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Bioactive Compounds and Bioactive Properties of Chaga (Inonotus obliquus) Mushroom: A Review

December 2020
Journal of Food Bioactives 12(8)

DOI:10.31665/JFB.2020.12245

Authors:

University of California, Davis

Chaga (Inonotus obliquus) is an edible herbal mushroom extensively distributed in the temperate to frigid regions of the Northern hemisphere, especially the Baltic and Siberian areas. Chaga parasites itself on the trunk of various angiosperms, especially birch tree, for decades and grows to be a shapeless black mass. The medicinal/nutraceutical use of chaga mushroom has been recorded in different ancient cultures of Ainu, Khanty, First Nations, and other Indigenous populations. To date, due to its prevalent use as folk medicine/functional food, a plethora of studies on bioactive compounds and corresponding compositional analysis has been conducted in the past 20 years. In this contribution, various nutraceutical and pharmaceutical potential, including antioxidant, anti-inflammatory, anti-tumor, immunomodulatory, antimutagenic activity, anti-virus, analgesic, antibacterial, antifungal, anti-hyperglycemic, and anti-hyperuricemia activities/effects, as well as main bioactive compounds including phenolics, terpenoids, polysaccharides, fatty acids, and alkaloids of chaga mushroom have been thoroughly reviewed, and tabulated using a total 171 original articles. However, only key bioactivities and bioactives are selectively discussed. Besides, the up-to-date toxicity concerns and risk assessment about the misuse of chaga, which limit its acceptance and use as medicinal/nutraceutical products, have also been clarified.

Known phenolic small molecules and polymers of chaga and their purification/identification

…

Bioactivities of phenolics purified from chaga

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Polysaccharides and other known compounds of chaga and their purification/identification

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Bioactivities of polysaccharides and other compounds purified from chaga

…

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Review J. Food Bioact. 2020;12:9–75

Journal of

Food Bioactives International Society for

Nutraceuticals and Functional Foods

Bioactive compounds and bioactive properties of chaga (Inonotus

obliquus) mushroom: a review

Han Peng and Fereidoon Shahidi*

Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9

*Corresponding author: Fereidoon Shahidi Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada

A1B 3X9. Tel: (709)864-8552; E-mail: fshahidi@mun.ca

DOI: 10.31665/JFB.2020.12245

Received: December 04, 2020; Revised received & accepted: December 24, 2020

Citation: Peng, H., and Shahidi, F. (2020). Bioactive compounds and bioactive properties of chaga (Inonotus obliquus) mushroom: a review.

J. Food Bioact. 12: 9–75.

Abstract

Chaga (Inonotus obliquus) is an edible herbal mushroom extensively distributed in the temperate to frigid regions

of the Northern hemisphere, especially the Balc and Siberian areas. Chaga parasites itself on the trunk of various

angiosperms, especially birch tree, for decades and grows to be a shapeless black mass. The medicinal/nutraceu-

cal use of chaga mushroom has been recorded in dierent ancient cultures of Ainu, Khanty, First Naons, and other

Indigenous populaons. To date, due to its prevalent use as folk medicine/funconal food, a plethora of studies on

bioacve compounds and corresponding composional analysis has been conducted in the past 20 years. In this con-

tribuon, various nutraceucal and pharmaceucal potenal, including anoxidant, an-inammatory, an-tumor,

immunomodulatory, anmutagenic acvity, an-virus, analgesic, anbacterial, anfungal, an-hyperglycemic, and

an-hyperuricemia acvies/eects, as well as main bioacve compounds including phenolics, terpenoids, polysac-

charides, fay acids, and alkaloids of chaga mushroom have been thoroughly reviewed, and tabulated using a total

171 original arcles. However, only key bioacvies and bioacves are selecvely discussed. Besides, the up-to-date

toxicity concerns and risk assessment about the misuse of chaga, which limit its acceptance and use as medicinal/

nutraceucal products, have also been claried.

Keywords: Phenolics; Terpenoids; Polysaccharides; Alkaloids; Nutraceutical/medicinal properties; Bioactives and bioactivities; Toxicity/

safety concerns.

1. Introducon

Chaga (Inonotus obliquus) is a terrestrial polypore fungus of Hy-

menochaetaceae family, which is mainly distributed in temper-

ate to frigid regions, including North/East Asia, North America,

and Central/Eastern/North Europe (Zhong et al., 2009). It is also

found in low-latitude areas such as Western/Southern Europe and

even Southeast Asia (Thailand) (Glamočlija et al., 2015). Chaga

parasites itself on the bark of various boreal broad-leaved decidu-

ous angiosperms such as birch (Betula spp.) and beech (Fagus

spp.). However, some other rare hosts such as maple (Acer spp.),

alder (Alnus spp.), oak (Quercus spp), and poplar (Populus spp.)

may also be available (Lee et al., 2008). The parasitized site on

the trunk would nally develop to be a white heart rot in the ap-

pearance of shapeless black mass, and these decays typically last

for more than ten years and result in the death of the host (Lee et

al., 2008). In Northern and Eastern Europe/Asia such as Russia,

Poland, Finland, Belarus, and Japan, this wood-destroying fungus

has been used as a functional beverage (tea) or folk medicine (de-

coction, ointment) for the treatment of stomach diseases, intesti-

nal worms, liver/heart ailments, dermatomycoses, joint pains, and

dierent kinds of cancers for centuries (Babitskaya et al., 2002;

Koyama, 2017; Lemieszek et al., 2011; Saar, 1991; Shashkina et

al., 2006; Shikov et al., 2014). In North America, the historical

use for medicinal purposes (including skin irritation and arthri-

tis) by Alaskan, First Nations and other Indigenous tribes such as

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Cree, Chipewyan, Gitxsan, Wet’suwet’en has also been recorded

(Cottesfeld, 1992; Kari, 1987; Rogers, 2012; Scerbak et al., 2016).

The binomial name of chaga is known as Inonotus obliquus, but

other names including Phaeoporus obliquus, Polyporus obliquus,

or Fuscoporia obliqua, have also been sporadically used (He et al.,

2001; Reid, 1976). Inonotus is a genus of fungi in the family Hy-

menochaetaceae that was rst described and given by Petter Adolf

Karsten and so far is estimated to have 101 species in its wider sense

(2005 data) (Ghobad-Nejhad and Kotiranta, 2008; Kirk et al., 2008;

Ren and Dai, 2018; Ryvarden, 2005; Wu et al., 2018; Zhou et al.,

2016). Interestingly, even though chaga has been clearly dened and

classied in nomenclature and taxonomy, the misuse of the original

data from the studies of others closely related species rather than the

real Inonotus obliquus has frequently happened in some previous

reviews (Duru et al., 2019; Zheng et al., 2010). To date, numerous

studies have claimed various bioactivities, together with related mo-

lecular mechanism of chaga, including antioxidant, antimicrobial,

anti-cancer, hypoglycemic, antilipidemic, anti-inammation, abirri-

tative, immunoregulatory, and cardioprotective eects (Koyama et

al., 2008; Patel, 2015; Shashkina et al., 2006; Zhong et al., 2009).

Apparently, such a broad spectrum of biological/pharmacological

functions implies the complexity of bioactive substances in chaga.

However, despite decades of eorts, the full scale of known bio-

active components of chaga and corresponding mechanisms of its

health eects upon oral ingestion or other administration approaches

is still uncertain. Meanwhile, several side eects associated with

specic cases are rarely discussed. This contribution intends to ll

the existing gap in previous works and to update the secondary me-

tabolites of chaga and their biological properties as well as safety

considerations based on the latest available studies.

2. Health claims for chaga (Inonotus obliquus) extracts

In East Asian countries, such as China, Japan, and Korea, the use

of medicinal mushrooms (e.g., Ganoderma lucidum and Grifola

frondosa) and their derived products (e.g., β-glucan and lentinan)

has continued in traditional therapies, but is now also supported by

the modern medicinal systems with the verication of phases I, II,

or even III clinical trials (Chatterjee et al., 2011; Deng et al., 2009;

Deng et al., 2008; Gao, 1993; Gao et al., 2004a; Gao et al., 2004b;

Gordon et al., 1998; Kidd, 2000; Ohno et al., 2011; Taguchi et al.,

1985; Xu et al., 2012; Zhang et al., 2019). Similarly, chaga is one

of the most important and popular medicinal mushrooms which has

been extensively used in the East European countries for centuries.

As already mentioned, the diversity of its bioactive compounds and

eects thereof have been gradually unveiled in the past decades,

even though related clinical data are relatively scarce. The recent ad-

vancement of health functions as well as the molecular mechanism

of chaga extracts is summarized (Table 1) and discussed

2.1. An-tumor eects

Among various pharmacological properties of crude extracts of

chaga, its anti-tumor eects have attracted the most attention. Ac-

cording to World Health Organization (WHO) (2018), cancer, the

second leading cause of death, led to an estimated 9.6 million death

globally in 2018; thus accounting for around one in six deaths. In the

United States, approximately 39.55% of men and women are diag-

nosed with cancer at some points during their lifetime (2015–2017

data), and estimated national expenditure for cancer care in 2017 was

$147.3 billion (NIH, 2020). As shown in Table 1, various extracts of

chaga mushroom present broad in vitro anti-proliferation activities on

various cancer cells. Baek et al. (2018) reported that the hexane and

dichloromethane fractions of methanolic extract of chaga showed sig-

nicant cytotoxicity on A549, H1264, H1299, and Calu-6 lung cancer

cell lines, with IC50 of 95.3–225.1 μg/ml. Water and 70% ethanolic

extracts of chaga inhibited the growth of MCF-7 human breast can-

cer cells, NCI-H460 human non-small cell lung cancer cells, HeLa

human cervical uteri tumor cells, and HepG2 human liver cancer

cells with IC50 ranging from 80.93 to 318.19 μg/ml (Glamočlija et

al., 2015). In in vitro models of PC3 human prostatic carcinoma

cells and MDA-MB-231 human breast carcinoma cells, petroleum

ether fraction of chaga showed a similar anti-proliferation activity to

doxorubicin (Ma et al., 2013). Chaga extracts were found to inhibit

the proliferation of cancer cells by inhibiting mitosis and arresting

the cell cycle. Jarosz et al. (1990) found that the culture medium of

chaga and its lower-molecular weight extracts (fractions from Se-

phadex G-25 chromatography) block the mitosis of Hela cells with

a signicant increase of catalase activity and impairment of chromo-

some and cellular membrane. Later, Mishra et al. (2013) showed that

water extract of chaga arrested DLD 1 and HCT116 cells at S phase.

While in B16-F10 cells, the water extract arrested cell cycle at G0/

G1 phase with down-regulation of pRb, p53, p27, cyclin D1/E and

CDK 2/4 expression levels (Youn et al., 2009). Likewise, the cell cy-

cle of HepG2 cells was arrested by water extract of chaga at the G0/

G1 phase associated with down-regulation of p53, pRb, p27, cyclins

D1/D2/E, and CDK 2/4/6 expression (Youn et al., 2008). However,

in HT-29 cells, the ethanol extract of chaga arrested it in the G1 phase

by inhibition of CDK2, CDK4, cyclin D1, and pRb, but with activa-

tion of p21, p27, and p53 (Lee et al., 2015a). This vital function of

p53 was proven to be unrelated to the pro-apoptotic eect of hexane

and dichloromethane fractions of methanolic extracts of chaga on

A549, H1264, H1299, and Calu-6 lung cancer cell lines (Baek et al.,

2018). Besides, several classic apoptotic pathways were reported to

be modulated by chaga extracts. For example, water extract of chaga

induced cell apoptosis through downregulation of antiapoptotic pro-

tein (Bcl-2) and upregulation of proapoptotic proteins (Bax and cas-

pase-3) in HT-29 cells (Lee et al., 2009). The apoptosis of HepG2

cells induced by water extract of chaga was coupled with the activa-

tion of caspase-3 (Youn et al., 2008). Meanwhile, both caspase 3 and

9 were activated in both extract-treated DLD 1 and HCT116 cells,

but caspase 8 was only partially activated in HCT116 cells (Mishra

et al., 2013). In these two in vitro studies of Youn et al. (2008) and

Mishra et al. (2013), water extract inhibited both cytoplasmic and

nuclear levels of NK-κB and β-catenin, as well as the cytosolic level

of a key inammatory mediator Cox-2 (cyclooxygenase-2). The in

vivo anti-tumor eects of chaga extracts were also assessed in various

animal models. The intraperitoneal administration of water extract of

chaga at a dose of 20 mg/kg/day for ten days signicantly inhibited

the growth of tumor mass in B16-F10 cells implanted mice (Youn et

al., 2009). A 14-days oral administration of water extract of chaga

at a dose of 20–100 mg/kg body weight/day regressed the tumors in

sarcoma 180 implanted mice by inhibiting the sarcoma 180-induced

reduction of splenic lymphocytes, stimulating TNF-α release in peri-

toneal macrophage, and eliciting the over-expression of Bax gene

in sarcoma 180 cells of mice (Chen, 2007). In addition, the water

extract of chaga showed inhibitory eects on the growth of intesti-

nal polyps in APCMin/+ mice and colon tumors in AOM/DSS-treated

mice. Supplement of the water extract of chaga suppressed the nu-

clear levels of β-catenin, inhibited its downstream targets (cyclin Dl

and c-Myc), reduced pro-caspase-3 and cleaved PARP, along with

CRC (colorectal cancer) oncogene CDK8 in APCMin/+ mice (Mishra

et al., 2013). Simultaneously, the inhibition of inammatory proteins

including iNOS and Cox-2 and mRNA levels of pro-inammatory

cytokines (IL-6, IL-1β, TNF-α and IFN-γ) was found in the intestine

Journal of Food Bioactives | www.isn-jfb.com 11

Peng et al. Bioactive compounds and bioactive properties of chaga

Table 1. Bioacvies of crude extracts of chaga (Inonotus obliquus)

Crude extract Bioacvity Model IC50/EC50/LC50 values or

experimental dosage (ED)

Specic mechanism or

manifestaon Reference

Hexane and

dichloromethane

fracons of

methanol extract

An-proliferaon

acvity

Calu-6 lung cancer cell IC50∼2.30 mg/ml Baek et al. (2018)

A549 lung cancer cell IC50∼2.03 mg/ml

H1264 lung cancer cell IC50∼2.03 mg/ml

H1299 lung cancer cell IC50∼2.40 mg/ml

Chloroform

extract

An-proliferaon

acvity

P388 mouse leukemia cell IC50∼13.9 μM Nomura et al. (2008)

HeLa human cervical

cancer cells

–

Ethanol extract An-proliferaon

acvity

NCI-H460 human lung

cancer cell line

ED∼50 μg/ml Sun et al. (2011)

Ethanol extract An-proliferaon

acvity

HT-29 human colon cancer cells ED∼2.5–10 µg/ml Arrested cell cycle in G1 phase;

decreased expression of CDK2,

CDK4, and cyclin D1; increased

expression of p21, p27, and

p53; inhibited phosphorylaon

of Rb and E2F1 expression.

Lee et al. (2015a)

Ethanol extract An-proliferaon

acvity

DLD-1 human colon cancer cell ED∼400 μg/ml Induced nuclear fragmentaon Hu et al. (2009)

Methanol extract An-proliferaon

acvity

HL-60 IC50∼32.2 μg/ml –Nguyen et al. (2018)

LU-1 IC50∼38.0 μg/ml –

SW480 IC50∼41.3 μg/ml –

HepG2 IC50∼51.3 μg/ml –

KB IC50∼57.0 μg/ml –

LNCaP IC50∼57.7 μg/ml –

80% Methanol

extract

An-proliferaon

acvity

A549, PA-1, U937, HL-60 IC50∼23.2–105.2 μg/ml –Nakajima et al. (2009)

Methanol extract An-proliferaon

acvity

HT1080 cells ED∼10–100 μg/ml –Ryu et al. (2017)

An-tumor eect B16F10 melanoma cell

implanted C57BL/6 mice

ED∼30 μM/mouse/day

(oral administraon)

–

Ethyl acetate and

petroleum ether

fracons of 100%

ethanol extracts

An-proliferaon

acvity

human 29 prostac cancer

cell PC3 and human breast

cancer cell MDA-MB-231

IC50∼19.22 and 46.49 μg/ml –Ma et al. (2013)

Ethyl ether and

water extracts

An-proliferaon

acvity

Human cervical

cancer HeLa cells

– Impaired the chromesome in

metaphase and lysis; impared the

cell membrane; no eects on CAT

Jarosz et al. (1990)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Crude extract Bioacvity Model IC50/EC50/LC50 values or

experimental dosage (ED)

Specic mechanism or

manifestaon Reference

70% ethanol and

water extracts

An-proliferaon

acvity

MCF-7 human breast

cancer cell

IC50∼92.65-239.43 μg/ml –Glamočlija et al. (2015)

NCI-H460 human non-

small cell lung cancer cell

IC50∼80.93–267.27 μg/ml –

HeLa human cervical

uteri tumor cell

IC50∼217.36–318.19 μg/ml –

HepG2 human liver cancer cell IC50∼94.24–281.12 μg/ml –

Culvaon broth An-proliferaon

acvity

Hela cells – Inhibited the cell mitosis

and increased the catalase

acvity; induced impairment

of chromosome/cellular

membrane and cell lysis

Jarosz et al. (1990)

unknown-

solvent extract

An-proliferaon

acvity

SCC-13 human malignant

keranocytes

ED∼10–200 μg/mL Down-regulated the

expression of NF-κB

Song et al. (2004)

Water extract An-proliferaon

acvity

A549 lung cancer cell –Higher toxicity on cancer-derived

cells A549 than on normal

transformed cells BEAS-2B

Géry et al. (2018a)

Water extract An-proliferaon

acvity

HepG2 human liver cancer cells ED∼750 μg/ml Arrested cells in G0/G1 phase;

up-regulated the expression of

capase-3; down-regulated the

expression of cell cycle modulators

(p53, pRb, and p27) and G0/G1

regulatory proteins (Cdk2, Cdk4,

Cdk6, and Cyclin D1, D2, and E)

Youn et al. (2008)

Water extract An-proliferaon

acvity

Hela human cervical

uteri tumor cells

– Decreased the cell protein amount

and mitoc index value; decreased

the acvity of LDH, HBDH, MDH, GGT

and increasing the acvity of CAT

Rzymowska (1998)

Water extract An-proliferaon

acvity

HCT-116 human

colorectal cancer cell

ED∼20 mg/ml Up-regulated Bax, bad, and

caspase-3 genes and mRNA

expression p53, p21WAF1/CIP1;

increased Bax/bcl-2 rao; increased

caspase-3 acvity and p53 protein

expression and decreased the

expression of NF-κB, p65 protein

and COX-2 gene; arrested cell at G0/

G1 phase ; downregulated CyclinD1

Tsai et al. (2017)

Water extract An-proliferaon

acvity

HT-29 human colon cancer cells ED∼0–1.0 mg/ml Arrested the cell cycle;

upregulated the level of Bax

and caspase-3 proteins and

down-regulated Bcl-2 protein

Lee et al. (2009)

Table 1. Bioacvies of crude extracts of chaga (Inonotus obliquus) - (connued)

Journal of Food Bioactives | www.isn-jfb.com 13

Peng et al. Bioactive compounds and bioactive properties of chaga

Crude extract Bioacvity Model IC50/EC50/LC50 values or

experimental dosage (ED)

Specic mechanism or

manifestaon Reference

Silver

nanoparcles of

water extract

An-proliferaon

acvity

A549 human lung cancer cell ED∼1 mM –Nagajyothi et al. (2014)

MCF-7 human breast

cancer cell

ED∼1 mM –

Fermented

meterials

An-proliferaon

acvity

HepG2 human liver cancer cells ED∼200 μg/ml Arrested cell cycle at G0/G1phase Hou et al. (2018)

Water extract An-proliferaon

acvity

Sarcoma 180 cells ED∼20–100 μg/ml Arrested the cell cycle

at G0-G1 phase

Chen (2007)

Water extract An-

proliferaon and

immunomodulatory

eect

Sarcoma 180 cell implanted

male ICR mouse tumor model

ED∼20–100 mg/kg BW/

day (oral administraon)

Restored splenic lymphocyte number

and proliferaon potenal; increased

the producon of TNF-α; inhibited

the expression of bcl-2 and bax gene

in tumors; reduced the tumor weight

Water extract An-proliferaon

eects

B16-F10 mouse melanoma cell ED∼750 μg/ml Formaon of dendrite-like

structures; arrested cell cycle in

(sub-)G0/G1 phase and acvated

caspase-3 acvity; down-regulated

expression of p53, p27, and pRb

proteins; decreased the expression

of Cdk2 Cdk4, Cyclin D1 and Cyclin E

Youn et al. (2009)

Water extract An-tumor eect B16-F10 cell implanted

Balb/c mice

ED∼20 mg/kg/

day (intraperitoneal

administraon)

–

Water extract An-tumor eect Lewis lung cancer cell-

implanted mouse tumor model

ED∼6 mg/kg BW/day

(oral administraon)

Promoted a decrease of body

weight in middle-aged and old

mice; slowed tumor progression;

decreased tumor vascularizaon;

suppressed lung metastasis;

prevented body temperature

decrease aer tumor implantaon

Arata et al. (2016)

Water extract An-proliferaon

ability

HT1080, Hep G2, CT-26

cancer cells and broblast

CRL-7250 normall cell

ED∼0.2–200 μg/ml Inhibited the viability of both

cancer and normal cells

Song et al. (2007)

An-tumor eects CT-26 cell-inoculated

BALB/c mice pulmonary

metastasis model

ED∼20 or 10 μg/ml

(oral and intravenous

administraon)

Decreased pulmonary metastasis

Pro-tumor eects CT-26 cell-inoculated

BALB/c mice pulmonary

metastasis model

ED∼100 μg/ml (intravenous

administraon)

