflammatory
mediators. Therefore, monitoring the expression of these mediators is vital for
understanding the inflammatory process and provides a measure to evaluate the effects of
anti- inflammatory agents (Taofiq et al., 2016).
Nitric oxide is a multi-functional mediator that plays an important role in cellular
signaling and a variety of physiological functions in many cells and tissues, including the
brain, the vasculature, and the immune system (Paige & Jaffrey, 2007). Evidence
indicates that overproduction of NO due iNOS is a significant contributor to the
inflammatory processes and may provide an indicator of the degree of inflammation
(Alderton et al., 2001; Barkett & Gilmore, 1999). Therefore, the inhibition of NO
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overproduction may be an essential measure for assessing the anti-inflammatory effects
of drugs (Kim et al., 2006).
Inflammation is characterized by the production of a wide variety of free radicals,
nitrogen reactive species, and cytokines—such as TNF-α, IL-6 and IL-1β—which act as
modulators throughout the inflammation process (Adams & Hamilton, 1984). TNF-α
stimulates the production of other cytokines such as IL-6, IL-1β. IL-6 is a multifunctional
cytokine with pro- and anti-inflammatory properties that plays a central role in immune
and inflammatory responses (Beutler & Cerami, 1989). IL-1β is also a multifunctional
cytokine that has been implicated in pain, fever, inflammation and autoimmune
conditions (Dinarello, 2002). High levels of these cytokines elicit number of
physiological effects including septic shock, inflammation, and cytotoxicity (Garlanda et
al., 2007; Sánchez-Miranda et al., 2013). Thus, inhibiting the expression of cytokines in
macrophage cells is very important during the anti-inflammatory response.
Chaga has been used for its medicinal properties throughout history in many parts
of the world. Numerous scientific reports have investigated the chemical composition and
the biological activities of Chaga from various geographical locations. However, no
previous study has utilized chaga from the United States of America. In this study, the
results showed that different extracts of Chaga sclerotia collected from Maine have
significant anti-inflammatory activity on LPS-stimulated RAW 264.7 cells. The
inhibitory effect was through a decrease in the production of the NO and a down-
regulation of TNF-α, IL-6, and IL-1β in RAW 264.7 macrophages; there was no effect on
cell viability at a concentration range of (50–150 µg/mL). The results also showed that
phenolic extracts obtained from different extraction methods have different anti-
inflammatory properties. All samples made by the accelerated solvent extraction method
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and conventional methods significantly reduce the level of NO. The results also showed
that ME extract did not affect the NO production at any concentration. The inhibitory
effect of Chaga extracts could be attributed to the content of phenolic compounds and
DPPH scavenging activity. Phenolic compounds have been largely recognized as natural
molecules with potential antioxidant activity. It has been demonstrated that oxidative
stress can activate a variety of inflammatory mediators that contribute to the
inflammation process; oxidative stress inhibited by compounds with high antioxidant
activity such as phenolic compounds. Previous studies have also reported that alcohol
extracts of Chaga are rich in polyphenolic compounds that possess strong antioxidant
activity and can protect cells against oxidative damage. Such extracts have been reported
to attenuate inflammation reactions and decrease the production of inflammatory
mediators in 264.7 macrophages.
Previous studies have reported small phenolic ingredients as a main constituent of
alcoholic extracts of Chaga that contribute significantly to the antioxidant activity.
However, no reports have examined the anti-inflammatory effect of these constituents
using RAW 264.7 cells. In our previous report, we increased the extraction of phenolic
acids from Chaga by optimizing an ASE extraction method. In this study, we investigated
the NO-inhibitory activities of small phenolic ingredients in stimulated RAW 264.7 cells.
Our results showed phenolic acids affected different inflammatory mediators; some of the
compounds tested did not affect the production of NO or the expression of the
inflammatory cytokines, while others had a significant effect. For example, both VA and
SA did not alter the production of NO and the expression of TNF-α, IL-6, and IL-1β at
any of the examined concentrations. However, PA, PCA, and CA significantly reduced
the production of NO and attenuated the expression of TNF-α, IL-6, and IL-1β at all the
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examined concentrations. This is in accordance with previous studies, which
demonstrated anti-inflammatory properties of PA, PCA, and CA (Juman et al., 2012).
However other studies have suggested that phenolic compounds with only one phenol
ring—such as the tested compounds—have less of an anti-inflammatory effect through
inhibition of cytokine production; it has been hypothesized that other mechanisms might
be involved in the anti-inflammatory action of phenolics (Miles et al., 2005). Our results
suggest that some of the small phenolic compounds present in Chaga might play a vital
role in anti-inflammatory activity.
Previous evidence demonstrates that polysaccharides from many sources have a
variety of therapeutic effects including antioxidant and anti-inflammation activities.
Polysaccharides in Chaga from Russia, China, and South Korea have been reported to act
as immune-modulators and possess anti-inflammation properties from in vivo and in vitro
studies. In this study, we examined the chemical structure of crude polysaccharides
extracted from Chaga tea, in both powder and bagged form, collected from Maine, USA.