Increased pulmonary metastasis

Immunomodulatory

acvity

RAW 264.7 cells ED∼0.2–20 μg/ml Increased NO producon and mRNA

expression of iNOS, IL-1β, IL-10;

Table 1. Bioacvies of crude extracts of chaga (Inonotus obliquus) - (connued)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Crude extract Bioacvity Model IC50/EC50/LC50 values or

experimental dosage (ED)

Specic mechanism or

manifestaon Reference

Freshly isolated splenocytes ED∼0.2–20 μg/ml Smulated proliferaon; up-

regulated mRNA expression

of IL-2, IL-4, IL-10, IL-12, IFN-γ,

TGF-β; increased expression

of IL-2, IL-10, TNF-α, IFN-γ;

NK cells ED∼0.2–20 μg/ml Smulated NK cytotoxic acvity

Culvaon broth Immunomodulatory

acvity

Vaccinated chickens ED∼0.8% of daily diet

(oral administraon)

Inhibited hemagglunaon

in negave group; enhanced

the neutralizing anbody

ters, proliferaon of PBMCs,

proporons of CD3+, CD3+CD8+,

and CD3+CD4+ T lymphocytes,

as well as the rao of Th1/Th2

Zhang et al. (2018)

Water extract An-proliferaon/

inammaon

acvies

HCT116 and DLD1 Human

colorectal cancer cell

ED∼0.2 and 0.5 mg/ml Arrested cell cycle in S phase;

acvated caspase-8, caspase-3,

caspase-9, pARP; inhibited the level

of NF-κB, c-Myc, β-catenin, and Cox-2

Mishra et al. (2013)

An-tumor eect/

an-inammaon

eect

APCMin/+ mouse colorectal

adenoma model

ED∼100 and 300 mg/

kg BW/0.5 day (oral

administraon)

Reduced the count of large polyps

in small/large intesne; surpassed

the overexpression of cyclin D1

and c-Myc in intesnal epithelial

cells; inhibited the expression

of β-catenin and CDK-8, pro-

caspase-3 and cleaved PARP;

Suppressed iNOS and Cox-2 level

An-cancer eect/

an-inammaon

eect

AOM/DSS-induced mouse

colon cancer model

ED∼100 and 300 mg/

kg BW/0.5 day (oral

administraon)

Maintained colonic epithelial cell

structures and improves histological

damage in response to AOMJDSS;

suppressed mRNA overexpression

of IL-6, IL-1β, TNF-α, IFN-γ

Ethyl acetate

fracon of

residue water

extract

Pro-inammatory

acvity

LPS-induced RAW 264.7

murine macrophage cells

ED∼50–500 μg/ml Increased (adverse eect)

TNF-α and IL-6 producon

Van et al. (2009)

80% ethanol

extract

An-inammatory

acvity

LPS-induced RAW 264.7

murine macrophage cells

ED∼50–500 μg/ml Inhibited NO producon;

down-regulated IL-6 and TNF-α

levels; no eect on IL-1β

70% Ethanol

extract

An-inammatory

acvity

LPS-induced RAW 264.7

murine macrophage cells

ED∼100 μg/ml Inhibited NO producon and iNOS

and COX-2 expression; inhibited

the phosphorylaon of IκB-α,

Akt, and MAPKs (JNK, p38, ERK)

Kim et al. (2007)

Table 1. Bioacvies of crude extracts of chaga (Inonotus obliquus) - (connued)

Journal of Food Bioactives | www.isn-jfb.com 15

Peng et al. Bioactive compounds and bioactive properties of chaga

Crude extract Bioacvity Model IC50/EC50/LC50 values or

experimental dosage (ED)

Specic mechanism or

manifestaon Reference

70% Ethanol

extract

An-inammatory

eect

DSS-induced BALB/c

mice colis model

ED∼50 mg/kg BW/day

(oral administraon)

Decreased TNF-α, COX-2, IL-4,

IFN-γ, STAT1, and STAT6; lowered

the levels of IgE and IgA in the

spleen and mesenteric lymph

node; suppressed the DSS-induced

colonic ssue destrucon

Debnath et al. (2012)

50% Ethanol and

water extract

An-inammatory

acvity

LPS-induced RAW 264.7

murine macrophage cells

ED∼250 μg/ml Inhibited TNF-α producon Javed et al. (2019)

Histamine-induced RAW 264.7

murine macrophage cells

ED∼250 μg/ml Inhibited TNF-α producon

Histamine-induced

microvascular inammaon

in male C57BL6 mice

ED∼12.5 μg/ml Reversed the histamine-induced

reducon of conducted vasodilaon

Ethyl acetate

and petroleum

ether fracon of

ethanol extracts

An-inammatory

acvity

LPS-induced RAW 264.7

macrophage cells

ED∼40 μg/ml Inhibited NO producon Ma et al. (2013)

NF-κB reporter gene-stably

transfected RAW264.7 cells,

ED∼40 μg/ml Inhibited acvaon of

NF-κB luciferase

Methanol extract An-inammatory

acvity

LPS-induced RAW 264.7

murine macrophage cell

ED∼45–135 μg/ml Suppressed NO and PEG2

producon; inhibited protein

and mRNA expression of LPS-

induced TNF-α, iNOS, COX-2,

NF-κB (p65/p50); inhibited the

degradaon of cytosol IκB-α;

reducing the level of nuclear p65

Park et al. (2005b)

An-inammatory

eect

Carrageenin-induced

paw edema in male

Sprague-Dawley rats

ED∼100/200 mg/kg

(oral administraon)

–

Water extract An-inammatory

acvity

LPS-induced RAW 264.7

macrophage cells

–Inhibited the producon of TNF-α,

STAT1, pSTAT1, STAT6, and pSTAT6

Choi et al. (2010)

An-inammatory

eect

DSS-induced male BALB/c

mouse acute colis model

ED∼100/200 mg/kg

(oral administraon)

Maintained the liver weight;

decreased the serum IgE level;

decreased the expressions of

TNF-α, IFN-γ, IL-4, STAT6, and

STAT1 proteins in the spleen;

Water extract An-inammatory DSS-induced female C57BL/6

mouse acute colis model

ED∼50 and 100 mg/

kg BW/12 h

Suppressed edema, mucosal

damage, and the loss ofcrypts

induced by DSS; inhibited iNOS

levels and myeloperoxidase

accumulaon in colon ssues;

suppressed mRNA overexpression

of TNF-α, IFN-γ, IL-1β, and IL-6

Mishra et al. (2012)

Table 1. Bioacvies of crude extracts of chaga (Inonotus obliquus) - (connued)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Crude extract Bioacvity Model IC50/EC50/LC50 values or

experimental dosage (ED)

Specic mechanism or

manifestaon Reference

Ethyl acetate,

butanol, water

fracons of 60%

ethanol extract

Anoxidant acvity DPPH, superoxide and hydroxyl

radical scavenging assays

EC50∼31.42–336.42 μg/ml –Liang et al. (2009)

Water and 70%

ethanol extracts

Anoxidant acvity DPPH, FRAP, TBARS and

β-carotene bleaching assays

EC50∼0.07–9.22 mg/ml –Glamočlija et al. (2015)

Water and 80%

ethanol extract

Anoxidant acvity DPPH, APPH and superoxide

scavenging assays

ED∼5 μg/ml –Cui et al. (2005)

Ethyl acetate

fracon of

water extract

Anoxidave

stress acvity

H2O2-treated human

HaCaT keranocytes

ED∼50 μg/ml –

Water extract Anoxidave

stress acvity

Female SKH-1 hairless mice

UV irradiaon model

ED∼1.0% (external use) Suppressed UV-induced morphologic

skin changes (thickening and wrinkle)

Yun et al. (2011)

H2O2-treated human

dermal broblasts

ED∼1–50 µg/ml Scavenged intracellular ROS and

prevented lipid peroxidaon;

increased collagen synthesis

through inhibion of MMP-

1 and MMP-9 acvies

95% Ethanol

extracts

Anoxidave

stress acvity

BJ normal human

skin broblast

ED∼1 mg/mL Increased SOD1, CAT and

KI67 mRNA expression and

decreased ROS producon

Szychowski et al. (2018)

An-proliferaon

eect/prooxidave

stress acvity

Caco-2 human colon cancer cell ED∼1 mg/mL Decreased SOD1, CAT and

KI67 mRNA expression and

increased ROS producon

Water extract Anoxidant acvity H2O2-treated lymphocyte from

gastroenterology paents

and healthy volunteers

ED∼50–500 μg/ml Alleviated oxidave DNA damage Najafzadeh et al. (2007)

Ethanol extract Anoxidant acvity H2O2-treated lymphocytes

from healthy volunteers

ED∼6.25–100 μg/ml Alleviated oxidave DNA damage Park et al. (2005a)

Water extract Anoxidant acvity H2O2-treated human

lymphocytes

ED∼10–500 μg/ml Alleviated oxidave DNA damage Park et al. (2004)

Subfracons of

Methanol extract

Anmutagenic

acvity

MNNG and 4NQO induced

Salmonella typhimurium

strains TA98 and TA100; Trp-P-1

and B(α)P induced Salmonella

typhimurium strains TA98

and TA100 in presence with

the S-9 rat enzyme system

ED∼50 g/plate –Ham et al. (2009)

Ethyl acetate

extract

Anmutagenic eect N-methyl-N′-nitro-N-

nitrosoguanidine induced mice

ED∼0–1.6 mg/mice/day –Ham et al. (2003)

Table 1. Bioacvies of crude extracts of chaga (Inonotus obliquus) - (connued)

Journal of Food Bioactives | www.isn-jfb.com 17

Peng et al. Bioactive compounds and bioactive properties of chaga

Crude extract Bioacvity Model IC50/EC50/LC50 values or

experimental dosage (ED)

Specic mechanism or

manifestaon Reference

Methanol extract Analgesic acvity Hot plate test in mice ED∼100 and 200 mg/kg

BW (oral administraon)

–Park et al. (2005b)

Acec acid-induced abdominal

constricon test in mice

ED∼100 and 200 mg/kg

BW (oral administraon)

–

Water and

aqueous water

extract

An-virus HIV-infected MT-4

lymphoblastoid cells

ED∼5.0 μg/ml –Shibnev et al. (2015)

Water extract An-virus Hepas C virus-infected

porcine embryo kidney cells

–Inhibited infecve properes of

virus more than 100-fold and the

producon of infecve virus

Shibnev et al. (2011)

Water extract An-virus HIV-infected MT-4

amd CD4 cell,

ED∼0.01–1,000 µg/ml Inhibited HIV infecon and

HIV-induced cell damage

Sakuma (2004)

HIV-infected and PHA-

smulated peripheral

blood mononuclear cells

ED∼0.01–1,000 µg/ml

70% Ethanol and

water extracts

Anbacterial acvity Staphylococcus aureus

(ATCC 6538), Bacillus cereus

(clinical isolate), Micrococcus

avus (ATCC 10240),

Listeria monocytogenes

(NCTC 7973), Pseudomonas

aeruginosa (ATCC 27853),

Salmonella typhimurium

(ATCC 13311), Escherichia coli

(ATCC 35210), Enterobacter

cloacae (human isolate)

– – Glamočlija et al. (2015)

Anfungal acvity Aspergillus fumigatus (human

isolate), Aspergillus versicolor

(ATCC 11730), Aspergillus

ochraceus (ATCC 12066),

Aspergillus niger (ATCC 6275),

Trichoderma viride (IAM

5061), Penicillium funiculosum

(ATCC 36839), Penicillium

ochrochloron (ATCC 9112)

Penicillium verrucosum var.

cyclopium (food isolate)

– –

Silver

nanoparcles of

water extract

Anbacterial acvity Escherichia coli, Proteus

mirabilis, Staphylococcus

epidermidis

Nagajyothi et al. (2014)

Table 1. Bioacvies of crude extracts of chaga (Inonotus obliquus) - (connued)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Crude extract Bioacvity Model IC50/EC50/LC50 values or

experimental dosage (ED)

Specic mechanism or

manifestaon Reference

Water extract Pro-adipocyte

dierenaon

3T3-L1 preadipocytes ED∼10, 25, 50, 100 μg/ml Acvated adipogenesis of 3T3-

L1 preadipocytes; increased TG

accumulaon; smulated gene

expression of CCAAT/enhancer-

binding protein α and PPARγ

during adipocyte dierenaon;

induced the expression of AP2,

LPL, and CD 36; increased the

expression of PPARγ and GLUT4

Joo et al. (2010)

Water extract Anhyperglycemic

acvity

3T3-L1 adipocytes ED∼100–2,000 μg/ml Increased both non-insulin-

smulated and insulin-smulated

glucose uptake; acvated PI

3-K and increased the Akt

phosphorylaon; increased mRNA

expression of lipogenic genes FAS;

increased the mRNA expression

of fay acid oxidaon genes

including CPT-1, AOX, and LCAD

Lee and Hyun (2014a)

HepG2 and C2C12 cells

incubated with the

condioned media from

3T3-L1 adipocyte cultures

–Increased the phosphorylaon

of AMPK

Subcellular membrane –Increased translocaon of

GLUT4 from cytoplasmic vesicles

to plasma membrane

Anhyperglycemic

eect

High fat-fed obese mice ED∼50 mg/kg BW/day

(oral administraon)

Improved insulin sensivity and

reduced adiposity; increased

mRNA expression of adiponecn

in epididymal adipose ssue;

increased the mRNA expression

of fay acid oxidave genes

(CPT-1, AOX, and PGC1α)

Chloroform

extract of

culvaon broth

An-hyperglycemic

acvity

Dipepdyl pepdase-4 assay ED∼200 μg/ml –Geng et al. (2013)

Dry material of

culvaon broth

An-hyperglycemic

and anoxidave

stress eects

Alloxan-induced type-

1 diabec mice

ED∼500 and 1,000

mg/kg BW/day (oral

administraon)

Decreased serum contents of FFA,

TC, TG and LDL-C; increased HDL-C,

insulin level and hepac glycogen

contents in liver; increased CAT, SOD

and GPx acvies, and decreased

MDA content in liver; restored the

damage of pancreac β-cells

Sun et al. (2008)

Table 1. Bioacvies of crude extracts of chaga (Inonotus obliquus) - (connued)

Journal of Food Bioactives | www.isn-jfb.com 19

Peng et al. Bioactive compounds and bioactive properties of chaga

Crude extract Bioacvity Model IC50/EC50/LC50 values or

experimental dosage (ED)

Specic mechanism or

manifestaon Reference

80 % Ethanol

extract of dry

material of

culture broth

An-hyperglycemic

and anoxidave

stress eects

Alloxan-induced type-

1 diabec mice

ED∼30 and 60 mg/kg BW/

day (oral administraon)

Decreased serum contents of FFA,

TC, TG and LDL-C; increased HDL-C,

insulin level and hepac glycogen

contents; increased CAT, SOD and

GPx acvies, and decreased

MDA content in liver; restored the

damage of pancreac β-cells

Xu et al. (2010a)

Ethyl acetate

extract

An-hyperglycemic

and anoxidave

stress eects

Alloxan-induced type-

1 diabec mice

ED∼500 mg/kg BW/day

(oral administraon)

Decreased serum contents of TC

and TG; increased serum HDL-C

and hepac glycogen contents;

increased GPx acvies, and

decreased MDA content in liver;

Lu et al. (2010)

Water extract An-hyperglycemic

eect

KK-Ay mice (Genecally

type-2 diabec mice)

ED∼100 and 300 mg/

kg (single dose, oral

administraon)

Reduced the blood glucose

and plasma insulin

Miura (2007)

Raw power An-

hyperglycemic and

hepatoprotecve

eect

Otsuka long-evans tokushima

fay rat (genecally diabec

rat oral administraon

ED∼50 g/kg BW/day Decreased serum contents of TC

and TG; reduced the serum ALT

level and liver fay accumulaon

Cha et al. (2006)

Ethanol extract Platelet aggregaon

inhibitory acvity

Human blood samples ED∼2.5 mg/ml –Hyun et al. (2006)

Water extract An-hypertension

eect

Stroke-prone spontaneously

hypertensive rats,

ED∼extracts of 0.03

g dry material/day

Decreased mean arterial pressure

and the rate of rise of mean arterial

pressure; decreased blood pressure

in the cross-seconal area of the

subendocardial cardiomyocytes;

increased the blood pressure in

the capillaries; decreased the

alkaline phosphatase and IL-6

expression in the capillaries;

lowered the HbA1c level

Koyama et al. (2006)

100% Ethanol An-hyperuricemia

eect

Potassium oxonate/

hypoxanthine-induced

hyperuricemic mice

ED∼30, 60, 120 mg/kg BW

(single oral administraon)

Suppressed xanthine oxidase acvity

in serum and liver; down-regulated

renal uric acid transporter 1

Yong et al. (2018)

50% Methanol

fracon of 100 %

ethanol extract

An-hyperuricemia

acvity

Xanthine oxidase

Inhibion assay

IC50∼20.5 µg/mL –Wold et al. (2020)

80% Methanol

extract

An-hyperuricemia

acvity

Xanthine oxidase

inhibitory assay

IC50∼34.37 µg/mL –Szychowski et al. (2018)

Table 1. Bioacvies of crude extracts of chaga (Inonotus obliquus) - (connued)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Crude extract Bioacvity Model IC50/EC50/LC50 values or

experimental dosage (ED)

Specic mechanism or

manifestaon Reference

80% Ethanol

extract

An-obesity and

probioc eects

High-fat diet fed C57BL6/J mice ED∼500 mg/kg BW per day Improved the obesity of mice,

including the adjustment of body

weight gain, energy intake, energy

eciency, liver glucose metabolism

and triglyceride metabolism,

tricarboxylic acid (TCA) cycle, and

degradaon of three major nutrients

(carbohydrate, lipid, and protein);

Increased cecal propionate based

on Bacteroides and Akkermansia,

thereby inhibing energy intake

and fat accumulaon in mice

Yu et al. (2020)

Cases related to paents/healthy volunteers

Raw power An-hyperglycemic

eect

Type-2 diabec paents ED∼100 mg (single dose,

oral administraon)

Decreased the postprandial peak

glucose, PPGE, AUC glucose;

improved the postprandial

endothelial dysfuncon

Maenaka et al. (2008)

Food product

containing

chaga extract

An-hypertension

eect

Healthy adults ED∼5 ml for two mes

or single dose of 15

ml/person/day

Lowered systolic blood pressure

and diastolic blood pressure

Yonei et al. (2007)

An-oxidave

stress eect

Suppressed lipid peroxide

Adverse eect Frequent micturion and

increased sweang

Water extract An-virus eect HIV-infected paents – One succeeded, one failed Sakuma (2004)

Ethanol extract An-psoriasis eect psoriasis paents – – Frost (2016); Dosychev

and Bystrova (1973)

Medicinal

product

An-pepc

ulcers eect

pepc ulcer paents – – Frost (2016); Fedotov

and Rodsolaĭnen (1981)

DSS: dextran sulfate sodium; PPARγ: peroxisome proliferator-acvated receptors γ; AP2: adipocyte protein 2; LPL: lipoprotein lipase; CD36: fay acid translocase; MDCK cell: Madin-Darby Canine Kidney cell; CRFK cell: Crandell-

Reese feline kidney cell; FPV: feline panleukopenia virus; FIPV: feline infecous peritonis virus; FHV-1: feline herpesvirus 1; FCV: feline calicivirus; MMP: matrix metalloproteinase; IκBα: nuclear factor of kappa light polypepde

gene enhancer in B-cells inhibitor, alpha; BW: body weight; HFD: high-fat diet; STZ: streptozotocin; MMP: matrix metalloproteinase; MSPKs: mitogen-acvated protein kinases; PI3K: phosphoinoside 3-kinase; AKT: protein

kinase B; ERK: extracellular signalregulated protein kinase; JNK: c-Jun N-terminal kinase; P38: Cytokinin Specic Binding Protein (CSBP); MAPKs: mitogen-acvated protein kinases; NF-κB: nuclear factor κB; COX: cyclooxyge-

nase, STZ: streptozocin; MDA: maleic dialdehyde; TC: total cholesterol; TG: triglyceride; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; CAT: catalase; SOD: superoxide dismutase; GPx:

glutathione peroxidase; TBARS: thiobarbituric acid-reacve species; PPGE: postprandial plasma glucose excursion; AUC: area under the curve; HBDH: hydroxybutyrate dehydrogenase; LDH: lactate dehydrogenase; MDH: malate

dehydrogenase; GGT: gamma-glutamyl transferase; MNNG: N-methyl-N′-nitro-N-nitrosoguanidine; C/EBPα: CCAAT/enhancer-binding protein α); PPARγ: peroxisome proliferator-acvated receptors γ; GLUT4: glucose transporter

4; aP2: adipocyte protein 2; LPL: lipoprotein lipase; CD36: fay acid translocase; STAT: signal transducers and acvators of transcripon; IFN: interferon: COX: cyclooxygenase; IL: interleukin; Ig: immunoglobulin; ALT: alanine

aminotransferase; ACC: acetyl-CoA carboxylase; FAS: fay acid synthase; AOX: acyl CoA oxidase; CPT1: carnine palmitoyltransferase 1; PGC1-α: peroxisome proliferator-acvated receptor gamma coacvator 1-α; LCAD: long-

chain acyl-CoA dehydrogenase; PI 3-K: phosphoinoside 3-kinase; SREBP1-c: sterol-regulatory-element-binding protein 1c.

Table 1. Bioacvies of crude extracts of chaga (Inonotus obliquus) - (connued)

Journal of Food Bioactives | www.isn-jfb.com 21

Peng et al. Bioactive compounds and bioactive properties of chaga

of these two tumor/cancer models, which demonstrated that the anti-

inammatory eect might be a key mechanism in anti-cancer eect

of chaga extracts (Mishra et al., 2013). Furthermore, a successful cure

for triple-negative breast cancer of a 49 years old female patient by

combined use of chaga and Ganoderma lucidum has been reported

(Tiziana et al., 2020). Even during radiation therapy, the inamma-

tory markers of this patient were still signicantly reduced by admin-

istrating low dosages of chaga. On the other hand, Song et al. (2007)

thought that the anti-tumor eect of chaga was associated with its

immunomodulatory ability. In their study, chaga extract simulated the

in vitro immunomodulatory activity of mouse splenocytes but also

inhibited the pulmonary metastasis in CT-26 cell-inoculated BALB/c

mice (Song et al., 2007). This view was strongly supported by further

anti-tumor studies of chaga polysaccharide, as discussed in section

4.3. Hence, these two mechanisms may be involved in inhibiting tu-

mor progression in dierent stages which are due to dierent com-

pounds. Particularly, it is noteworthy that either short period (4-days)

oral administration (20 or 200 mg/kg BW/day; high/low doses,) or

short period (4-days)/low dose (10 mg/kg BW/day) intravenous ad-

ministration of water extract of chaga could signicantly inhibit pul-

monary metastasis in CT-26 inoculated mice. However, when mice

were treated for a long period (14-days) oral administration (20 or

200 mg/kg BW/day) or a short period (4-days)/high dose (100 mg/

kg BW/day) intravenous administration, their tumor metastasis was

signicantly stimulated (Song et al., 2007). This contradictory result

may imply the adverse eect of long-term/high-dose use of chaga, as

discussed in section 3.

2.2. An-inammatory eects

Inammation is a vital part of the immune system’s response to dam-

aged cells, pathogens, and irritants. During inammation, the cy-

tokines released by injured cells signal the damaged sites for the im-

mune system, which further helps to defend the body against foreign

invaders such as pathogens, irritants, and toxins. However, chronic

inammation can contribute to the development of diseases, espe-

cially cardiovascular disease and tumor progression (Coussens et al.,

2002; Pahwa et al., 2019). On the one hand, inammation promotes

the apoptosis of injured cells and tries to eliminate the cause of in-

ammation through activating immune cells to release pro-apoptotic

cytokines and free radicals (Haanen and Vermes, 1995). On the other

hand, to replace the necrotic tissue, it constantly stimulates the pro-

liferation of adjacent cells until repair is completed (Coussens et al.,

2002). The abnormal repetition of cell proliferation in microenviron-

ments rich in inammatory cells (e.g. dendritic cells, macrophages,

eosinophils, mast cells, and lymphocytes, but chiey neutrophils),

growth factors (e.g. platelet-derived growth factor, platelet-derived

angiogenesis factor (PDGF), transforming growth factor-α (TGF-α),

TGF-β and basic broblast growth factor), activated stroma (e.g. en-

dothelial cells, nerve cells, immune cells, and extracellular matrix),

and DNA-damage-promoting agents (e.g. UV light, gastric acids, sil-

ica, reactive oxygen/nitrogen species (ROS/RNS), alcohol, viruses,

parasites, and bacteria) potentiates the in vivo DNA damage-induced

mutations, in other words, neoplastic risk (Coussens et al., 2002;

Kiraly et al., 2015). Therefore, prevention of chronic inammation

may be regarded as an anti-cancer therapeutic opportunity. There

are numerous herb/food products containing functional components

with proven excellent anti-inammatory properties, one of them be-

ing chaga (Azab et al., 2016; Muszyńska et al., 2018). The aqueous

alcohol extracts of chaga can eectively inhibit inammation by

lowering NO (nitrite oxide) production in LPS (lipopolysaccharide)-

induced RAW 264.7 murine macrophage (Ma et al., 2013; Park et

al., 2005b; Van et al., 2009). The NO inhibition ability of methanol

or 80% ethanolic extract of chaga at 50 μg/ml is close to celastrol

at 25 μg/ml but better than that of L-N6-(1-iminoethyl) lysine at 10

μM (Ma et al., 2013; Van et al., 2009). Besides, in an in vitro in-

ammation model, dierent inammation signaling proteins such

as MAPKs (mitogen-activated protein kinases), ILs (interleukins),

STATs (signal transducer and activator of transcription proteins),

IFN-γ (interferons), NF-κB (nuclear factor kappa-light-chain-en-

hancer of activated B cells), and TNF (tumor necrosis factor) were

modulated by chaga extracts. Luciferase has been used as a measure

of the activation (high uorescence incidence) or inhibition (low

uorescence incidence) of NF-κB. A cell line stably expressing lu-

ciferase reporter gene under the transcriptional control of the NF-κB

response element, known as NF-kB luciferase reporter cell line, is

widely used for screening signaling activators or inhibitors related

to TLR (toll-like receptors) signaling pathways and activation of the

transcription factor NF-κB in pharmaceutical studies (Battin et al.,

2017). Ma et al. (2013) reported that 70% ethanolic extract of chaga

inhibited the activation of NF-κB-dependent luciferase in (lucif-

erase reporter gene) stably transfected RAW264.7 cells. Meanwhile

in LPS-induced RAW 264.7 inammation model, the 80% alcohol

extract (100 μg/ml) exhibited a similar or higher inhibition activ-

ity of pro-inammatory factors compared with salicin (500 μg/ml),

which down-regulated expression of IL-6, TNF-α, iNOS, COX-2

and inhibited the phosphorylation of IκB-α, Akt, and MAPKs (JNK,

p38, ERK) (Van et al., 2009). In the same model, pure methanolic

extract and water extract of chaga not only decreased the production

of PEG2, STAT1, pSTAT1, STAT6, and pSTAT6 but also suppressed

the degradation of cytosol IκB-α and the protein/mRNA levels of

TNF-α, iNOS, COX-2, NF-κB (p65/p50), and nuclear p65 (Choi et

al., 2010; Park et al., 2005b). Most recently, 50% methanolic and

water extracts of chaga were found to inhibit TNF-α production in

either LPS- or histamine-induced RAW 264.7 cells. Meanwhile,

simultaneous treatment of 50% methanolic extract and histamine

could attenuate histamine-induced microvascular inammation by

reversing the reduction of conducted vasodilation of second-order

arterioles in the gluteus maximus muscle of C57BL/6 mice (Javed et

al., 2019). Furthermore, anti-inammatory eects have been further

veried in in vivo inammation models. Park et al. (2005b) exam-

ined the anti-inammatory eect of a methanolic extract of chaga

in a carrageenin-induced mouse edema model. They found that this

extract exhibited a preventative inhibitory eect on inhibiting carra-

geenin-induced edema for 2–4 h if it was administered orally for 7

consecutive days prior to injecting carrageenin, even if eectiveness

of extract (100/200 mg/kg) was much lower than that of the positive

control (ibuprofen, 100 mg/kg). In addition, in DSS (dextran sul-

fate sodium)-induced mouse acute colitis model, oral administration

of water extract of chaga after inducing colitis maintained the liver

weight, it decreased the serum level of IgE, decreased the expression

of TNF-α, IFN-γ, IL-4, STAT6, and STAT1 proteins in the spleen

(Choi et al., 2010). Moreover, in another DSS-induced mouse acute

colitis model, both preventative and therapeutic treatment of water

extract of chaga suppressed edema, mucosal damage, and the loss of

crypts, inhibited iNOS levels and myeloperoxidase accumulation,

and suppressed mRNA overexpression of TNF-α, IFN-γ, IL-1β, and

IL-6 induced by DSS in colon tissues (Mishra et al., 2012).

2.3. Anoxidant eects

In aerobic organisms, oxygen consumption is essential for ecient

energy metabolism but, paradoxically, produces ROS (reactive ox-

ygen species) and free radicals(Reuter et al., 2010). The detrimen-

tal environmental factors, including radiation and toxins as well as

adverse physiological/psychological status such as tension, sleep

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Bioactive compounds and bioactive properties of chaga Peng et al.

deprivation, hyperglycemia, and obesity, can excessively induce free

radicals. The overload of free radicals leads to chronic inammatory

reactions and molecular damage in cells, which then progresses to a

broad spectrum of diseases, especially type-1/2 diabetes and cancers

(Hapuarachchi et al., 2003; Limón-Pacheco and Gonsebatt, 2009;

Tsuboi et al., 2008; Zhang et al., 2013a). Thus, endogenous antioxi-

dant enzymes such as CAT (catalase), SOD (superoxide dismutase),

GPx (glutathione peroxidase), thioredoxin and endogenous/exog-

enous antioxidants such as GSH (glutathione), ascorbic acid, uric

acid, tocopherols, bilirubin, phenolics play crucial roles in prevent-

ing in vivo free radical-induced oxidative damage. Similar to other

medicinal mushrooms and plant-based herbs, polar-solvent extracts

of chaga were found to exert intense antioxidant activity. Various

antioxidant activities of water/alcohol extracts of chaga have been

evaluated in DPPH (2,2-diphenyl-1-picrylhydrazyl), FRAP (ferric

reducing antioxidant power), superoxide/hydroxyl radical scaveng-

ing, TBARS (thiobarbituric acid reactive substances) formation inhi-

bition, and β-carotene bleaching assays (Cui et al., 2005; Glamočlija

et al., 2015; Liang et al., 2009). The eectiveness of these extracts in

scavenging free radicals and reducing transition metal ions is close

to that of ascorbic acid at the same concentration (Cui et al., 2005).

These antioxidant abilities of chaga mushroom provides the chemi-

cal basis in preventing oxidative stress and damage. For instance,

water and ethanolic extracts of chaga protected H2O2-treated human

lymphocyte from DNA damage (Najafzadeh et al., 2007; Park et al.,

2005a; Park et al., 2004). Besides, water extracts of chaga also pre-

vented H2O2-induced apoptosis and premature senescence in human

broblasts, it functioned through scavenging intracellular ROS (reac-

tive oxygen species), preventing lipid peroxidation, and increasing

collagen synthesis through inhibition of MMP-1 and MMP-9 activi-

ties (Yun et al., 2011). Recently, Szychowski et al. (2018) found that

chaga extract enhanced the antioxidative stress ability of normal cells

but induced oxidative stress to cancer cells. Thus, treatment of 1 mg/

ml ethanolic extract on BJ normal human skin broblast induced in-

crease of SOD1, CAT and KI67 mRNA expression along with de-

crease of ROS production. However, the same dose gave opposite

results in Caco-2 human colon cancer cells, decreased SOD1, CAT

and KI67 mRNA expression and increased the ROS production (Szy-

chowski et al., 2018). Apart from the anti-inammation and antioxi-

dant activities, the antimutagenic activity of chaga could also attenu-

ate cancer initiation and progression processes (Chung et al., 2010).

The data of Park et al. (2005a) and Ham et al. (2003) support the

protective eects of chaga extracts against oxidative DNA damage in

H2O2-treated human lymphocytes and MNNG-induced genotoxicity

in mice. In another study, Ham et al. (2009) later found that two sub-

fractions of methanolic extract mainly contained 3β-hydroxylanosta-

8,24-dien-21-al and inotodiol, respectively; these strongly inhibited

the mutagenesis of Salmonella typhimurium strain TA100 induced

by the directly acting mutagen MNNG (N-methyl-N-nitro-N-nitro-

soguanidine) by 77.3–80.0%. At the same concentration, they also

inhibited another directly acting mutagens 4NQO (D-biotin, 4-nit-

roquinoline-1-oxide)-induced mutations in Salmonella typhimurium

strain TA98 and TA100 by 52.6–62.0%. Besides, the mutagenesis in

strain TA98 induced by the indirectly acting mutagens Trp-P-1 (tryp-

tophan-P-1) and B(a)P (benzo[a]pyrene) was reduced by 47.0–68.2%

by these subfractions, while the mutagenesis in TA100 induced by

Trp-P-1 and B(α)P was reduced by 70.5–87.2%.

2.4. An-diabec eects

As emphasized in the most recent WHO (2017) statistics report,

8.5% of global adults had diabetes which resulted in an estimated 1.6

million deaths in 2016. In 2015 in Canada, diabetes and prediabetes

rates were 9.3% (3.4 million) and 22.1% (5.7 million), respectively

(Houlden, 2018). These data in 2018 in the United State of America

were around 10.4% (34.2 million) and 26.8% (88 million), respec-

tively (Centers-for-Disease-Control-Prevention, 2020). One of the

most direct results of pre-diabetes and diabetes is hyperglycemia.

Without treatment, hyperglycemia can further cause severe compli-

cations, including ketoacidosis, infection (immune dysfunction), and

various tissue/organ damage. Chaga and its extracts showed an out-

standing anti-hyperglycemic eect in both in vivo type-1 and type-2

diabetic models. The clinical data of Maenaka et al. (2008) showed

that prior use of chaga improved postprandial endothelial dysfunc-

tion and various indicators of blood sugar in type-2 diabetic patients.

Besides, in genetically type-2 diabetes KK-Ay mice, either a single

or repeated 6 weeks oral administration of water extract of chaga

could signicantly reduce blood glucose, as well as plasma insulin,

which demonstrate that chaga extract could alleviate insulin resist-

ance (Miura, 2007). In addition, hypoglycemic eects of chaga were

conrmed in a type-1 diabetic model. Sun et al. (2008) and Xu et

al. (2010a) reported that 2-weeks oral administration of cultured or

wild chaga extracts could decrease mice serum contents of FFA (free

fatty acids), TC (total cholesterol), TAG (triacylglycerols), LDL-C

(low-density lipoprotein-cholesterol), and liver MDA (malondialde-

hyde) content in alloxan-induced diabetes models. Meanwhile, the

treatment also increased mice HDL-C (high-density lipoprotein-

cholesterol), insulin level, and hepatic glycogen contents as well as

CAT, SOD and GPx activities in the liver (Sun et al., 2008; Xu et

al., 2010a). The histopathological examination of these mice showed

that the damage to pancreatic β-cells was restored in the treated dia-

betic mice compared to the untreated group, in other words chaga

stimulated regeneration of the β-cells and thus normalized the level

of insulin (Sun et al., 2008; Xu et al., 2010a). Later, other potential

mechanisms on regulating insulin and blood lipid levels by using

chaga were found. For example, alkaloids and terpenoids isolated

from chaga extract were found eective in inhibiting the DPP-4 (di-

peptidyl peptidase 4), an important enzyme and as a new therapeutic

target for diabetes (Geng et al., 2013). The dierentiation of 3T3-L1

preadipocytes was induced by chaga extract via signaling pathway

of C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ (per-

oxisome proliferator-activated receptors γ) (Joo et al., 2010). Wa-

ter extract of chaga also increased both non-insulin-stimulated and

insulin-stimulated glucose uptake of 3T3-L1 adipocytes through

activating PI 3-K (phosphoinositide 3-kinase) and phosphorylation

of its downstream protein the Akt, and increasing mRNA expression

of lipogenic genes FAS (fatty acid synthase) and fatty acid oxidation

genes including CPT-1 (carnitine palmitoyltransferase 1), AOX (acyl

CoA oxidase), and LCAD (long-chain acyl-CoA dehydrogenase)

(Lee and Hyun, 2014a). A similar result was conrmed through high

fat-fed obese mice, the oral administration of water extract of chaga

at a dose of 50 mg/kg BW/day improved insulin sensitivity and re-

duced adiposity with increasing mRNA expression of adiponectin

and fatty acid oxidative genes including CPT-1, AOX, PGC1α (per-

oxisome proliferator-activated receptor gamma coactivator 1-α) in

epididymal adipose tissue (Lee and Hyun, 2014a).

2.5. Other health eects and their potenal relevance with

chemistry of chaga extracts

Beyond the health eects mentioned above, other bioactivity stud-

ies have been carried out as summarized in Table 1. The chaga

extract exhibited a broad-spectrum of antiviral, anti-bacterial and

anti-fungal activities in various in vitro trials (Glamočlija et al.,

2015; Shibnev et al., 2015; Shibnev et al., 2011). The ethanolic

extract of chaga showed platelet aggregation inhibitory activity

Journal of Food Bioactives | www.isn-jfb.com 23

Peng et al. Bioactive compounds and bioactive properties of chaga

in whole blood and platelet-rich plasma, from which Hyun et al.

(2006) isolated a novel tripeptide and conrmed its anti-aggrega-

tion eect in mice. In addition, the alcohol extracts showed anti-

hyperuricemic eect by inhibiting xanthine oxidase in both in vitro

and in vivo trials (Szychowski et al., 2018; Wold et al., 2020; Yong

et al., 2018). Yonei et al. (2007) published a clinical study about

chaga which veried several health claims of foods containing

chaga by a double-blind trial. The parameters including systolic/

diastolic blood pressure, lipid peroxide, and the mental/physical

symptoms such as “cold skin” and “inability to sleep because of

worries” were signicantly improved. However, several adverse

eects were also found (see section 3).

Normally extraction means concentrating certain groups of

functional ingredients from a specic material. Compared to the

hypoglycemic ecacy of the materials used in the studies of Sun

et al. (2008) and Xu et al. (2010a), 80% ethanolic extract of the

cultured broth of chaga was almost 100-fold more ecient than the

simple cultured broth of chaga. Regarding dierent extraction and

preliminary purication approaches, variations exist in the compo-

sition of extracts. The dry chaga contains around 2–2.76% protein,

0.04–6.0% phenolics, 11.63–15% ash, 0.51–8% terpenoids, 0.2–2%

melanin, 2.76% lipid, 25–37.56% lignin, 2% cellulose, and 12.5%

hemicellulose (Glamočlija et al., 2015; Ju et al., 2010; Kim et al.,

2008b; Koyama et al., 2008; Rhee et al., 2008; Shashkina et al.,

2006; Si, 2018). Regardless of the actual proportion of various com-

pounds in chaga, the main bioactive components in various chaga

extracts are polysaccharides, terpenoids, phenolics/lignin, melanin,

peptides/protein, and their covalent complexes; some compounds

such as alkaloids have also been reported. The data of Mishra et

al. (2012) revealed signicant anti-inammation ability of water

extract (40 °C, 3 h) of chaga which contained 57, 204, 127 μg/mg

of phenolics, polysaccharides, and protein, respectively. In another

comparative study, the water extract prepared by 2 h process at 80

°C showed the presence of 247.5 μg/mg extract of polysaccharides

and 136.9 μg/mg extract of protein, while these were not detected

in the pure-ethanolic extract (Hu et al., 2009). The latter, however,

possessed a much stronger pro-apoptotic eect on human colorectal

cancer cell line DLD-1 in a time-dependent manner. The presence

of higher concentrations of terpenes and/or phenolics is regarded

as being the contributor. Along with the anti-proliferation ability,

simlilar comparative data between water and organic solvent ex-

tracts also corresponded with their in vitro anti-inammatory and

enzyme inhibition activity (Baek et al., 2018; Nomura et al., 2008;

Van et al., 2009; Wold et al., 2020). The presence of a high amount

of terpenoids and sometimes even alkaloids in organic-solvent ex-

tracts was deemed as the main cause (Baek et al., 2018; Geng et al.,

2013; Ma et al., 2013; Nomura et al., 2008; Wold et al., 2020). On

the other hand, the crude polysaacharide fraction (water fraction)

of 80% ethanolic extract of chaga was found to render stronger

anti-inammatory activity than its crude phenolic/terpene fraction

(ethyl acetate fraction) (Van et al., 2009). Meanwhile, Lee et al.

(2009) showed that anti-proliferation activity of the 70% ethanolic

extract on HT-29 cells was signicantly lower than that of the water

extract. Hyun et al. (2006) screened the anti-platelet aggregation

activity of water/ethanol extracts from nine chaga samples. The en-

thanolic extract of one sample showed the highest platelet aggrega-

tion inhibitory activity compared to the other ethanol/water extracts

but platelet aggregation inhibitory activity of water extracts was

found in more samples. The platelet aggregation inhibitory activ-

ity was eventually attributed to a tripeptide isolate (Trp-Gly-Cys).

In short, the exact ecacies of biactivities of chaga extracts varied

with dierent samples employed. Meanwhile, the combined eects

of dierent pure compounds also needs to be considered although

certain compounds may mainly contribute to some specic health

eects. To verify the exact contributors and the specic mechanism

of these bioactivity dierences, further studies of the bioactivity of

isolated pure compounds from the extracts are necessary, as dis-

cussed further in section 4.

3. Safety of chaga products and oxalate-associated side eects

of chaga decocon

Based on their long folk therapy history, the use of chaga and its

products is generally deemed safe. Although clinical or animal

studies have not suciently investigated the acute toxicity, subtox-

icity, or chronic toxicity of chaga crude extracts, some preliminary

studies have incidentally assessed their toxicity/safety in in vitro

cellular assays and murine animal trials. In terms of cellular test,

the ethanol and water extracts of chaga were only toxic at concen-

trations of 100 and 400 μg/ml, respectively, to human HaCaT ke-

ratinocytes (Cui et al., 2005). Similarly, normal Chang-liver cells

and primary porcine liver cells PLP2 were not markedly aected

by alcohol and/or water extracts of chaga at a concentration of less

than 400 μg/ml (Glamočlija et al., 2015; Youn et al., 2008). There

are also studies that show the general cytotoxicity in both the nor-

mal and cancer cell lines. Song et al. (2007) reported that the water

extract of chaga at high concentrations of over 100 μg/ml inhibited

the viability of HT1080, Hep G2, CT-26 as well as CRL-7250 nor-

mal human broblast after a 6-days culture (much longer than the

treating duration in other studies). Nakajima et al. (2009) found the

water extract of chaga was more toxic on IMR90 normal human

lung cells (IC50/LD50∼18.7–29.8 µg/ml) than on cancer cell lines

(A549, PA-1, U937, and HL-60, IC50/LD50∼23.2–105.2 µg/ml).