We evaluated the anti-inflammation effects of these extracts using LPS-stimulated RAW
264.7 cells. The yield and chemical characteristics of the polysaccharide samples are
summarized in Table 1. The results indicate that more components of Chaga could be
extracted from the powder form than from bagged form. For example, P6 yielded 30%
polysaccharide containing 17.56% carbohydrate, 10.23% protein, 4.12% uronic acid, and
17.61% phenolic content; while B6 yielded 17.33% polysaccharide containing 11.81%
carbohydrate, 7.23% protein, 2.84% uronic acid, and 8.33% phenolic content. Our data
are in agreement with previous reports suggesting that more bioactive constituents can be
extracted from different raw sources in powders forms than other forms. We attribute the
higher extraction efficiency to the greater surface area of powdered form, which allows
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better penetration of the solvents to target analytes and thus higher extraction efficiency.
The results show that the crude polysaccharides of Chaga extracts in both forms have
high phenolic content, indicating that these phenolic compounds are bound to
macromolecules in Chaga such as polysaccharides and melanin. Chaga contains high
amounts of water-soluble macromolecule pigments known as melanin. The dark color of
the extracts suggests that a relatively high amount of melanin is present. Other reports
also suggested that crude polysaccharide extract from chaga have melanin and melanin-
associated phenolic compounds.
Our results demonstrate that crude polysaccharides obtained from both the
powder and the bagged form significantly inhibit LPS-induced NO production in RAW
264.7 cells. We observed higher NO inhibitory effect of polysaccharides obtained from
Chaga tea in the powdered form, in comparison to those in bagged form at the same
concentration. Polysaccharides obtained from the powdered form significantly inhibited
the production of TNF-α and IL-6; however, none of the crude polysaccharide extracts
obtained in either form altered the expression of IL-1β. However, since the extract is
crude, it contains high values of phenolic compounds and melanin, which may contribute
to the anti-inflammation effect of the extracts.
4.5 CONCLUSION
With increasing interest in research on the health-promoting effects of Chaga
(Inonotus obliquus), we have shown here that Chaga collected in Maine, USA can exhibit
significant anti-inflammatory properties against LPS-activated 264.7 RAW macrophages.
Our results suggest that ASE extract and Chaga tea extract obtained from powder form
may be a promising method in the development of new anti-inflammatory supplements.
Thus, intake of Chaga as a tea might help to attenuate inflammatory reaction. The data
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also showed that the metabolites in Chaga such as phenolic acids inhibited NO
production in macrophages.
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APPENDIX: OVERALL CONCLUSIONS
To our knowledge, there has been no prior study that optimizes extraction
parameters for phenolic compound isolation from Maine-harvested Chaga or assessed the
effects of the extraction methodology on anti-inflammation benefits. A simple, precise,
and accurate HPLC method for the simultaneous quantification of five phenolic acids
extracted from Chaga was developed and validated. Using base and acid hydrolysis
conditions, with and without AA and EDTA protection, we determined that the majority
of phenolic acids occur bound to the cell wall components of the fungus. Thus, a
hydrolysis procedure is required to allow for maximum extraction and quantification of
total phenolic acid content. The study also highlighted the protective effect of AA and
EDTA on phenolic acids during acid and base hydrolysis.
An Accelerated Solvent Extraction (ASE) based green extraction method was also
developed to obtain Chaga-derived phenolic compounds. The response surface
methodology (RSM) was utilized for optimizing parameters relative to the extraction of
phenolic-rich extracts. Maximum TPC (39.32 mg GAL/g DW) and DPPH scavenging
activity (76.59%) yields were produced when an extraction temperature of 170 °C and
ethanol: water composition of 66% were used in phenolic compound recovery.
Additionally, the total phenolic acids content of the extract was increased at these
optimized conditions, supporting our findings of the previously mentioned HPLC work.
Chaga extracts produced under several extraction conditions also resulted in
varying degrees of phenolic compound isolation, which subsequently affected the degree
of anti-inflammatory benefits. This finding confirms that the bioactive advantage of the
fungal extract may be attributed, at least in part, to some of the phenolic acids within the
Chaga extracts. Therefore, Maine Chaga demonstrates potential for therapeutic value.
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FUTURE WORK:
As an outcome of this work, other future studies became evident and are cited
below.
● The extracts from different extraction methods require further
characterization to identify the amounts and types of phytochemicals
present in each, which most likely resulted in different extraction models
for TPC and antioxidants.
● The extracts must be characterized for individual TP to determine if the
phenols are acting alone, synergistically, or additively to impact
(negatively and positively) both antioxidant and anti-inflammatory effects.
● The crude extracts of Chaga contain carbohydrates and minerals
along with the phenolic compounds, necessitating further purification to remove
these impurities for further studies and characterization of individual phenolic
compounds.
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BIOGRAPHY OF THE AUTHOR
Weaam Alhallaf was born in Baghdad, IQ, on April 12, 1983. Weaam was raised in
Baghdad, IQ, where she lived for 18 years and graduated from Al-Butoolah High School
in 2000. Following high school, Weaam attended Science College in Baghdad, IQ, for
four years and graduated in May of 2004 with a Bachelor of Science in Chemistry. In
2005, Weaam attended the graduate school of Science College in Baghdad, IQ, for two
years and graduated with a Master of Science in Biochemistry. She appointed in April of
2008 as a teaching assistant at Baghdad University for five years, and in 2013 she was
awarded a scholarship from the Iraqi government to pursue her Ph.D. degree in the Food
Science Department at the University of Maine with a minor in Food Chemistry. After
receiving her degree, Weaam wants to continue her career in academia. Weaam is a
candidate for the Doctoral of Philosophy degree in Food Science and Human Nutrition
from the University of Maine in May of 2020.