As for in vivo trials, the pro-tumor eect as well as toxic appear-

ance in liver to the naked eye in the CT-26 cells-inoculated mice

induced by intravenous administration of water extract of chaga

were noticed (Song et al., 2007). In the case of non-intravenous ad-

ministration, Park et al. (2005b) did not nd any toxic syndromes

based on the body weight change of male Sprague-Dawley rats

which were orally administrated 100 or 200 mg/kg body weight of

chaga methanolic extract for 7 consecutive days. There were also

no life-threatening toxic eect and body weight loss in the mice

administrated 30 mg/kg/day, intraperitoneally, or 300 mg/kg/day,

orally, of the extract for 60 consecutive days (Kim et al., 2006). A

single dosage of ethanol extract of chaga at 30–120 mg/kg body

weight had no toxic impact on kidney and liver functions of male

SPF Kunming mice (Yong et al., 2018). Another anti-tumor study

on pathogen-free female ICR mice showed that 20-weeks consec-

utive external use of chaga-origin inotodiol had no inuence on

their body weight (Nakata et al., 2007). The review of Koyama et

al. (2008) reported that oral administration of dried raw chaga at 1

g/day for 2–3 weeks did not cause any problem in human subjects.

However, other reports indicated the side eects, including di-

etary hyperoxaluria, oxalate-induced acute/chronic nephropathy,

and liver injury, upon oral administration of chaga over a moder-

ate/long-term and high dose use (Kim et al., 2005; Lee et al., 2020;

Lumlertgul et al., 2018; Maenaka et al., 2008; Yonei et al., 2007).

The most recent clinical case came from the emergency room of a

Korean hospital in 2016. A 49 years-old male without any family

medical history and history of kidney stone, diabetes, hyperten-

sion, and operation, was conrmed with kidney failure (oxalate

nephropathy) and eventually underwent kidney transplantation

after 18-months maintenance with hemodialysis. His regular ex-

amination result of renal function and urine analysis were both

normal until hospitalization. After looking into his drug history,

the kidney failure caused by oxalate nephropathy was associated

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Bioactive compounds and bioactive properties of chaga Peng et al.

with his 5-years continuous use of chaga powder (for treating at-

opic dermatitis) (Lee et al., 2020). The dosage he took was 3 g/day

(two times/day) in the rst 4 years and 9 g/day in the fth year.

Back to 2014, a 72-year-old Japanese female was diagnosed with

liver cancer and had to undergo hepatectomy after 15 months. For

alleviating the cancer, she ingested chaga powder (4–5 teaspoons/

day) from the sixth month to the twelfth month after diagnosis, but

eventually turned to be oxalate nephropathy with detectable oxa-

late crystals in her kidney tubules and urinary sediment (Kikuchi

et al., 2014). As early as 2007 in Japan, a double-blind study of

chaga food product using 60 healthy human volunteers showed un-

favorable eects including frequent urination and increased sweat-

ing after oral administration at doses of 5 or 15 ml/person/day for

8 weeks even if no specic attention was paid to the concentration

of blood/urine oxalate (Yonei et al., 2007). The potential cause of

adverse results in these cases was thought to be related to the ex-

tremely high quantity of oxalic acid in chaga. Lee et al. (2020)

reported a 14.2% oxalate (0.142 g oxalate/g chaga) in chaga pow-

der. Glamočlija et al. (2015) reported oxalic acid content of chaga

water extracts at 3.29% (Thailand), 5.57% (Finland), and 9.76%

(Russia), while 70% ethanolic extracts possessed a lower percent-

age at 0.67% (Thailand), 0.95% (Finland), 2.42% (Russia). It is

worth noting that less than 100 mg of oxalate daily is considered

safe for preventing kidney stone even if typical diets contain 200

to 300 mg of oxalate daily, and the daily oxalate intake of patient

(9g×3–14%) in the rst case is close to its lethal dose of 2–30 g/

day (Lee et al., 2020). On the other hand, there is no related study

about the oxalate levels in cultured chaga materials.

The above cases should make people consider the susceptibility

of Asians to chaga-origin oxalate nephropathy because potential ra-

cial dierence in handling dietary oxalate truly exists (Lewandowski

et al., 2001; Lewandowski et al., 2005). However, around 12 other

chaga-related cases including two nephropathy cases have also been

noted by British Columbia, Drug and Poison Information Centre

(BC-DPIC), as reported by Toxicology Committee chair of NAMA

(North American Mycological Association) (BC-DPIC, 2016; Beug,

2019; Takikawa, 2006). In the nephropathy case of BC-DPIC, hep-

atitis as well as renal failure happened in patient at the same time

and dialysis was still required on last follow-up 2 months later but

fortunately the patient was recovered. Another case is an unocial

personal narrative from NAMA, the patient was a regular chaga user

(a cup of chaga decoction daily) for over 10 years, nothing wrong

happened to him until the resumption of using chaga after a prostate

surgery. Then he had quite heavy hematuria, followed by excruciat-

ingly painful bladder spasms which was suspected to be due to us-

ing chaga, even 3 weeks post surgery (Beug, 2019). Therefore, even

though the content of oxalic acid in chaga and excretion capacity of

absorbed oxalate is circumstantial, long-term administration of chaga

or its decoctions/tincture will undoubtedly increase the plasma con-

centration of oxalate and corresponding risk of oxalate nephropathy.

In addition, liver damage induced by the arbitrary use of tradi-

tional herbs as well as chaga has become a worldwide medical issue

(Douros et al., 2016; Jing and Teschke, 2018; Lee et al., 2015b; Lin

et al., 2019; Takikawa, 2006). It is understandable that conducting

expensive clinical studies are impractical for every herb especially

for niche market products. However, specic safe guideline and

healthy limitation for the use of non-mainstream herbal products

should be followed. Regardless of clinical studies, the scientic

basis of the safety assumption including acute/chronic animal tri-

als and subsequent blood/urine/histoanatomy analysis is reasonable

to be requested before their commercialization. Furthermore, suf-

cient chemical analysis not only helps demonstrating the bioactive

sources of natural herb products but would also reveal their risk fac-

tors before tragedies happen to vulnerable individuals. Sometimes

the so-called bioactive compound is the risk itself as it is the dose

that makes the poison. Meanwhile, the safety and chemical compo-

sition of wild mushroom supplements are largely inuenced by their

nutritional host. In the following section, a retrospect of the known

organic constituents especially the bioactive compounds of chaga

and their potential safety concerns are discussed.

4. Main bioacves/medicinal constuents of chaga and their

bioacvies

4.1. Terpenoids

Based on numerous comparative studies of the structure-function

relationship of dierent components in chaga, it was found that the

anti-cancer eect of chaga extracts is remarkably inuenced by their

content of terpenoid/terpene derivatives (Kim et al., 2011; Liu et al.,

2014; Zhao et al., 2016a; Zheng et al., 2011b). Terpenoid/terpene de-

rivatives are a major class of chemical compounds found in natural

plants which normally function as signaling chemicals (e.g. gibber-

ellin and abscisic acid), attractants (e.g. carotenoids, caryophyllene,

limonene), repellents (e.g. linalool, farnesene), as well as crucial

structural components of biomembranes (e.g. phytosterols) (Sharma

et al., 2017; Theis and Lerdau, 2003). Terpenes are a plentiful and di-

verse group of hydrocarbon compounds categorized by their number

of isoprene units and include hemiterpene, monoterpene, sesquiter-

pene, and diterpene, among others. Even if the mixed-use of ter-

penoids and terpenes is common, the term “terpenoids” is dierent

from “terpenes”, the latter compounds are simply unsaturated hy-

drocarbons polymerized by isoprene units while the former belongs

to terpene derivatives structured with various elements or functional

groups such as oxygenated and nitrogenated branches. According to

the number of cyclic structures, the triterpenoids can be divided into

linear triterpenoids (squalene), monocyclic triterpenoids (e.g. achil-

leol A and camelliol C), bicyclic triterpenoids (e.g. myrrhanol C and

myrrhanone A), tricyclic triterpenoids (e.g. arabidiol and achilleol

B), pentacyclic triterpenoids (ceanothanolic and rosamultic acid),

as well as the two most common categories in the study of chaga

terpenoids, namely tetracyclic triterpenoids and steroids (Daniel

and Mammen, 2016; Grishko et al., 2015; Kimura et al., 2001; Per-

veen, 2018; Xiang et al., 2006; Xu et al., 2018). The steroids and

tetracyclic triterpenoids both contain four cycloalkane rings joined

mutually, therefore it is sometimes dicult to conceptually sepa-

rate them from each other. Some structural characteristics such as

methyl groups on the C-4 and 14 positions may help to distinguish

some steroids from terpenoids (Tong, 2013). Biosynthetic routes can

also help to dierentiate them. The tetracyclic triterpenoids derivate

from 2,3-oxidosqualene or/and squalene involving various syn-

thetic reactions such as hydroxylation, cyclization, hydrogenation/

dehydrogenation, epoxidation/peroxidation, and hydride/methyl

shift (Rascon-Valenzuela et al., 2017). Then the produced lanosterol

(animals/yeast) or cycloartenol (plants) can further be metabolized

into steroids. The derivation of steroids involves demethylation,

ketonization, and hydrogenation/dehydrogenation (Bishop and

Yokota, 2001). Therefore, some tetracyclic triterpenoids, including

lanosterol and cycloartenol, can also be classied as steroids. Figure

1 displays various core skeletons of tetracyclic and pentacyclic trit-

erpenoids (Bishop and Yokota, 2001; Biswas and Dwivedi, 2019;

Hamid et al., 2015; Rascon-Valenzuela et al., 2017; Stanczyk,

2009; Xiao et al., 2018). The lanostane-type terpenoids are main

triterpenoids/steroids isolated from mushrooms which also apply

to terpenoid composition of chaga. As summarized in Table 2, 57

out of 108 known triterpenoids/steroids are lanostane-type tetracy-

Journal of Food Bioactives | www.isn-jfb.com 25

Peng et al. Bioactive compounds and bioactive properties of chaga

clic terpenoids/steroids. Some triterpenoids such as fuscoporianols

A-D, inoterpene A-F, inonotsuoxodiol A, spiroinonotsuoxodiol,

inonotsudiol A, inonotusane A-G, inotolactone A-C, inonotsuox-

ide A and B, inonotsutriol A-E, fuscoporianol A-C, obliquic acid,

and inotodiol are exclusive and can only be found in chaga (Figure

2). The isolation and identication methods of these compounds

are also briey given in Table 2. Moreover, the review of Nikitina

et al. (2016), which summarized the original Russian articles, may

provide dierent structural information about chaga terpenoids

from those given in this contribution that is built upon using the

English source.

Lanostane-type terpenoids are well known for their potential

in cancer treatment (Duru and Çayan, 2015). As Table 3 summa-

rizes, numerous in vitro anti-proliferation studies of lanostane-type

terpenoids isolated from chaga extracts have been published. In

this table, only the results with signicant inhibitory ability at the

experimental dosage (ED) employed or the results with IC50 (half

maximal inhibitory concentration) less than 40 μM are shown.

For example, the ergosterol peroxide puried from chaga exerted

moderate-high cytotoxicity on various cancer cell lines such as

PC3, MDA-MB-231, A549, L1210, HepG2, MCF-7, HCT116,

HT-29, SW620, DLD-1 cells (Kang et al., 2015; Kim et al., 2011;

Ma et al., 2013). In HT-29 and HCT116 colorectal cancer cell

models, ergosterol peroxide could induce subG1 arresting, inhibit-

ing the nuclear levels of of β-catenin, and ultimately resulting in

reduced transcription of c-Myc, cyclin D1, and CDK-8 (Kang et

al., 2015). The inotodiol also showed cytotoxicity on many cancer

cell lines including L1210, A549, P388, AGS, MCF-7, and Hela

cells (Chung et al., 2010; Nomura et al., 2008; Tanaka et al., 2011;

Zhong et al., 2011). In A549 lung cancer cell model, inotodiol ar-

rested cell cycle in S phase, decreased expression of Ki-67 and

Bcl-2 proteins, and increased expression of p53 and bax proteins

(Zhong et al., 2011). Furthermore, the in vivo anti-cancer eect

of chaga terpenoids has been conrmed in animal trials. Taking

ergosterol peroxide (isolated from chaga), as an example, the oral

administration of ergosterol peroxide at 15 mg/kg body weight/12

h for 8 or 14 weeks helped maintaining colonic epithelial cell

structures, improving histological damage in response to AOM/

DSS, and suppressing tumor growth in the colon colorectal cancer

in mice (Kang et al., 2015). More in vivo anti-cancer trials can be

found in the studies of chaga-origin triterpenoids such as lanoster-

ol, inotodiol, and 3β-hydroxylanos-8,24-dien-21-al (Table 3). Oth-

er bioactivities of chaga-origin terpenoids including α-glucosidase

inhibitory activity, EBV-EA activation inhibitory activity, PTKs

(protein tyrosine kinases) inhibitory activity, hepatoprotective ac-

tivity, antioxidant, and anti-inammatory activity have also been

reported (Table 3). Intriguingly, chaga ethanolic extract was found

to have signicant in vivo anti-hyperuricemic eect, and the trit-

erpenoids such as 3β-hydroxylanosta-7,9(11),24-trien-21-oic acid,

inonotusic acid, trametenolic acid, and betulin were considered as

the main contributors due to their ecient xanthine oxidase in-

hibitory activity (Yong et al., 2018). However, this standpoint was

challenged later due to the failed repetition in the study of Wold et

al. (2020) who suggested the non-terpenoid compounds inhibit the

xanthine oxidase activity. This property was therefore not included

in Table 3. Besides, a quantity of medicinal potential of common

triterpenoids such as ergosterol peroxide, β-sitosterol, betulin-

ic acid, and oleanolic acid, which are extractable from not only

chaga but also various other fungi/plants, have been prevalently

reported (Chhikara et al., 2018; Merdivan and Lindequist, 2017;

Moghaddam et al., 2012; Yogeeswari and Sriram, 2005). Such

bioactivity studies may provide additional information in inves-

tigating pharmaceutical properties of chaga products. Apart from

the various bioactivities published, the terpenoids also attract the

toxicity concerns. Some terpenoids are known to cause detrimental

eects on skin, digestive tract and even central nervous system

with various adverse syndrome such as irritation, gastrointestinal

disorders, hallucination, seizure, and coma (Mbaveng et al., 2014).

However, to date, there is no specic toxicity studies on the unique

terpenoids of chaga.

The growth rate of wild chaga is extremely slow. To satisfy the

increased commercial requirement of chaga products, the articial

culture of chaga has attracted much attention. However, the di-

versity and content of chaga terpenoids are quite distinct between

wild and cultured types. For example, instead of the two dominant

sterols in wild chaga, namely lanosterol and inotodiol, ergosterol

becomes the main sterol in the cultured mycelium. Meanwhile,

other trace sterols in wild chaga such as episterol, 24-methylene

dihydrolanosterol, and ergosterol peroxide can not be found in

cultured mycelium (Zheng et al., 2007a). A similar phenonmenon

was found for the terpenoids composition of chaga among dierent

wild types. Géry et al. (2018a) compared chaga samples collected

from Canada, Ukraine, and France. The betulin and betulinic acid

contents of French chaga were almost 100-fold and 10-fold higher

than the Canadian/Ukrainian ones, respectively. Furthermore, the

collected raw materials of wild chaga were sometimes divided into

sclerotium and fruits/mycelia parts. The simliarity or dierence of

the composition of these two parts have frequently been reported

but are beyond the scope of this review and hence are not described

here (Kim et al., 2005; Song et al., 2008; Sun et al., 2011). These

results directly implicate that the growth environment is one of

the critical factors determining the composition and proportion of

chaga terpenoids. Optimizing the nutritional condition of articial

medium, including pH, the concentration of minerals, carbon, and

nitrogen sources, or even nitrogen to oxygen ratio, could eec-

tively enhance terpenoids’ production such as betulin, inotodiol

and betulinic acid in cultured chaga (Bai et al., 2012; Chen et al.,

2020a; Wei et al., 2018). Meanwhile, supplementing Ag+, Cu2+

and Ca2+ could stimulate accumulation of lanosterol and ergosterol

(Zheng et al., 2008a). Adding methyl jasmonate or linoleic acid

could enhance more than 50 % of the total triterpenoid production

as well as its phenolic content and diversity (Xu et al., 2015b; Xu

et al., 2016a). Besides, cultivating the mycelium with the aqueous

extracts or methanolic extracts of birch bark, birch core or chitosan

could signicantly enhance the steroid production of inotodiol, er-

gosterol peroxide, betulin, ergosterol, cholesterol, lanosterol, stig-

masterol, and sitosterol (Kahlos, 1994; Wang et al., 2014). Similar-

ly, the addition of betulin, or various spent substrates such as bark

residues of white birch, birch extracts, corn grain and mulberry

powder in the medium, or exposing into light at certain wave-

lengthes to mimic the wild nutritional or host condition could e-

ciently stimulate the growth of chaga mycelium as well as its poly-

saccharide yield (Chen et al., 2020b; Fradj et al., 2019; Poyedinok

et al., 2015; Wang et al., 2019). Other stimulants such as colloidal

metal nanoparticles, AgNPs (silver nanonparticles), could inhibit

polysaccharide and avonoid synthesis but may stimulate melanin

synthesis while MgNPs (magnesium nanonparticles) colloid was

eective in stimulating the accumulation of endopolysaccharides,

avonoids, and melanin pigments (Poyedinok et al., 2020).

4.2. Phenolic compounds in chaga

Naturally occurring phenolics could be found in most plant and

other sources. They play vital roles in chemical defense, pigmenta-

tion, signals delivery, and even structure building in the organisms

especially the plants and microorganisms (Mandal et al., 2010;

Zhang et al., 2016). In our daily diet, natural phenolics have been

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Journal of Food Bioactives | www.isn-jfb.com 27

Peng et al. Bioactive compounds and bioactive properties of chaga

Figure 1. Various skeleton cores of pentacyclic, tetracyclic triterpenoids, and steroids. (a) Types of pentacyclic triterpenoid; (b) Types of tetracyclic triterpenoid; (c) Types of steroid.

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Table 2. Known terpenes and terpenoids of chaga and their puricaon/idencaon

Terpenoid Molecular

formula Extracon Method Qualicaon

Method Puricaon mMethod Reference

Lanosterol/lanosta-8,24-dien-3β-olaC30H50OMethanol, six mes MS and

1H-NMR/13C-NMR

Liquid-liquid extracon,

silica gel column/

RP-HPLC/Sephadex

LH-20 column

Kim et al. (2011)

β-Sitosterol/24R-ethylcholesta-5-en-3β-ol IC29H50O

3β-Hydroxylanosta-8,24-dien-21-alaC30H48O2

Ergosterol peroxide/5,8-epidioxyergosta-6,22-dien-3β-olbC28H44O3

Inotodiol/Lanost-8,24-dien-3β,22R-diolaC30H50O2

Trametenolic acid/3β-hydroxylanosta-8,24-dien-21-oic acidaC30H48O3

BetulindC30H50O2

Betulin-3-O-caeatedC39H56O5Dichloromethane,

48 h, reux

MS and

1H-NMR/13C-NMR

Silica gel column, RP-

HPLC (C18 column)

Wold et al. (2020)

Lanosta-7,9(11),24-trien-3β,22-diolaC30H50O3n-Hexane IR spectra, MS, and

1H-NMR/13C-NMR

Alumina column Kahlos and

Hiltunen (1986)

Lanosta-8,23E-dien-3β,22R,25-triol/3β,22R,25-

trihydroxylanosta-8,23E-dienea

C30H50O3Chloroform, 20

days, 60 °C

IR spectra, MS, and

1H-NMR/13C-NMR

Silica gel column and

RP-MPLC/HPLC

Taji et al. (2008b)

Lanosta-7,9(11),23E-trien-3β,22R,25-triol/3β,22,25-

trihydroxylanosta-7,9(11),23E-trienea

C30H48O3

Lanosta-8,24-dien-3β,21-diol/3β,21-dihydroxylanosta-

8,24-diene/uvariol/21-hydroxylanosterola

C30H50O2

Inonotusol A/(−)-(3R,5S,10S,11R,15S,17R,20R,21S,24S)-21,24-

cyclopenta-3,11,15,21,25-pentahydroxylanosta-8-en-7-onea

C30H48O695% Ethanol, 2

h, three mes

IR spectra, MS, and

1H-NMR/13C-NMR

Liquid-liquid extracon,

silica gel column, RP-

HPLC (C18 column)

Liu et al. (2014)

Inonotusol B/(−)-(3R,5S,10S,11R,15S,17S,20R,21S,24R)-21,24-

cyclopenta-3,11,15,21,25-pentahydroxylanosta-8-en-7-onea

C30H48O6

Inonotusol C/(17α,20β,24α)-21,24-cyclopenta1α,3β,21α,25,28-

pentahydroxy-5α-lanosta-7,9(11)-dienea

C30H48O5

Inonotusol D/(17β,20β,24β)-21,24-cyclopenta-

1α,3β,21α,25,28-pentahydroxy-5α-lanosta-7,9(11)-dienea

C30H48O5

Inonotusol E/(−)-(3R,5S,10S,11R,17S,20R,21S,24R)-21,24-

cyclopenta-3,11,21,25-tetrahydroxylanosta-8-en-7-onea

C30H48O5

Inonotusol F/(17α,21α,23α)-24-methyl-3β-

hydroxy-5α-lanosta-8,24-dien-21,23-lactonea

C31H48O3

Inonotusol G/3β,22-dihydroxy-5α-lanosta-8,25-dien-24-oneaC30H48O3

Inonotusic acid/(−)-(5S,10S)-13-isopropyl-7-

oxo-abieta-8,11,13-trien-20-oic acide

C21H28O2

3β,22-Dihydroxylanosta-8,24-dien-7-oneaC30H48O3

Ergosta-7,22-dien-3β-olbC28H46O

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Peng et al. Bioactive compounds and bioactive properties of chaga

Terpenoid Molecular

formula Extracon Method Qualicaon

Method Puricaon mMethod Reference

Lawsaritol/sgmast-4-en-3β-oliC29H50O

Fungisterol/ergosta-7-en-3β-olbC28H48O

Ergone/ergosta-4,6,8(14),22-tetraen-3-onebC28H40O

ErgosterolbC28H44O

3β-Hydroxylanosta-8,24-dien-21,23-lactoneaC30H46O395% Ethanol, 24 h,

room temperature,

5 mes

MS and

1H-NMR/13C-NMR

Liquid-liquid extracon,

silica gel column

Shin et al. (2000)

Methyl trametenolateaC31H50O3– – –

21,24-Cyclopentalanosta-8-en-3β,21,25-triolaC30H50O395% Ethanol, 24 h,

room temperature,

5 mes

MS and

1H-NMR/13C-NMR

Liquid-liquid extracon,

silica gel column

Shin et al. (2001b)

Lanosta-8-en-3β,22,25-triolaC30H52O395% Ethanol, 24 h,

room temperature,

5 mes

MS and

1H-NMR/13C-NMR

Liquid-liquid extracon,

silica gel column

Shin et al. (2002)

Inonotsutriol D/lanosta-8-en-3β,22R,24R-triolaC30H50O3Chloroform, 7

days, 50 °C

IR spectra, MS, and

1H-NMR/13C-NMR

Silica gel column and RP-

MPLC (silica gel column)/

HPLC (C18 column)

Tanaka et al. (2011)

Inonotsutriol E/lanosta-8-en-3β,22R,24S-triolaC30H50O3

Oleanolic acidcC30H48O395% Ethanol, 1 h,

reux, 5 mes

IR spectra, MS, and

1H-NMR/13C-NMR

Liquid-liquid extracon,

silica gel column,

Sephadex LH-20 and

RP-HPLC (C18 column)

Zhao et al. (2015a)

Betulinic aciddC30H48O3

Inonotusane A/(21S, 24R)-24-cyclolanost-8-en-3β,21,25-triolaC30H50O3

Inonotusane B/(21S, 24S)-24-cyclolanost-8-en-3β,21,25-triolaC30H50O3

Inonotusane C/3β-hydroxy-4,4,14-

trimethylchola-8,22E-dien-24-alo

C27H42O4

Obliquic acid/3β-hydroxy-25,26,27-

trinorlanosta-8,22E-dien-24-oic acida

C27H42O3

3β-Hydroxylanosta-7,9(11),24-trien-21-oic acidaC30H46O3

(+)-Fuscoporianol C/3β,22α,25-trihydroxylanosta-8,23E-dieneaC30H50O3

Inonotsutriol A/(20R,21R,24S)-21,24-

cyclopentalanosta-8-en-3β,21,25-triola

C30H50O3Chloroform, 20

days, 60 °C

IR spectra,

1H-NMR/13C-

NMR, and MS

Silica gel column and RP-

MPLC (silica gel column)/

HPLC (C18 column)

Taji et al. (2008a)

Inonotsutriol B/(20R,21R,24R)-21,24-

cyclopentalanosta-8-en-3β,21,25-triola

C30H50O3

Inonotsutriol C/(20R,21R,24S)-21,24-

cyclopentalanosta-7,9(11)-dien-3β,21R,25-triola

C30H48O3

Inonotsulide A/(20R,24S)-3β,25-

dihydroxylanost-8-en-20,24-olidea

C30H48O4Chloroform, 20

days, 60 °C

IR spectra,

1H-NMR/13C-

NMR, and MS

Silica gel column and RP-

MPLC (silica gel column)/

HPLC (C18 column)

Taji et al. (2007)

Inonotsulide B/(20R,24R)-3β,25-

dihydroxylanost-8-en-20,24-olidea

C30H46O4

Table 2. Known terpenes and terpenoids of chaga and their puricaon/idencaon - (connued)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Terpenoid Molecular

formula Extracon Method Qualicaon

Method Puricaon mMethod Reference

Inonotsulide C/(20R,24S)-3β,25-dihydroxylanosta-

7,9(11)-dien-20,24-olidea

C30H46O3

Inonotsuoxide A/22R,25-epoxylanost-8-en-3β,24S-diolaC30H50O3Chloroform, 7

days, 50 °C

IR spectra,

1H-NMR/13C-

Silica gel column

and RP-MPLC (silica

Nakata et al. (2007)

Inonotsuoxide B/22S,25-epoxylanost-8-en-3β,24S-diolaC30H50O3

Inotolactone B/3β-hydroxy-24-methyl-

lanosta-8,24(25)-dien-26,22R-olidea

C31H48O395% Ethanol, 3 days,

room temperature

IR spectra,

1H-NMR/13C-

NMR, and MS

Silica gel column and

RP-HPLC (C8 column)

Ying et al. (2014)

Inotolactone A/3β-hydroxy-24-methyl-

lanosta-7,9,24(25)-trien-26,22R-olidea

C31H46O3

Inotolactone C/3β-hydroxydriman-12,11-olidefC15H24O3

6β-Hydroxydriman-12,11-olidefC15H24O3

3β-HydroxycinnamolidefC15H22O3

17-Hydroxy-ent-asan-19-oic acidgC20H32O3

Saponaceoic acid I/3β,25-dihydroxy-4,4,14-

trimethyl-5α-cholesta-8,23-dien-21-oic acida

C30H48O495% Ethanol, 1 h,

reux, 5 mes

IR spectra, MS, and

1H-NMR/13C-NMR

Liquid-liquid extracon,

silica gel column,

Sephadex LH-20 and

RP-HPLC (C18 column)

Zhao et al. (2016a)

Ganodecochlearin A/22R,25-epoxylanost-7,9-dien-3β,24S-diolaC30H48O3

9,11-Dehydroergosterol peroxidebC28H42O3

Inonotusane D/3β-hydroxy-24,25,26,27-

tetranorlanosta-8-en-22-onea

C26H42O2

Inonotusane E/3β,12β,15α,21R,25-pentahydroxy-

21,24S-cyclopentalanosta-7,9(11)-dienea

C30H48O5

Inonotusane G/lanosta-8-en-3β,22,24,25-

tetraol-25-methyl oxidea

C31H54O4

Inonotusane F/Chagabusone A/3β-hydroxylanosta-

8,25-dien-24-on-21-oic acida

C30H46O480% Methanol, 2

days, twice, room

temperature

IR spectra, MS, and

1H-NMR/13C-NMR

Liquid-liquid extracon,

silica gel column/RP-

HPLC (C18 column)

Baek et al. (2018)

Spiroinonotsuoxodiol/3S,7S-dihydroxy-

7(8→9R) abeo-lanost-24-en-8-onea

C32H52O4Chloroform IR spectra, MS, and

1H-NMR/13C-NMR

Silica gel column, MPLC

(silica gel column) and

RP-HPLC (C18 column)

Handa et al. (2010)

Inonotsuoxodiol A/3β,22-dihydroxylanosta-8,24-dien-11-oneaC30H48O3

Inonotsudiol A/lanosta-8,24-dien-3 β,11β-diolaC38H48O2

5,8,22-ErgostatrienolbC28H44OPetroleum, 14 h,

room temperature

GC-MS –Sun et al. (2011)

5,7-ErgostadienolbC28H46O

Inoterpene AaC30H52O3Methanol, 3 h,

reux, 3 mes

IR spectra, MS, and

1H-NMR/13C-NMR

Liquid-liquid extracon,

silica gel column, and

HPLC (C18 column)

Nakamura et

al. (2009)

Inoterpene BaC30H52O3

Table 2. Known terpenes and terpenoids of chaga and their puricaon/idencaon - (connued)

Journal of Food Bioactives | www.isn-jfb.com 31

Peng et al. Bioactive compounds and bioactive properties of chaga

Terpenoid Molecular

formula Extracon Method Qualicaon

Method Puricaon mMethod Reference

Inoterpene CaC30H52O3

Inoterpene DaC30H50O3

Inoterpene EaC30H50O4

Inoterpene FaC30H48O2

(3R,5S,8R,9S,10S,13S,14S,17S)-21-Methylidyne-pregn-3-

ol/(3R,5S,8R,9S,10S,13S,14S,17S)-17-(1-hydroxyprop-2-

ynyl)-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-

tetradecahydro-1H-cyclopent-a[a]phenanthren-3-olk

C22H33O2Chloroform, 12 h,

room temperature,

three mes

UPLC-Q-TOF-MSnSilica gel column/RP-

HPLC (C18 column)

Geng et al. (2013)

(2S,4aR,10bR)-1,1,4a,10b-Tetramethyl-1,2,3,4,4a,4b,5,

6,10b,11,12,12a-dodecahydrochrysen-2-ol

C22H31O

(5α,20S)-3β,20-Bis-(dimethylamino)-4-(hydroxylmethyl)-

4,14-dimethyl-9β,19-cyclopregn-6-en-16α-olk

C28H47N2O2

(22E)-Sgmasta-7,22,25-trien-3-yl acetateiC31H47O2

(3β)-Olean-12-en-3-yl-(4-hydroxyphenyl)propanoatecC39H57O3

LigudentatolvC14H17O

24-Methylene dihydrolanosterolaC31H52O80% Ethanol, 24 h,

room temperature

GC-MS –Zheng et al. (2007a)

4,4-Dimethyl fecosterolbC32H50O

4-Methyl fecosterolbC31H48O

Fecosterol/δ-8(24),28-ErgostadienolbC30H46O

Episterol/ergosta-7,24(28)-dien-3-olbC28H46O

Ergosta-5,7,9(11),22-tertraen-3-olbC28H42O

Ergosta-5,7,9(11),22-tertraen-3-ol benzoatebC35H46O2

Fuscoporianol D/3β,22α-dihydroxy-lanosta-

8,25(27)-dien-24-peroxidea

C30H50O480% Ethanol, 24 h,

room temperature

GC-MS,

1H-NMR/13C-

NMR, X-ray, and

IR spectra,

Silica gel column and

macroporous resin

Fuscoporianol A/25-methoxy-21,

22-cyclolanosta-8-en-3β,21α-diola

C31H52O3Petroleum

ether, reux

IR spectra, MS and

1H-NMR/13C-NMR

Silica gel column He et al. (2001)

Fuscoporianol B/3β,22α-dihydroxy-

lanosta-8,23E-dien-25-peroxidea

C30H50O4

Fuscoporianol C/3β,22α,25-trihydroxy-lanosta-8,23E-dieneaC30H50O3

LupeoldC30H50O – GC and GC-MS –Kahlos and

iltunen (1987);

Kahlos (1994)

LupenonedC30H48O

Table 2. Known terpenes and terpenoids of chaga and their puricaon/idencaon - (connued)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Terpenoid Molecular

formula Extracon Method Qualicaon

Method Puricaon mMethod Reference

Sgmastanol/sitostanoliC29H52O

CholesterolhC27H46O

β-SelinenelC15H24 –GC and GC-MS –Ayoub et al. (2009)

cis-BergamotenenC15H24

trans-BergamotenenC15H24

α-SantalenemC15H24

β-Sesquifenchene C14H22

epi-β-SantalenemC15H24

PhotosantalolmC15H24O

β-EudesmollC15H26O

γ-EudesmollC15H26O

p-CymenetC10H14 Hydrodisllaon GC and GC-MS –Kahlos et al. (1992)

α-BisabolenesC15H24

δ-CadinolpC15H26O

(Z)-β-FarnesenerC15H24

α-CurcumeneuC15H22

α-CedreneqC15H24

α-TurmeroneuC15H22O

alanostane-type triterpenoids and steroids; bergostane-type steroids; coleanane-type triterpenoids; dlupane-type triterpenoids; eabietane-type diterpenoids; fdrimane-type sesquiterpenoids; gasane-type diterpenoids; hcholes-

tane-type steroids; isgmastane-type steroids; jcycloartane-type triterpenoids and steroids; kpregnane-type steroids; leudesmane-type sesquiterpenoids; msantalane-type sesquiterpenoids; nbergamotane-type sesquiterpe-

noids; ocholane-type triterpenoids; pcadinane-type sesquiterpenoids; qcedrane-type sesquiterpenoids; rfarnesane-type sesquiterpenoids; sbisabolane-type sesquiterpenoids; tmenthane-type monoterpenoid; ucurcumane-type

sesquiterpenoids; vnoreudesmane-type sesquiterpenoids

Table 2. Known terpenes and terpenoids of chaga and their puricaon/idencaon - (connued)

Journal of Food Bioactives | www.isn-jfb.com 33

Peng et al. Bioactive compounds and bioactive properties of chaga

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Bioactive compounds and bioactive properties of chaga Peng et al.

Journal of Food Bioactives | www.isn-jfb.com 35

Peng et al. Bioactive compounds and bioactive properties of chaga

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Figure 2. Terpenoids in chaga. (a) Lanostane-type terpenoids in chaga; (b) Other terpenoids in chaga.

Journal of Food Bioactives | www.isn-jfb.com 37

Peng et al. Bioactive compounds and bioactive properties of chaga

Table 3. Bioacvies of the terpenoids puried from chaga

Terpenes Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

Osmundacetone An-proliferaon

acvity

Bel-7402 cell line IC50∼4.7 μM –Liu et al. (2014)

PTKs inhibitory

acvity

ELISA assay IC50∼7.7 μM –

Ergosterol An-proliferaon

acvity

PC3 IC50∼9.82 μM –Ma et al. (2013)

An-inammatory

acvity

LPS-induced RAW

264.7 macrophages

ED∼40 μg/ml, inhibion

rate∼6% and 23.46%

Inhibited the NO producon

and NF-κB luciferase acvity

Ergosterol peroxide An-inammatory

acvity

LPS-induced RAW

264.7 macrophages

ED∼40 μg/ml, inhibion

rate∼36.88% and 53.63%

Inhibited the NO producon

and NF-κB luciferase acvity

Ma et al. (2013)

An-proliferaon

acvity

PC3 human prostac

carcinoma cell

IC50∼38.19 μM –

MDA-MB-231 breast

carcinoma cell

IC50∼30.23 μM –

A549 human lung cancer cell IC50∼17.04 μM –Kim et al. (2011)

L1210 mouse lymphocyc

leukemia cell

IC50∼36.40 μM –

HepG2 human liver cancer IC50∼13.19 μM –

MCF-7 breast cancer cell IC50∼9.06 μM –

HCT116 human

colorectal cancer cell

ED∼10 μg/ml Induced subG1 arrest; increased

cleaved PARP and decreased uncleaved

caspase-3; reduced expression of

β-catenin, c-Myc, cyclin D1 and CDK-8

Kang et al. (2015)

HT-29 human colorectal

cancer cell

ED∼5 μg/ml

SW620 human

colorectal cancer cell

ED∼10 μg/ml –

DLD-1 human colorectal

cancer cell

ED∼10 μg/ml –

An-tumor eect AOM/DSS-induced

colorectal cancer in mice

ED∼15 mg/kg/12 h

(oral administraon)

Suppressed colon tumor growth and total

tumor count but not the tumor incidence

in mice; suppressed the overexpression

of β-catenin, c-Myc and cyclin D1

Lanosterol Hepatoprotecve

acvity

D-galactosamine-induced

toxicity in WB-F344 cells

ED∼10 μM Protecon rate∼74.8% Liu et al. (2014)

An-cancer

acvity

TPA-induced Raji cell ED∼10–1,000 rao/TPA Inhibited EBV-EA acvaon Nakata et al. (2007)

An-proliferaon

acvity

L1210 cell line IC50∼37.15 μM –Zhao et al. (2015a)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Terpenes Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

HT1080 cells ED∼10–100 μg/ml –Ryu et al. (2017)

A549 ED∼62.5–250 μg/ml –Chung et al. (2010)

AGS ED∼62.5–250 μg/ml –

MCF-7 ED∼62.5–250 μg/ml –

Hela ED∼62.5–250 μg/ml –

An-tumor eect Sarcoma-180 cells

implanted Balbc/c mice

ED∼0.1/0.2 mg/mice/day –

Pro-proliferaon

acvity

human follicle dermal

papilla cells

ED∼1–25 μg/ml –Sagayama et

al. (2019)

Trametenolic acid Hepatoprotecve

acvity

D-galactosamine-induced

toxicity in WB-F344 cells

ED∼10 μM Protecon rate∼75% Liu et al. (2014)

An-cancer

acvity

TPA-induced Raji cell ED∼10–1,000 rao/TPA Inhibited EBV-EA acvaon Nakata et al. (2007)

An-inammatory

acvity

LPS-induced RAW

264.7 macrophages

ED∼40 μg/mL Inhibited the NO producon and

NF-κB luciferase acvity, inhibion

rate∼50.04% and 18.42%

Ma et al. (2013)

Pro-proliferaon

acvity

human follicle dermal

papilla cells

ED∼1–25 μg/ml –Sagayama et

al. (2019)

Inonotusol F Hepatoprotecve

acvity

D-galactosamine-induced

toxicity in WB-F344 cells

ED∼10 μM Protecon rate∼71.9% Liu et al. (2014)

3β,22-

Dihydroxylanosta-

8,24-dien-11-one

Hepatoprotecve

acvity

D-galactosamine-induced

toxicity in WB-F344 cells

ED∼10 μM Protecon rate∼81.2% Liu et al. (2014)

Inonotusol G An-proliferaon

acvity

KB cell line IC50∼9.9 μM –Liu et al. (2014)

Inonotsutriol A An-proliferaon

acvity

A549 cell line IC50∼2.34 μM –Zhao et al. (2015a)

Inonotsutriol D An-proliferaon

acvity

Hela cell IC50∼29.56 μM –Zhao et al. (2015a)

A549 cell line IC50∼8.39 μM –

P388 cell line IC50∼10.20 μM –Tanaka et al. (2011)

L1210 cell line IC50∼10.00 μM –

KB cell line IC50∼11.60 μM –

Inonotsutriol E An-proliferaon

acvity

HT29 IC50∼37.43 μM –Zhao et al. (2015a)

Hela IC50∼32.08 μM –

Table 3. Bioacvies of the terpenoids puried from chaga - (connued)

Journal of Food Bioactives | www.isn-jfb.com 39

Peng et al. Bioactive compounds and bioactive properties of chaga

Terpenes Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

L1210 IC50∼38.23 μM –

A549 cell line IC50∼1.63 μM –

3β,22α-

Dihydroxylanosta-

8,25-diene-24-one

An-proliferaon

acvity

A549 cell line IC50∼5.39 μM –Zhao et al. (2015a)

Hela cell line IC50∼20.20 μM –

Betulin An-proliferaon

acvity

NCI-H460 lung cancer cell IC50∼2.8 μM –Wold et al. (2020)

HT29-MTX colon cancer cell IC50∼1.6 μM –

A549 IC50∼28.81 μM –Zhao et al. (2015a)

Betulinic acid An-proliferaon

acvity

NCI-H460 lung cancer cell IC50∼2.10 μM –Wold et al. (2020)

HT29-MTX colon cancer cell IC50∼0.80 μM –

Hela IC50∼30.30 μM –Zhao et al. (2015a)

Inonotsuoxide A An-proliferaon

acvity

Hela cell line IC50∼12.15 μM –Zhao et al. (2015a)

L1210 cell line IC50∼19.40 μM –Tanaka et al. (2011)

An-cancer

acvity

TPA-induced Raji cell ED∼10–1,000 rao/TPA Inhibited EBV-EA acvaon Nakata et al. (2007)

Inonotsuoxide B An-proliferaon

acvity

Hela cell line IC50∼14.22 μM –Zhao et al. (2015a)

HT29 cell line IC50∼22.27 μM –

L1210 cell line IC50∼16.30 μM –Tanaka et al. (2011)

Inonotusane C An-proliferaon

acvity

human lung cancer

A549 cell line

IC50∼22.50 μM –Zhao et al. (2015a)

Hela cell line IC50∼29.18 μM –

Inotodiol An-proliferaon

acvity

NCI-H460 lung cancer cell IC50∼3.8 μM –Wold et al. (2020)

HT29-MTX colon cancer cell IC50∼3.8 μM –

L1210 cell line IC50∼12.40 μM –Tanaka et al. (2011)

human lung cancer A549 cell – Down-regulated the expression of

Ki-67 and Bcl-2 protein; up-regulated

the expression of p53 and bax protein;

arrested A549 cells in S phase

Zhong et al. (2011)

mouse leukemia P388 cell ED∼30 μM Up-regulated the expression

of caspase-3/7

Nomura et al. (2008)

HT1080 cells ED∼10–100 μg/ml –Ryu et al. (2017)

A549 ED∼62.5–250 μg/ml –Chung et al. (2010)

AGS ED∼62.5–250 μg/ml –

Table 3. Bioacvies of the terpenoids puried from chaga - (connued)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Terpenes Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

MCF-7 ED∼62.5–250 μg/ml –

Hela ED∼62.5–250 μg/ml –

An-tumor eect mouse leukemia P388-

bearing female CDF1 mice

ED∼3 and 10 mg/kg for

day 1 and 4, respecvely

–Nomura et al. (2008)

Sarcoma-180 cells

implanted Balbc/c mice

ED-0.1/0.2 mg/mice/day –Chung et al. (2010)

DMBA/TPA-induced skin

carcinogenesis in pathogen-

free female ICR mice

ED∼85 nmol/0.1

ml acetone/day

for 20 weeks

–Nakata et al. (2007)

An-cancer

acvity

TPA-induced Raji cell ED∼10–1,000 rao/TPA Inhibited EBV-EA acvaon

An-inammatory

acvity

LPS-induced RAW

264.7 macrophages

ED∼40 μg/ml Inhibited the NO producon,

inhibion rate∼3.13%

Ma et al. (2013)

Pro-proliferaon

acvity

human follicle dermal

papilla cells

ED∼1–25 μg/ml –Sagayama et

al. (2019)

3β-Hydroxylanos-

8,24-dien-21-al

An-proliferaon

acvity

NCI-H460 lung cancer cell IC50∼33.00 μM –Wold et al. (2020)

L1210 cell line IC50∼10.70 μM –Tanaka et al. (2011)

KB cell line IC50∼14.70 μM –

MDA-MB-231 IC50∼36.5 μM –Ma et al. (2013)

HT1080 cells ED∼10–100 μg/ml –Ryu et al. (2017)

A549 ED∼62.5–250 μg/ml –Chung et al. (2010)

AGS ED∼62.5–250 μg/ml –

MCF-7 ED∼62.5–250 μg/ml –

Hela ED∼62.5–250 μg/ml –

An-tumor eect Sarcoma-180 cells

implanted Balbc/c mice

ED-0.1/0.2 mg/mice/day –

3β-Hydroxylanos-

8,24-dien-21-ol

An-proliferaon

acvity

L1210 cell line IC50∼10.40 μM –Tanaka et al. (2011)

KB cell line IC50∼32.1 μM –

Pro-proliferaon

acvity

human follicle dermal

papilla cells

ED∼1–25 μg/ml –Sagayama et

al. (2019)

Inonotusane D An-proliferaon

acvity

HT29 cell line IC50∼24.23 μM –Zhao et al. (2016a)

L1210 cell line IC50∼19.93 μM –

MCF-7 cell line IC50∼19.20 μM –

4T1 IC50∼9.40 μM –

Table 3. Bioacvies of the terpenoids puried from chaga - (connued)

Journal of Food Bioactives | www.isn-jfb.com 41

Peng et al. Bioactive compounds and bioactive properties of chaga

Terpenes Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

Inonotusane E An-proliferaon

acvity

HT29 IC50∼37.72 μM –Zhao et al. (2016a)

HepG2 IC50∼24.29 μM –

4T1 IC50∼26.67 μM –

Inonotusane F An-proliferaon

acvity

HT29 IC50∼31.31 μM –Zhao et al. (2016a)

Hela IC50∼26.99 μM –

L1210 cell line IC50∼27.70 μM –

HepG2 IC50∼35.83 μM –

MCF-7 cell line IC50∼15.20 μM –

4T1 IC50∼24.10 μM –

Inonotusane G An-proliferaon

acvity

Hela IC50∼31.88 μM –Zhao et al. (2016a)

HepG2 IC50∼36.32 μM –

4T1 IC50∼20.90 μM –

Inotolactone B An-proliferaon

acvity

MCF-7 cell line IC50∼36.34 μM –Zhao et al. (2016a)

4T1 IC50∼39.39 μM –

α-Glucosidase

inhibitory acvity

PNPG hydrolysis assay – –

Inotolactone A An-proliferaon

acvity

MCF-7 cell line IC50∼30.72 μM –Zhao et al. (2016a)

α-Glucosidase

inhibitory acvity

PNPG hydrolysis assay – –

Ganodecochlearin A An-proliferaon

acvity

A549 cell line IC50∼35.11 μM –Zhao et al. (2016a)

HepG2 IC50∼35.98 μM –

4T1 IC50∼10.91 μM –

Saponaceoic acid I An-proliferaon

acvity

A549 cell line IC50∼39.39 μM –Zhao et al. (2016a)

HT29 IC50∼12.78 μM –

Hela IC50∼24.23 μM –

L1210 cell line IC50∼37.98 μM –

MCF-7 cell line IC50∼8.35 μM –

4T1 IC50∼7.79 μM –

Inonotusol A An-proliferaon

acvity

4T1 IC50∼33.80 μM –Liu et al. (2014)

Inonotusol C An-proliferaon

acvity

HepG2 IC50∼30.56 μM –Liu et al. (2014)

Table 3. Bioacvies of the terpenoids puried from chaga - (connued)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Terpenes Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

4T1 IC50∼34.29 μM –

Inonotusol B An-proliferaon

acvity

HepG2 IC50∼31.37 μM –Liu et al. (2014)

4T1 IC50∼30.45 μM –

9,11-Dehydroergosterol

peroxide

An-proliferaon

acvity

A549 cell line IC50∼10.77 μM –Zhao et al. (2016a)

HT29 IC50∼30.76 μM –

Hela IC50∼35.82 μM –

L1210 cell line IC50∼29.31 μM –

HepG2 IC50∼10.93 μM –

MCF-7 cell line IC50∼8.40 μM –

4T1 IC50∼9.31 μM –

Spiroinonotsuoxodiol/

(3S,7S,9R)-3,7-

dihydroxy-7(8→9)abeo-

lanost-24-en-8-one

An-proliferaon

acvity

P388 IC50∼29.5 μM –Handa et al. (2010)

L1210 IC50∼12.5 μM –

HL-60 IC50∼30.1 μM –

KB IC50∼21.2 μM –

Inonotsuoxodiol A/

lanosta-8,24-dien-

3β,11β-diol

An-proliferaon

acvity

P388 IC50∼23.8 μM –Handa et al. (2010)

L1210 IC50∼23.8 μM –

HL-60 IC50∼27.2 μM –

KB IC50∼14.5 μM –

Inonotsudiol A/(22R)-

3β,22-dihydroxylanosta-

8,24-dien-11-one

An-proliferaon

acvity

P388 IC50∼15.2 μM –Handa et al. (2010)

L1210 IC50∼19.7 μM –

HL-60 IC50∼17.7 μM –

Betulin-3-O-caeate An-inammatory

acvity

LPS + IFNγ-acvated

C57BL/6 primary

macrophages

IC50∼17.6 μM Reduced NO producon Wold et al. (2020)

Anoxidant

acvity

DPPH radical

scavenging assay

IC50∼52 μM –

Inotolactone A α-Glucosidase

inhibitory acvity

PNPG hydrolysis assay IC50∼0.24 mM –Ying et al. (2014)

Inotolactone B IC50∼0.24 mM –

3β-Hydroxycinnamolide IC50∼3.39 mM –

PTKs: protein tyrosine kinases; EBV-EA: Epstein–Barr virus early angen acvaon; AOM: Azoxymethane; DSS: Dextran sulfate sodium; PNPG: p-nitrophenyl-α-D-glucopyranoside.

Table 3. Bioacvies of the terpenoids puried from chaga - (connued)

Journal of Food Bioactives | www.isn-jfb.com 43

Peng et al. Bioactive compounds and bioactive properties of chaga

considered as contributors of various health benets with relative-

ly few toxic/side eects based on extensive cellular, animal, and

molecular biology experiments, as well as intervention and epi-

demiological studies (Scalbert et al., 2005). The small-molecule

phenolics could be classied according to the number of carbons

on their skeleton cores, such as simple phenolics (C6), phenolic

acids (C6-C1, C6-C2, C6-C3), coumarins (C6-C3), naphthoqui-

nones (C6-C4), xanthones (C6-C1-C6), stilbenes and anthraqui-

nones (C6-C2-C6), chalconoids and avonoids (C6-C3-C6), and

lignans (C6-C3)2 (Vermerris and Nicholson, 2008). As summa-

rized in Table 4, there are a total of 64 small-molecule phenolics

in chaga. Along with several small-molecules, common phenolics

such as coumarins, phenolic acids, and avonoids, a rare phenolic

group, namely styrylpyrones (C6-C2-C5), was also reported in

chaga. They are inonoblin A-C, inoscavin B-C, phelligridin C-H,

methylinoscavin A-C, davallialactone, and methyl davallialactone

(Figure 3). The styrylpyrones are mainly produced from Hymeno-

chaetaceae family such as Phellinus and Inonotus genus macro-

mycetes or primitive angiosperm families including Piperaceae,

Lauraceae, Annonaceae, Ranuculaceae and Zingiberaceae (Lee

and Yun, 2011). More data of the bioactivities and corresponding

molecular mechanism of styrylpyrones are given in a review by

Lee and Yun (2011). The bioactive studies of chaga-isolated phe-

nolics are relatively few, as listed in Table 5. The puried 3,4-dihy-

droxybenzaldehyde, 4-(3,4-dihydroxyphenyl)-(E)-3-buten-2-one,

and 3,4-dihydroxybenzalacetone exhibited considerable in vitro

anti-proliferation activity on various cancer cell lines (Liu et al.,

2014; Nakajima et al., 2009; Zhao et al., 2016a). The antioxidant

activities of dierent chaga-isolated styrylpyrones are expressed

as the ratios of IC50 values of these styrylpyrones (μM) to IC50

values of Trolox (μM) using DPPH and ABTS assays, which are

0.43 and 1.45 for inonoblin A, 0.58 and 1.42 for inonoblin B, 0.65

and 0.82 for inonoblin C, 0.33 and 1.51 for phelligridin D, 0.40

and 1.57 for phelligridin E, and 0.43 and 1.48 for phelligridin G,

respectively (Lee et al., 2007). Besides, dierent fractions of lignin

were recently isolated and identied from wild chaga in the form

of lignin-carbohydrate complex and assessed using in vitro anti-

oxidant, anti-proliferation, immunomodulatory, and anti-inam-

matory activity studies (Niu et al., 2016; Wang et al., 2015).

Similar to other secondary metabolites, the specic diversity

and quantity of phenolics in chaga are inuenced by their nutri-

tional and environmental conditions. Zheng et al. (2008b) com-

pared the phenolic content of wild chaga and its submerged cul-

tures, the predominant phenolics or their derivatives in wild chaga

including phelligridin A, phelligridin D, inoscavin A, inoscavin

B, and melanins could hardly be detected in the cultured product.

Meanwhile, the main phenolics of cultured chaga such as narin-

genin, epicatechin gallate (ECG), and kaempferol barely existed

in the wild group. This dierence was assumed to be the cause

of the less in vivo immune-stimulating eects by phenolic com-

pounds of cultured chaga than wild chaga (Zheng et al., 2008b). In

another cultivation study of chaga, davallialactone and inscavin B

were only synthesized when using the culture medium consisting

of lignocellulosic biomass, while the group cultured in medium

containing no lignin did not show these two phenolics (Xu et al.,

2014a). Besides, the lignocellulose-added medium gave a signi-

cantly higher production level of other avonoids including ECG,

epigallocatechin gallate (EGCG), phelligridin G, and a lower level

of simple phenolic acids such as gallic acid and ferulic acid, which

resulted in a signicant enhancement of the total antioxidant abil-

ity of chaga extracts (Xu et al., 2014a; Zhu and Xu, 2013). Based

on the functional nature of phenolics, the disadvantaged environ-

mental factors may act as elicitors and skew certain pathways and

aect their production. For example, instead of using lignocellu-

losic medium during submerged cultures, the coculture of chaga

with other white-rot fungi such as Phellinus punctatus or Phellinus

morii leads to an increased accumulation of phenolic compounds

including phelligridin C, phelligridin H, methyl inoscavin A, in-

oscavin C, inoscavin B, davallialactone, methyl davallialactone,

as well as melanins and various lanostane-type triterpenoids, even

though production of mycelial biomass will be inhibited (Zheng et

al., 2011c). Furthermore, imposing oxidative stress by moderately

supplementing with H2O2 or Na2[Fe(CN)5NO] (sodium nitro-

prusside), or using other stimulatory agents such as γ-irradiation,

Tween-20, Tween-80, jasmonic acid, L-tyrosine, linoleic acid,

heavy metal ions (Mg2+, Cu2+, Co2+, Zn2+, and Mn2+) and extracts

or cell debris of Alternaria alternata, Aspergillus avus and Mucor

racemosus in mycelia medium of chaga can also signicantly in-

crease the production, accumulation and/or diversity of phenolics,

and corresponding antioxidant ability of extracts thereof (Kim et

al., 2009; Poyedinok et al., 2020; Xu et al., 2015a; Xu et al., 2015b;

Xu et al., 2019b; Xu et al., 2016b; Yang and Zheng, 1994; Zhao

et al., 2009; Zheng et al., 2007b; Zheng et al., 2009a; Zheng et al.,

2009b). More insights into the regulatory machinery that controls

biosynthesis of chaga phenolics, especially styrylpyrones, are dis-

cussed below. Dierent from mechanism of higher plants, the elic-

itors-induced increase of phenolic production in chaga is mediated

by boosted NO (nitric oxide) synthesis via a signalling pathway

independent of oxylipins or jasmonic acid (Zheng et al., 2009a).

Later, the higher cellular NO-mediated homeostasis between S-

nitrosylation and denitrosylation of SNO (S-nitrosothiols) was

found to play an important role in the biosynthesis of styrylpyrones

in chaga (Zheng et al., 2011a). Suppressing GSNOR (S-nitroso-

glutathione reductase)-mediated S-nitrosylation enhanced TrxR

(thioredoxin reductase) activity and biosynthesis of phelligridins

C and H, inoscavin C, and methyl inoscavin B while reducing that

of phelligridin D, methyl inoscavin A, davallialactone and methyl

davallialactone. Conversely, inhibiting TrxR-induced denitrosyla-

tion increased production of phelligridin D, methyl inoscavin A,

davallialactone, and methyl davallialactone, but decreased that of

phelligridins C and H, methyl inoscavin B and inoscavin C (Zheng

et al., 2011a). Besides, the elicitors-stimulated NO gerneration

was followed with increased gene transcription and/or protein

expression of phenylalanine ammonia lyase (PAL), 4-coumarate

CoA ligase (4CL), inducible NO synthases-like protein (iNOSL),

and styrylpyrone synthase (SPS), the key enzymes involved in

styrylpyrone biosynthesis (Zhao et al., 2015b). Furthermore, the

S-nitrosylation of these enzymes was also found with NO accumu-

lation (Zhao et al., 2016b).

4.3. Polysaccharides and their derivaves

Polysaccharides are known for their role as structural and energy-

related elements in plants, animals, and microorganisms. They are

formed through polymerization of at least 10 monosaccharides that

are connected by glycosidic bonds in linear or branch sequence.

The polysaccharides can be divided into homopolymers if they

are composed of identical monosaccharides and heteropolymers

if they contain more than one monosaccharide (Rodrigues et al.,

2011). The structural complexity of polysaccharides involving the

molecular weight, sequence and composition, anomeric congura-

tion, type of glycosidic linkage, and presence of substituents can

be used in demonstrating the discrepancies of their bioactive func-

tions (Rodrigues et al., 2011; Wasser, 2002). In the studies of phys-

icochemical properties of chaga polysaccharides, various fractions

with dierent monosaccharide composition and molecular weight

(MW) are achieved through various purication methods (Table

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Bioactive compounds and bioactive properties of chaga Peng et al.

6). For example, the puried chaga polysaccharide reported by Liu

et al. (2019), is a proteoglycan with a MW of 40 kDa and contains

57.17% carbohydrate and 32.53% protein. The carbohydrate part

is comprised of D-galactose, D-glucose, D-xylose, and D-mannose

in a 2.0:3.5:1.0:1.5 mole ratio. In the study by Xiang et al. (2012),

the crude polysaccharide reported was composed of rhamnose,

arabinose, xylose, mannose, glucose, galactose with a mole ratio

of 2.64:5.09:3.03:24.8:10.3:54.1. Six fractions were puried from

it with MW of 19–36 kDa and consisted of 7.12–38.3% protein.

Meanwhile, in the study of Hu et al. (2016), the puried polysac-

charide contained 98.6% polysaccharide which was composed of

mannose, rhamnose, glucose, galactose, xylose and arabinose in a

mole ratio of 9.8:13.6: 29.1:20.5:21.6:5.4, and its molecular weight

was 32.5 kDa. Besides, ve homogeneous fractions were puried

from chaga crude polysaccharides by Huang et al. (2012), the MWs

of which were 150, 93, 230, 44, 100 kDa, respectively. Herein, the

fraction with MW at 230 kDa was a glycoprotein and the fractions

with MW of 44 and 100 kDa were acidic polysaccharides com-

posed of 21.2 and 23.3% uronic acid. Kim et al. (2006) reported 5

puried fractions from chaga, among which one was identied as

fucoglucomannan with a molecular weight of approximately 1,000

kDa. This fraction consisted of 70.8% mannose, 1.6% glucose,

0.8% fucose, 0.1% glucosamine, and 26.8% protein. At the same

time, some puried polysaccharide fractions of chaga also con-

tained little or no protein (Chen et al., 2015; Fan et al., 2012; Hu

et al., 2017a; Huang et al., 2012; Ma et al., 2012). In the study of

Ma et al. (2012), 8.45% protein, 30.01% neutral sugar, and 14.47%

uronic acid were present in one puried fraction of chaga poly-

saccharide (122 kDa); around 48% of this puried polysaccharide

were unknown compounds (neither protein nor polysaccharide).

Its carbohydrate content was composed of 2.67% rhamnose, 3.20%

arabinose, 6.57% xylose, 21.60% mannose, 48.00% glucose, and

17.90 % galactose. More recently, Wold et al. (2018) isolated and

fully analyzed three relatively pure polysaccharide fractions from

chaga, neutral polysaccharides (60–73 kDa), alkaline polysaccha-

rides (>450 kDa) and acidic polysaccharides (10–31 kDa). The

neutral polysaccharides consisted of a (1→3)-linked β-Glc back-

bone with branches of (1→6)-linked β-Glc, in addition to substan-

tial amounts of (1→6)-linked α-Gal with 3-O-methylation at about

every third Gal residue, and alkaline polysaccharides consisted

mainly of (1→3)- and (1→6)-linked β-Glc and (1→4)-linked β-

Xyl. The protein content of these fractions was less than 0.1% but

the content of phenolics (trace-9.7%) and unknown non-carbo-

hydrate compounds was still remarkably high (11–58%). Hence,

it is worth noting that regardless of being “crude” or “puried”,

polysaccharides of chaga in these studies are, to a large extent,

covalently linked to non-polysaccharide components. It is possible

that these non-polysaccharide components could enhance certain

bioactivities through their linkage with polysaccharides or simply

function independently. Therefore, to clarify and dierentiate the

exact mode of action of various fractions of chaga “polysaccha-

rides” in in vitro/in vivo bioactivity studies, a thorough purication

and comprehensive analysis of their physicochemical properties

also deserves further investigation.

For mushroom, a plethora of studies have shown that polysac-

charides possess immense biological properties, especially im-

mune-stimulating and anti-cancer/tumor activities (Rodrigues et

al., 2011; Singdevsachan et al., 2016; Yu et al., 2018). Through

intraperitoneal administration, mushroom polysaccharides were

treated as antigens in the body of higher animals, although the

analogy of cellular specicities between them and LPS is frequent-

ly made even if their structure and action mechanism are quite dis-

tinct (Hua et al., 2007; Kim et al., 2010; Kim et al., 2008a; Kim

et al., 2005; Kim et al., 2006; Pan et al., 2015; Park et al., 2003;

Shao et al., 2004; Wang et al., 2018d; Won et al., 2011; Yang et

al., 2015). Meanwhile, the diversity of the origin of these polysac-

charides gives them a vast variability of regulatory mechanisms

of various cell-cell interactions in higher organisms. For instance,

polysaccharides of Platycodon grandiorum only stimulate B cells

and macrophages instead of T cells, while lentinan and schizophyl-

lan stimulate T cells and macrophages but not B cells (Han et al.,

2001). Earlier, It was reported that using chaga polysaccharides

enhances the nitrite production and expression of IL-1 h, IL-6,

TNF-α, and iNOS in macrophages as well as the in vitro pro-pro-

liferation activity on fractioned B cells but no eects on T cells

(Kim et al., 2005). However, later on, dose-dependent activation

between chaga polysaccharide and enhancement of Th1/Th2 cell-

related cytokine secretion (IFN-γ and IL-4) in in vitro model of

mouse splenocytes demonstrated that T cells were also aected

(Won et al., 2011). Other studies indicating the in vitro and in vivo

pro-proliferation ability of chaga polysaccharides on immune cells

are given in Table 7.

The polysaccharides of mushroom were deemed to function as

anti-cancer/tumor agents which strive to inhibit or eliminate the

growth of cancer cells by activating and reinforcing the immuno-

logical functions of the host instead of directly attacking cancer

cells (Ooi and Liu, 2000). In fact, direct and indirect anti-tumor/

cancer ability was reported for both chaga polysaccharides and

other mushroom polysaccharides. Kim et al. (2006) reported a

puried fraction composed of α-linked fucoglucomannan-protein

complex (∼1,000 kDa) from chaga that exerted pro-proliferation

activity on Raw264.7 murine macrophages, albeit, no anti-prolif-

eration activity on HEC-1B, B16F10, A549, KATO-III, SW156,

and SK-OV3 cancer cell lines nor normal human cells HUVEC/

HEK293T at an even high concentration level of 200 μg/ml. Mean-

while, intraperitoneal administration of this fraction signicantly

inhibited tumor incidence, prolonging the survival rate of B16F10-

implanted mice at a dose of 30 mg/kg/day (Kim et al., 2006). A

similar result was reported by Chen et al. (2015), in in vitro assay

showing that one puried chaga polysaccharide (48.82 kDa) had

no toxicity in Jurkat cells, but intake of this polysaccharide frac-

tion not only inhibited the growth of transplantable Jurkat tumor

in mice signicantly, but also enhanced the splenocyte prolifera-

tion and lymphocyte proliferation induced by concanavalin A and

LPS in a dose-dependent manner. However, the opposite results,

showing signicant toxicity of puried polysaccharide fractions

(13.6–200 kDa), were observed in HepG2 cells at high concentra-

tions of 80–240 μg/ml (Liu et al., 2018; Xue et al., 2018). Lee et

al. (2014c) demonstrated the direct in vitro anti-migration and anti-

proliferation abilities of the crude polysaccharides by decreasing

the expression levels and activity of MMP-2 (matrix metallopro-

teinase-2)/MMP-9, the phosphorylation levels of MAPKs (mito-

gen-activated protein kinases), PI3K (phosphoinositide 3-kinase)/

AKT (protein kinase B) and COX-2 (cyclooxygenase-2), as well

as inhibiting the nuclear translocation of NF-κB (nuclear factor

κB) in A549 human lung cancer cells. The same authors proposed

a similar mechanism (MAPKS, COX-2 and NF-κB pathway) on

direct in vitro anti-migration, anti-invasion, and anti-proliferation

activity on B16-F10 mouse melanoma cells (Lee et al., 2014b).

However, the same authors later refuted their own proposed anti-

migration ability (Lee et al., 2016). While in vitro trial of LLC1

cell, the puried chaga polysaccharide showed direct cytotoxic-

ity for activating AMPK via phosphorylation of threonine 172 by

LKB1, downregulating Bcl-2 and upregulating Bax, as well as en-

hancing cleavage of Caspase-3 and PARP, leading to the opening

of mitochondrial permeability transition pore, and reducing MMP,

eventually resulting in an inhibition of ATP production and cellular

proliferation (Jiang et al., 2019). The inhibitory eect of LLC1

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Peng et al. Bioactive compounds and bioactive properties of chaga

Table 4. Known phenolic small molecules and polymers of chaga and their puricaon/idencaon

Phenolics Molecular formula Extracon Method Qualicaon Method Puricaon Method Reference

Gallic acid C7H6O5Water or 70% ethanol,

70–80 °C, 2–24 h

LC –Zheng et

al. (2008b);

Glamočlija et

al. (2015)

Protocatechuic acid C7H6O4Water or 70% ethanol, 70–80

°C, 2–24 h/boiling water, 1 h

LC, LC-MS and

GC-MS, MS and

1H-NMR/13C-NMR

Liquid-liquid extracon, HP-20

column and RP-HPLC (C18 column)

Ju et al. (2010);

Nakajima et

al. (2007);

Glamočlija et

al. (2015)

p-Hydroxybenzoic acid C7H6O3Water or 70% ethanol,

70–80 °C, 2–24 h

LC –Glamočlija et

al. (2015)

Vanillic acid C8H8O4High-pressure steam, 35%

methanol, 35% acetone, 30% water

LC-MS and GC-MS Liquid-liquid extracon Ju et al. (2010)

2,5-Dihydroxyterephthalic acid C8H6O6High-pressure steam, 35%

methanol, 35% acetone, 30%

water or Water boiling, 1 h

LC-MS and GC-

MS, MS and

1H-NMR/13C-NMR

Liquid-liquid extracon or/

and HP-20 column and RP-

HPLC (C18 column)

Nakajima et

al. (2009);

Nakajima et

al. (2007); Ju

et al. (2010)

Caeic acid C9H8O4Water boiling, 1 h MS and 1H-NMR/13C-

NMR

Liquid-liquid extracon, HP-20

column and RP-HPLC (C18 column)

Nakajima et

al. (2007)

3,4-Dihydroxybenzalacetone C10H10O3Methanol, six mes or

water boiling, 1 h

MS and 1H-NMR/13C-

NMR

Liquid-liquid extracon, HP-

20 column and RP-HPLC (C18

column) or Sephadex LH-20

column/silica gel column

Kim et al.

(2011);

Nakajima et

al. (2007)

3,4-Dihydroxybenzaldehyde C7H6O3Methanol, two mes,

room temperature

IR spectra, MS and

1H-NMR/13C-NMR

Liquid-liquid extracon, HP-20

column and RP-HPLC (C18 column)

Nakajima et

al. (2007); Liu

et al. (2014)

6,7-Dihydroxycoumarin C9H6O4High-pressure steam, 35%

methanol, 35% acetone, 30% water

GC-MS Liquid-liquid extracon Ju et al. (2010)

4-Hydroxy-3,5-dimethoxy

benzoic acid 2-hydroxy-1-

(hydroxymethyl) ethyl ester

C12H16O7Water boiling, 1 h MS and 1H-NMR/13C-

NMR

Liquid-liquid extracon, HP-20

column and RP-HPLC (C18 column)

Nakajima et

al. (2007)

2,5-Dihydroxybenzaldehyde C7H6O3Methanol, six mes MS and 1H-NMR/13C-

NMR

Liquid-liquid extracon, silica

gel column, MPLC, RP-HPLC/

Sephadex LH-20 column

Kim et al. (2011)

Inonoblin A/phelligridin I C33H20O13 Methanol, two mes,

room temperature

MS and 1H-NMR/13C-

NMR

Liquid-liquid extracon,

Sephadex gel LH-20 column

Lee et al. (2007)

Inonoblin B C23H14O10

Inonoblin C C25H18O9

Phelligridin D C20H12O8

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Bioactive compounds and bioactive properties of chaga Peng et al.

Phenolics Molecular formula Extracon Method Qualicaon Method Puricaon Method Reference

Phelligridin E C25H14O10

Phelligridin G C32H18O12

Methylinoscavin A C26H20O9Petroleum ether, chloroform,

ethyl acetate, acetone, ethanol

and water, reux for three mes

1H-NMR –Zheng et al.

(2011b)

Methylinoscavin B C25H22O8

Methylinoscavin C C24H18O8

Phelligridin C C20H12O7

Phelligridin H C33H18O13

Phelligridin F C26H22O9

2,3-Dihydroxy-1-(4-hydroxy-3-

methoxyphenyl)propan-1-one

C10H12O595% Ethanol, 2 h,

reux, three mes

IR spectra, MS and

1H-NMR/13C-NMR

Liquid-liquid extracon, silica

gel column, Sephadex gel LH-20

and RP-HPLC (C18 column)

Liu et al. (2014)

2,3-Dihydroxy-1-(4-hydroxy-3,5-

dimethoxyphenyl)-1-propanone

C11H14O6

4-(3,4-Dihydroxyphenyl)-

(E)-3-buten-2-one

C11H14O6

Davallialactone C25H20O9–LC and 1H-NMR/13C-

NMR

–Zhao et al.

(2015b)

Methyl davallialactone C26H22O9

Inoscavin C C23H16O8

p-Coumaric acid C9H8O370% Aqueous acetone, 24 h,

room temperature, three mes

LC –Zheng et al.

(2009b)

Rhoifolin/apigenin-7-O-

neohesperidoside

C27H30O14

Isorhoifolin/apigenin-

7-O-runoside

C27H30O14

Naringin/naringenin

7-O-neohesperidoside

C27H32O14

Isorhamnen-3-O-runoside C28H32O16

Run C27H30O16

Narirun C27H32O14

Kaempferol C15H10O6

Quercen C15H10O7

Isohamnen C16H12O7

Luteolin C15H10O6

Naringenin C15H12O5

Apigenin C15H10O5

Table 4. Known phenolic small molecules and polymers of chaga and their puricaon/idencaon - (connued)

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Peng et al. Bioactive compounds and bioactive properties of chaga

Phenolics Molecular formula Extracon Method Qualicaon Method Puricaon Method Reference

Fortunelen/5,7-dihydroxy-

3′-methoxyavone

C16H12O5

EGCG C22H18O11

ECG C22H18O10

Inoscavin B C24H20O8

Homogensic acid C8H8O4HCl-acetonitrile, 2 h,

room temperature

LC –Kim et al.

(2008b)

Ferulic acid C10H10O4

o-Coumaric acid C9H8O3

Resveratrol C14H12O3

2,6-Dimethoxyphenol C8H10O3HCl-water, 5 h, reux; then hot

ethyl acetate and methanol

IR spectra and GC-MS –Mazurkiewicz

(2006)

Resorcinol C6H6O2

3-Hydroxy-4,5-

dimethoxybenzoic acid

C9H10O5

3-Hydroxy-2-oxo-2Hchromene-

4,6-dicarboxylic acid

C11H6O770% Methanol, 12 h, 60 °C IR spectra,

MS, UV and

1H-NMR/13C-NMR

Liquid-liquid extracon, silica

gel column, Sephadex gel

LH-20/ODS-Sepak cartridge

and RP-HPLC (C18 column)

Hwang et

al. (2016)

6,6′-Dihydroxy-(1,1′-biphenyl)-

3,3′-dicarboxylic acid

C14H10O6

4-Hydroxy-3,5-

dimethoxybenzoic

acid/syringic acid

C9H10O5

4-Hydroxyisophthalic acid C8H6O5

Eriocitrin C27H32O15 50% Methanol, 24 h,

room temperature

LC –Zheng et al.

(2008b)

Isorhamnen C16H12O7

EGC C15H14O7

2,3-Dihydroxybenzaldehyde C7H6O5

(2′R)4-[1-(Hydroxymethyl)-

2-methoxy-2-oxoethoxy]-

3,5-dimethoxy benzoic

acid methyl ester

C14H18O8–MS and 1H-NMR/13C-

NMR

Chiralpak IG column Zou et al. (2019)

(2′S)4-[1-(Hydroxymethyl)-

2-methoxy-2-oxoethoxy]-

3,5-dimethoxy benzoic

acid methyl ester

C14H18O8

4-Hydroxy-3,5-dimethoxy-

2-butoxy-2-oxoethyl ester

C15H20O7

Table 4. Known phenolic small molecules and polymers of chaga and their puricaon/idencaon - (connued)

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Bioactive compounds and bioactive properties of chaga Peng et al.

allograft tumor in mice was also veried through intraperitoneal

injection of puried chaga polysaccharide at 50 mg/kg BW/day

(Jiang et al., 2019). The more interesting point is that these chaga

polysaccharide fractions render in vivo anti-tumor/cancer eects

through not only intraperitoneal injection but also via oral admin-

istration (Kim et al., 2006; Mizuno et al., 1999; Won et al., 2011).

Specically, the oral treatment of puried fraction of chaga poly-

saccharides at a dose of 50/100 mg/kg/day showed an excellent in

vivo tumor-inhibitory eect in mice and in vitro immunoregulatory

activities on spleen lymphocyte and macrophage without direct in

vitro cytotoxicity on SGC-7901 human gastric cancer cells (Fan et

al., 2012). A daily oral ingestion of chaga crude polysaccharides at

a dose of 200 mg/kg body weight for 6 days could also eectively

inhibit the growth of melanoma solid tumor (Won et al., 2011).

Furthermore, Chen et al. (2010) found that crude polysaccharides

can inhibit both the proliferation of in vitro tumor cells and tu-

mor growth in orally-treated Balb/c-nu/nu nude mice. However,

the results which include enhancement of NO, ROS, TNF-α and

phagocytosis via regulating MAPKS (JNK, p38, ERK) and NF-κB

signaling pathways in macrophages still imply that the anti-tumor

eect of orally administrated chaga polysaccharides was also con-

tributed by activating immune response systems. The exact mecha-

nism of how the anti-tumor eect worked via oral administration

is not yet clear. It was suggested that a major component of chaga

polysaccharides, β-glucan, was taken up by intestinal macrophag-

es, after which it was transported to lymph nodes, spleen and bone

marrow, therefore upregulating and activating the intestinal im-

mune system (Rhee et al., 2008; Rop et al., 2009; Won et al., 2011).

Overall, the anti-cancer/tumor eects of chaga polysaccharides

may be through oral or intraperitoneal treatments with both direct

and indirect inhibitory eects.

Coinciding with those of chaga extracts, chaga crude polysac-

charides exerted excellent in vivo antihyperglycemic and antihy-

perlipidemic eects in dierent diabetic models. Xu et al. (2010b)

reported that oral administration of crude polysaccharide extract of

cultured chaga in alloxan-induced type-1 diabetic mice, at 150 and

300 mg/kg body weight for 21 days, signicantly decreased blood/

liver glucose level, liver malondialdehyde (MDA) and serum con-

tents of free fatty acids (FFA), total cholesterol (TC), triacylglyc-

erols (TAG), and low-density lipoprotein cholesterol (LDL-C).

Meanwhile, it eectively increased high-density lipoprotein cho-

lesterol (HDL-C), insulin levels, and hepatic glycogen contents,

along with the enhancement of the catalase (CAT), superoxide

dismutase (SOD), and glutathione peroxidase (GPx) activities in

the liver of diabetic mice (Xu et al., 2010b). In the STZ-induced

diabetic mice model, constant oral administration of chaga poly-

saccharides at 50 mg/kg BW/day for 4 weeks down-regulated IL-

2R and MMP-9, and enhanced IL-2 level, and decreased the ex-

pression of phosphorylated NF-κB in the kidneys, thus, inhibiting

inammatory inltrate and extracellular matrix deposit injuries in

the mice kidneys (Wang et al., 2017b). Meanwhile, in STZ/high-

fat-diet-induced type-2 diabetic mice model, the high oral dose of

polysaccharides at 900 mg/kg BW/day for 4 weeks alleviated the

STZ-lesioned organ tissues (liver, kidney, and pancreas), up-regu-

lated protein expressions of PI3K, p-Akt, GLUT4 if mice adipose

tissues (Wang et al., 2017c). In a subsequent paper (Wang et al.,

2017a), the polysaccharides-Cr(III) complex orally administrated

at 300, 600 and 900 mg/kg BW/day improved glucose tolerance

capacity, promoted the metabolism of glucose and synthesis of

glycogen, ameliorated severe pathological damages in kidneys

including mesangial expansion, glomeruli partly sclerosis and glo-

merular hypertrophy in STZ/high-fat-diet-induced type-2 diabetic

mice. Meanwhile, the improvement of antioxidant enzymes (CAT,

SOD, GPx) and various blood/liver parameters (insulin, MDA,

Phenolics Molecular formula Extracon Method Qualicaon Method Puricaon Method Reference

Lignin-carbohydrate

complexes (37.9 and 24.5

kDa, 75–80% lignin)

–Water, 4 h, 60 °C HPSEC Anion-exchange chromatography

(DEAE-cellulose column); SEC

(Sephadex G-25 column); dialysis

Wang et al.

(2015)

Lignin-carbohydrate complexes

(29, 35, and 61 kDa, 64% lignin)

–NaOH-water, 12 h, 4 °C HPSEC Anion-exchange chromatography

(DEAE-cellulose column); SEC

(Sephadex G-25 column); dialysis

Niu et al. (2016)

SEC: size exclusion chromatography; HPSEC: high performance size exclusion chromatography.

Table 4. Known phenolic small molecules and polymers of chaga and their puricaon/idencaon - (connued)

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Peng et al. Bioactive compounds and bioactive properties of chaga

Figure 3. Styrylpyrones in chaga.

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Bioactive compounds and bioactive properties of chaga Peng et al.

Table 5. Bioacvies of phenolics puried from chaga

Compounds Bioacvity Model IC50 value or experi-

mental dosage (ED)

Mechanism or

manifestaon Reference

Phenolics

3,4-dihydroxybenzaldehyde An-proliferaon acvity A549 cell line IC50∼23.63 μM

or 3.1 μM

–Liu et al. (2014);

Zhao et al. (2016a)

An-proliferaon acvity 4T1 IC50∼16.40 μM –Zhao et al. (2016a)

An-proliferaon acvity Bel-7402 cell line IC50∼3.7 μM –Liu et al. (2014)

PTKs inhibitory acvies ELISA assay IC50∼24.6 μM –

4-(3,4-dihydroxyphenyl)-

(E)-3-buten-2-one

An-proliferaon acvity A549 cell line IC50∼24.23 μM –Zhao et al. (2016a)

MCF-7 cell line IC50∼30.71 μM –

4T1 IC50∼26.67 μM –

3,4-dihydroxybenzalacetone An-proliferaon acvity PA -1 IC50∼12.2 μM –Nakajima et al. (2009)

HL-60 IC50∼32.9 μM –

A549 IC50∼23.6 μM –Kim et al. (2011)

HL-60 IC50∼21.8 μM –

HCT116 ED∼10 and 100 μM –Kuriyama et al. (2013)

3,4-dihydroxybenzaldehyde An-proliferaon acvity PA -1 IC50∼12.1 μM –Nakajima et al. (2009)

caeic acid An-proliferaon acvity HL-60 IC50∼27.4 μM –Nakajima et al. (2009)

HCT116 ED∼10 and 100 μM –Kuriyama et al. (2013)

(2′S)4-[1-(hydroxymethyl)-2-

methoxy-2-oxoethoxy]-3,5-

dimethoxy benzoic acid methyl ester

An-proliferaon acvity Hep3B cells ED∼25 μM –Zou et al. (2019)

4-hydroxy-3,5-dimethoxy-2-

butoxy-2-oxoethyl ester

–

Caeic acid An-proliferaon acvity DNA topoisomerase

II inhibitory assays

IC50∼15.0 μM –Kuriyama et al. (2013)

3,4-Dihydroxybenzalacetone IC50∼10 μM –

Gallic acid IC50∼50 μM –

Syringic acid IC50∼175 μM –

Protocatechuic acid IC50∼80 μM –

3,4-Dihydroxybenzaldehyde IC50∼150 μM –

2,5-Dihydroxy-terephthalic acid IC50∼170 μM –

Inonoblin A/Phelligridin I Anoxidant acvity ABTS and DPPH

scavenging assays

IC50∼0.43 and 1.45 μM –Lee et al. (2007)

Inonoblin B IC50∼0.58 and 1.42 μM –

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Peng et al. Bioactive compounds and bioactive properties of chaga

Compounds Bioacvity Model IC50 value or experi-

mental dosage (ED)

Mechanism or

manifestaon Reference

Inonoblin C IC50∼0.65 and 0.82 μM –

Phelligridin D IC50∼0.33 and 1.51 μM –

Phelligridin E IC50∼0.40 and 1.57 μM –

Phelligridin G IC50∼0.43 and 1.48 μM –

Caeic acid IC50∼0.66 and 0.41 μM –

Lignin fracon An-virus acvity HIV-protease IC50∼1.4 μg/ml –Ichimura et al. (1998)

Lignin–carbohydrate complex Immunomodulatory acvity RAW 264.7 macrophages ED∼50 or 100 μg/ml Smulated NO

producon and

phagocyc acvity

Niu et al. (2016)

Anoxidant acvity DPPH, hydroxyl radical

scavenging and FRAP assays

ED∼0.25–1.00 mg/ml –

Lignin–carbohydrate complex An-proliferaon acvity A549, LO2, Bel-

7402 or HEK 293

IC50∼150 and 200

µg/mL (for A549)

Arrested cells at S

phase of A549;

Wang et al. (2015)

An-inammatory acvity LPS-induced HEK 293/

NF-B-Luc cells

ED∼1 mg/ml Inhibited the

acvaon of NF-κB

Puried phenolic extract Immunomodulatory ability CYP-induced ICR mice ED∼50 mg/kg BW/day

(oral administraon)

Inhibited the CYP-

induced reducon

of body weight,

spleen index

and the viability

of peripheral

lymphocytes

Zheng et al. (2008b)

Table 5. Bioacvies of phenolics puried from chaga - (connued)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

FFAs, TC, TG, LDL-C, and HDL-C) were mentioned in all mod-

els described above (Wang et al., 2017a; Wang et al., 2017b; Wang

et al., 2017c).

Thus far, many studies have veried various signicant in vitro

antioxidant results of chaga polysaccharides including radical/

peroxide scavenging and metal reduction ability (Table 7). Clas-

sically, the “crude polysaccharides” are prepared through sev-

eral primary steps such as water extraction/alcohol precipitation,

deproteinization, and/or dialysis. However, for “puried poly-

saccharides”, combinations of several chromatographic isolation

techniques are necessary. The mixtures so produced are believed

to be predominately composed of polysaccharides and therefore

labeled as “crude/puried polysaccharides” in most of chaga stud-

ies. However, several studies have suggested that polysaccharides

were not the only main constituents of chaga “crude/puried poly-

saccharides”. The data of Chen et al. (2009) showed that 42.50%

of chaga deproteinated “crude polysaccharides” was protein while

only 18.50 and 6.10% of those were neutral sugar and uronic acid,

respectively. More data about the protein content of chaga “crude

polysaccharides” can be found in other articles citated here, rang-

ing from 6.28 to 30.2% (Mu et al., 2012; Wang et al., 2018c; Xu

et al., 2011a). As for puried polysaccharides, a high proportion of

protein content was also found. Xiang et al. (2012) reported 6 puri-

ed fractions consisting of 7.12–38.3% protein, and the fraction

with highest protein content (38.3%) exerted the highest radical

scavenging activity. Kim et al. (2006) obtained 5 puried fractions

of chaga polysaccharides with a total protein content ranging from

22.1 to 59.3%. While in another study, 5 fractions were puried

through dierent columns (DEAE-Sepharose column and gel per-

meation column, Sepharose CL-6B), which contained relatively

less protein (0–21.4%) (Huang et al., 2012). In some cases, the

deproteination process can even enhance protein content of chaga

crude polysaccharides which demonstrates that the protein compo-

nents are covalently bound to the polysaccharide matrix (Xiang et

al., 2012; Xu et al., 2014a; Xu et al., 2014b). This result is consist-

ent with the fact that polysaccharides, proteins and glycoproteins/

proteoglycans, are the shared main constituents of the fungi cell

wall (Beauvais and Latgé, 2018; Ma et al., 2018; Peberdy, 1990).

More compositional data of dierent “polysaccharides” fractions

of chaga were later updated which helped to challenge its anti-

oxidant results. Mu et al. (2012) once attributed moderate-strong

radical scavenging ability to the polysaccharide components in

chaga “crude polysaccharides”, but later they found that lignin-

carbohydrate complex was also present in the puried fraction of

this “crude polysaccharides”, containing 64–80% lignin but only

16–28% neutral sugars and uronic acid (Wang et al., 2015). Be-

sides, melanin is another group of aromatic copolymers rich in wa-

ter extract of chaga. It possesses a similar polysaccharide hydro-

philicity and range of molecular weight (ranging from less than 10

kDa to more than 120 kDa). Therefore it was rarely distinguished

and identied during the analysis of chaga polysaccharides (Babit-

skaya et al., 2002; Wold et al., 2018). In the study of Wold et al.

(2018), in spite of purifying through anion-exchange and size-ex-

clusion (gel ltration) chromatography, three protein-free (<0.1%)

fractions of chaga polysaccharides were successfully produced but

they still contained 4.2–9.7% phenolics due to the existence of

covalently bound melanin pigment on polysaccharides. However,

none of the quantication methods of protein (Bradford, BCA and

Lowry assays) and sugar (sulfuric acid-phenol assay) used in these

studies could entirely avoid the interference of abundant phenol

groups in lignin or melanin (Dalilur Rahman and Richards, 1987;

Owusu-Apenten, 2002; Redmile-Gordon et al., 2013). It should be

noted that whether the health eects of natural “polysaccharides”

is related to the existence or the synergistic eect of non-polysac-

charide components especially the protein therein that has been

proposed for decades remains controversial (Cruz et al., 2016; Cui

and Chisti, 2003; Ng, 2003; Xu et al., 2011b; Zhang et al., 2011).

However, there are insucient studies on the exact structures and

proportions of chaga melanin-, protein-, and lignin-polysaccharide

complexes that allow full understanding of the authentic origin

of the chemical mechanism underlying the antioxidant ability of

chaga “polysaccharides”. On the other hand, as mentioned before,

the “puried chaga polysaccharides” indeed show signicant in

vivo antioxidant and anti-inammatory eects, which further po-

tentiate various tissue-protective eects, especially for pancreas/

liver/kidney protection (Diao et al., 2014; Hu et al., 2016; Wang et

al., 2017a; Wang et al., 2017b; Xu et al., 2010b). Recently, Hu et

al. (2016) found that ingestion of chaga polysaccharides alleviated

DDC (diethyldithiocarbamate)-induced pancreatic acinar atrophy

and weight loss by increasing pancreatic SOD, and decreasing LDH

(lactate dehydrogenase), hydroxyproline, AMS (amylase), IFN-γ,

IL-1 levels in serum of chronic pancreatitis mice. Later in the same

model, the increase of pancreatic levels of GPx and TAOC (total

antioxidant capacity) and the decrease of serum levels of TNF-α,

TGF-β as well as lipase and trypsin, were also detected (Hu et al.,

2017b). The improved gut microbiota composition through ingest-

ing chaga polysaccharides were found to be positively correlated

with relief of inammation and oxidative stress (Hu et al., 2017b).

Furthermore, the activation of the Nrf2/HO-1 signaling pathway

by chaga polysaccharide also protected the mitochondrial damage

and neuronal cells apoptosis in L-glutamic acid-damaged HT22

cell model and APP/PS1 transgenic mice model (Han et al., 2019).

Similarly, the nding of Xu et al. (2019a) suggests that chaga poly-

saccharides protect mice against the T. gondii-induced liver injury,

partially due to inhibition of the TLRs/NF signaling axis and the

activation of the antioxidant response such as increasing the con-

tents of serum/liver SOD and GSH, by inducing the Nrf2/HO-1

signaling.

In general, compared with the organic solvent extracts rich in

phenolics or terpenoids, the polysaccharide-rich water extract/

decoction contains much higher amount of oxalic acid. However,

the purication process of polysaccharide, including precipitation

and dialysis, can eectively remove small-molecule compounds.

However, there are limited investigations on oral safety of chaga

polysaccharides. Chen et al. (2009) reported a single oral dose of

chaga crude polysaccharide at 5,000 mg/kg body weight exerted

no acute-toxicity damage on the liver, kidney, heart, thymus or

spleen of male Kunming mice. Hu et al (2017a; 2017b) found that

oral administration of puried fractions of chaga polysaccharide at

a dose of 1,000 mg/kg BW three times in one day showed no acute

symptoms in pathogen-free male ICR mice including external

morphological, behavioral, neurological, and autonomic changes.

Another test conducted for 20 consecutive days of oral administra-

tion with a dose of 1,500 mg/kg body weight/day also showed no

sub-acute-toxicity damage to the liver, pancreases, kidney, heart,

thymus and spleen of male Kunming mice (Wang et al., 2017a).

However, except for safe short-term doses, more toxicological tri-

als of long-term administration are much desired.

4.4. Other components

As mentioned before, melanin is another antioxidant source in cha-

ga. The natural melanin is polymerized by either aromatic amino

acids or phenolics via C-C linkage, hence, could be divided into

nitrogenous melanin (eumelanin, pheomelanin) and non-nitroge-

nous melanin (allomelanin, pyomelanin), respectively (Ahmad et

al., 2016; Plonka and Grabacka, 2006). Therefore, the structure as

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Peng et al. Bioactive compounds and bioactive properties of chaga

Table 6. Polysaccharides and other known compounds of chaga and their puricaon/idencaon

Compound Molecular

formula Extracon Method Qualicaon Method Puricaon Method Reference

Polysaccharides

Glycoprotein (230 kDa) –Water, 3 h, 80 °C SEC Alcohol precipitaon, AEC (DEAE-

Sepharose fast ow column), SEC

(SepharoseCL-6B column), dialysis

Huang et al. (2012)

Proteoglycan (40 kDa) –Water, 2 h, 100

°C, two mes

HPSEC (refracve

index, UV, and

MALLS detectors),

AEC, and FT-IR

Liquid-liquid extracon Liu et al. (2019)

α-Linked fucoglucomannan

(1,000 kDa)

–Water, 6 h, 121 °C SEC Alcohol precipitaon, AEC (DEAE-

cellulose column), SEC (Toyopearl

HW65F column), dialysis

Kim et al. (2006)

Puried fracons of

polysaccharide (93 kDa)

–Water, 3 h, 80 °C GC and HPSEC Alcohol precipitaon, AEC (DEAE-

Sepharose CL-6B column), SEC

(sepharose CL-6B column), dialysis

Fan et al. (2012)

Puried fracons of

polysaccharide (122 kDa)

–Water, 80 min, 75 °C,

ultrasonicaon

SEC Deproteinaon (Sevag reagent),

alcohol precipitaon, DEAE-52

cellulose column, dialysis

Ma et al. (2012);

Zhang et al. (2013b)

Puried fracons of

polysaccharide (32.5 kDa)

–Water, 2.5 h, 60 °C SEC Anion-exchange DEAE cellulose

column and SEC (Sephadex G-200)

Hu et al. (2016)

Puried fracons of

polysaccharide (111.9 kDa)

–Water, 2 h, 90 °C UV, IR spectra, HPSEC Alcohol precipitaon, DEAE-52

column, SEC (Sephadex G-100)

Han et al. (2019)

Puried homogeneous

polysaccharide fracon

(37.354 kDa)

–Water, 2.5 h, 60 °C FT-IR, HPSEC,

1H-NMR/13C-NMR

Deproteinaon (Sevag reagent),

alcohol precipitaon, AEC (DEAE

cellulose column), Sephadex G-200 gel

Hu et al. (2017a)

Neutral polysaccharides

(60–73 kDa)

–Water, 2 h, 100

°C, two mes

SEC-MALLS, IR

spectra, 1H-NMR/13C-

NMR, and GC-MS

AEC (ANX Sepharose™ 4 Fast Flow),

SEC (Superose® 6 column), dialysis

Wold et al. (2018)

Acidic polysaccharides

(melanin-polysaccharide

complex) (10–31 kDa)

–AEC (ANX Sepharose™ 4 Fast

Flow), SEC (Hiload™ 16/60

Superdex™ 200 column), dialysis

Alkaline polysaccharides

(>450 kDa)

–AEC (ANX Sepharose™ 4 Fast

Flow), SEC (Sephacryl S-500

HR column), dialysis

Alkaloids

3,3-Dimethyl-9-(propylamino)-

3,4-dihydro-1(2H)-acridinone

C18H21N2OChloroform, 12 h, room

temperature, three mes

UPLC-Q-TOF-MSnSilica gel column/RP-

HPLC (C18 column)

Geng et al. (2013)

2-Butyl-3-(3-methylphenyl)-

4(3H)-quinazolinone

C19H19N2O

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Bioactive compounds and bioactive properties of chaga Peng et al.

Compound Molecular

formula Extracon Method Qualicaon Method Puricaon Method Reference

1-(4-Methyl-1-piperazinyl)-2-

{[3-(2-methyl-1-piperidinyl)

propyl]amino}ethanone

C16H31N4O

1-{[2-(Diethylamino)ethyl]

amino}-3-(4-methyl-1-

piperazinyl)-2-propanol

C14H31N4O

N-{(1S,2S)-1-benzyl-3-

[1-(cyclohexylmethyl)

hydrazino]-2-hydroxypropyl}-

N2-[(2-methoxyethoxy)

carbonyl]-L-valinamide

C26H43N4O5

1,1-Dimethyl-3,3-bis(2,2,6,6-

tetramethyl-1-prop-2-en-

1-ylpiperidin-4-yl)urea

C27H49N4O

1-(3,6-Dihydropyridin-1(2H)-yl)-3-

[3-(dimethylamino)propyl]urea

C11H21N4O

(2R,4S,5S,7S)-5-Amino-N-butyl-

7-{4-[4-(dimethylamino)-butoxy]-

3-(3-methoxypropoxy)benzyl}-4-

hydroxy-2,8-dimethylnonanamide

C32H57N3O5

2,2-Bis[2,2,6,6-tetramethyl-

1-(octyloxy)piperidin-

4-yl]-hexanedioate

C40H73N2O6

3-(4-Cyclohexylbutyl)-6,11-

dimethyl-1,2,3,4,5,6-hexahydro-

2,6-methano-3-benzazocine

C24H36N

Other organic compounds

2-(1,4,4-Trimethylcyclohex-

2-en-1-yl)ethyl acetate

C13H22O2HCl-water, 5 h, reux;

then hot ethyl acetate

and methanol

IR spectra and GC-MS –Mazurkiewicz (2006)

4-Oxopentanoic acid C5H8O3

Docosane C22H46

Hexatriacontane C36H74

O-Acetyl-all-trans-Renol C22H32O2

Hexadecanoic acid C16H32O2

Heneicosane C21H44

Benzyl alcohol C7H8O

Table 6. Polysaccharides and other known compounds of chaga and their puricaon/idencaon - (connued)

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Peng et al. Bioactive compounds and bioactive properties of chaga

Compound Molecular

formula Extracon Method Qualicaon Method Puricaon Method Reference

Oxalic acid C2H2O4Water or 70% ethanol,

2–24 h, 70-80 °C

LC None Glamočlija et

al. (2015)

Cinnamic acid C9H8O2

Isocitric acid C6H8O7High-pressure steam,

35% methanol, 35%

acetone, 30% water

LC-MS and GC-MS Liquid-liquid extracon Ju et al. (2010)

1-Dodecanol C12H26OPetroleum, 14 h, room

temperature,

GC-MS –Sun et al. (2011)

2,10-Dimethyl-9-undecenol C13H26O

Ethyl octadecanoate C20H40O2

Isopropyl linoleate C20H38O2

Ethyl oleate C20H38O2

Ethyl hexadecanoate C18H36O2

Ethyl dodecanoate C14H28O2

Ethyl tetradecanoate C16H32O2

Di-isobutyl phthalate C16H22O4

Di-iso-octyl phthalate C24H38O4

Ethyl pentadecanoate C17H34O2

Ethyl Heptadecanoate C19H38O2

2,6,10,14-Tetramethyl

heptadecane

C21H44

2,6,10,14-Tetramethyl

pentadecane

C19H40

Hexadecane C16H34

Octadecane C18H38

Heptadecane C17H36

Nonadecane C19H40

Dibutyl phthalate C16H22O4

Methyl-8,11-octadecadienoate C19H34O2

Ethyl linoleate C20H36O2

Pentadecanal C15H30O

Linoleic acid C18H32O2

Benzaldehyde C7H6OHCl-water, 5 h, reux;

then hot ethyl acetate

and methanol

IR spectra and GC-MS –

Table 6. Polysaccharides and other known compounds of chaga and their puricaon/idencaon - (connued)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Compound Molecular

formula Extracon Method Qualicaon Method Puricaon Method Reference

(2S)-2-[(1S)-1-Phenylethyl]-

3,6-dihydro-2H-pyran

C13H15OChloroform, 12 h, room

temperature, three mes

LC-Q-TOF-MSnSilica gel column/RP-HPLC Geng et al. (2013)

1-Octen-3-ol C8H16OHydrodisllaon GC and GC-MS –Kahlos (1994)

Linolenic acid C18H30O2Hexane TLC, GLC, and GC-MS –Kahlos et al. (1989)

1,6-Dideoxy-3,4-O-(1,5,9-

trimethyl-decylidene)-Dmannitol

C19H37O4Chloroform, 12 h, room

temperature, three mes

LC-Q-TOF-MSnSilica gel column/RP-HPLC Geng et al. (2013)

(1S,4aR,5R,8aS)-5-[(1R)-5-

Hydroxy-1,5-dimethylhexyl]-4a-

methyldecahydronaphthalen-1-ol

C19H35O2

Glucitol C6H14O695% Ethanol, 24 h, room

temperature, 5 mes

MS and 1H-NMR/13C-

NMR

Liquid-liquid extracon,

silica gel column

Shin et al. (2001a)

Trp-Gly-Cys C16H20N4O4SHyun et al. (2006)

Phenylalanine C9H11NO250% Ethanol, 24 h,

room temperature

HPLC Sephadex LH-20 column Zheng et al. (2008b)

Tyrosin C9H11NO3

Puried melanin fracons (56–60

kDa or 100–120 kDa or more)

–NaOH-water, 2 h, boiling SEC (Toyopearl HW-

65 resin column)

SEC (Sephadex G-75 column) Babitskaya et

al. (2000)

Puried melanin fracons (2–20

kDa or 90–100 kDa or more)

–50–95% ethanol, 2 h, 100 °C;

then water, 1 h, 100 °C; then

KOH-water, 1–3 h, 20 °C

IR spectra, 13C NMR Ethanol precipitaon, acid

precipitaon, Sephadex G-100 column

Olennikov et al. (2012)

Puried melanin-polysaccharide

(<10 kDa, ∼5% polysaccharide)

–Water, 2 h, boiling,

three mes

HPSEC (diol-

300 column)

Ethanol precipitaon, dialysis,

acid precipitaon

Wold et al. (2020)

Puried polysaccharide-

melanin complex (10–31

kDa, ∼4.2–9.7% melanin)

–Water, 2 h, boiling, 2 mes SEC-MALLS, IR

spectra, 1H-NMR/13C-

NMR, and GC-MS

AEC (ANX Sepharose™ 4 Fast

Flow); SEC (Hiload™ 16/60

Superdex™ 200 column); dialysis

Wold et al. (2018)

Crude melanin – Water, 10 h, 70 °C or

microwave-assisted

extracon

–Acid precipitaon Burmasova et al.

(2019); Parfenov

et al. (2019)

UPLC-Q-TOF-MS/MS: ultra-high-performance liquid chromatography-quadrupole me-of-ight tandem mass spectrometry; RP-HPLC: reverse-phase high performance liquid chromatography; MALLS: mul-angle laser light scat-

tering; SEC: size exclusion chromatography; HPSEC: high performance size exclusion chromatography; AEC: anion-exchange chromatography; HPAEC: high-performance anion-exchange chromatography.

Table 6. Polysaccharides and other known compounds of chaga and their puricaon/idencaon - (connued)

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Peng et al. Bioactive compounds and bioactive properties of chaga

Table 7. Bioacvies of polysaccharides and other compounds puried from chaga

Compounds Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

Polysaccharides

Crude polysaccharides Anoxidant acvity DPPH test, hydroxyl

radical/superoxide anion

radical scavenging test

IC50∼0.27–7.0 mg/ml –Mu et al.

(2012)

Anoxidave

stress acvity

H2O2-induced cell

death of PC12 cell

ED∼5, 10, 20 µg/ml

Polysaccharides-

chromium (III)

complex (115 kDa)

Anoxidave

stress acvity

H2O2-induced oxidave

damage in hepac L02 cells

ED∼500 μg/mL Improved the cell viability; inhibited the

morphology alteraon and maintained

the integrity of mitochondria

Wang et al.

(2018a)

Puried polysaccharide

(97.12 kDa)

Anoxidave

stress acvity

H2O2-induced oxidave

damage in hepac L02 cells

ED∼50–500 μg/mL Improved the cell viability; restored

the morphology alteraons of cells and

maintained the integrity of mitochondria

Wang et al.

(2018c)

Anoxidant acvity DPPH radical

scavenging test

IC50∼498.35 μg/mL –

Crude protein-

polysaccharide complex

Anoxidant acvity DPPH assay IC50∼1.33–4.35

mg/ml

–Xiang et

al. (2012)

Crude exo/endo-

polysaccharide from

submerged cultures

Anoxidant acvies DPPH, TBARS assays ED∼0.5–3 mg/ml –Xu et al.

(2011a)

Crude exo-polysaccharide

from submerged cultures

Anoxidant acvies Hydroxyl and superoxide

radicals scavenging abilies

IC50∼1.08 mg/ml

and 174.1 µg/ml

–Chen et al.

(2011)

Crude polysaccharide Anoxidave

stress acvity

Fe2+-Cys-induced lipid

peroxidaon in fresh

mouse liver homogenate

ED∼100, 200, 300,

and 400 μg/ml

–Song et al.

(2008)

Fe2+-VC-induced

mitochondria swelling

ED∼100, 200, 400,

and 800 μg/ml

–

Puried polysaccharide

(40 kDa)

Anoxidant acvies DPPH radicals scavenging,

TEAC, and FRAP assays

ED∼50–1,000 μg/ml –Liu et al.

(2019)

Puried polysaccharide

(32.5 kDa)

Anoxidant acvies DPPH and hydroxyl radicals

scavenging assays

IC50∼1.3–3.2 mg/ml –Hu et al.

(2016)

Puried polysaccharide

(122 kDa)

Anoxidant acvies FRAP and an-liver-lipid

peroxidaon model

ED∼0.5–5 mg/ml –Ma et al.

(2012)

Puried polysaccharide

(122 kDa)

Anoxidant acvies FRAP and an-liver-lipid

peroxidaon model

ED∼0.5–5 mg/ml –Zhang et al.

(2013b)

Puried homogeneous

polysaccharide

(37.354 kDa)

Anoxidant acvies DPPH and hydroxyl

radical Scavenging

ED∼1.0–5.0 mg/mL –Hu et al.

(2017a)

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Bioactive compounds and bioactive properties of chaga Peng et al.

Table 7. Bioacvies of polysaccharides and other compounds puried from chaga - (connued)

Compounds Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

Puried homogeneous

selenized polysaccharide

(28.071 kDa)

–

Puried homogeneous

polysaccharide

(37.354 kDa)

Anoxidave

stress acvity

D-gal-induced oxidant

damage in mice

ED∼100 mg/kg DW Increased SOD and GPx levels coupled

with reducon in MDA level

Puried homogeneous

selenized polysaccharide

(28.071 kDa)

Unknown

polysaccharides

Anoxidant

protecve acvity

Tacrine induced apoptosis

of HepG2 cells

–Reduced tacrine-induced apoptosis; Inhibited

tacrine-induced ROS generaon, 8-OHdG

formaon in mitochondrial DNA, and loss of

the mitochondrial transmembrane potenal;

decreased tacrine-induced the cytochrome

c release and acvaon of caspase-3

Li et al.

(2019)

Puried polysaccharide Anoxidave stress and

an-proliferaon acvity

Zebrash embryos ED∼1–5 mg/mL Reduced levels of intracellular ROS and

apoptosis in the developing embryos;

arrested the cells at G1 stage

Eid and Das

(2020a)

Puried polysaccharide An-genotoxic eects UVB-exposed

zebrash embryos

ED∼2.5 mg/mL Reduced DNA damage and ameliorated

the deformed structures; upregulated

mRNA expressions of XRCC-5, XRCC-

6, RAD51, P53, and GADD45

Eid et al.

(2020b)

Puried polysaccharide Anoxidave

stress acvity

H2O2-treated RINm5F

pancreac β-cells

ED∼1–100 µg/ml Decreased DNA fragmentaon and

the rate of apoptosis; upregulated

phosphorylaon of MAPK (JNK, ERK, and

p38); Suppressed cleaved caspase-3

Sim et al.

(2016)

Puried polysaccharide

(42 kDa)

An-inammatory

and an-oxidave

stress eects, and

protecve eect of

reproducve funcon

Toxoplasma gondii-

induced male mouse

ED∼100, 200, and

400 mg/kg BW/day

(oral administraon)

Improved the spermatogenic capacity and

ameliorated pathological damage of tess;

increased serum testosterone, luteinizing

hormone and follicular-smulang hormone

levels; Decreased the levels of MDA and NO, but

increased the acvies of SOD and GSH; Up-

regulated tescular StAR, P450scc and 17β-HSD

expressions; up-regulated the expressions of

Nrf2, HO-1 and NQO-1, and suppressed the

apoptosis of tescular cells by decreasing Bax

and cleaved caspase-3 expresisions; enhanced

tescular PI3K, p-AKT and p-mTOR expression

Ding et al.

(2020)

Journal of Food Bioactives | www.isn-jfb.com 59

Peng et al. Bioactive compounds and bioactive properties of chaga

Compounds Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

Puried polysaccharide

(42 kDa)

An-inammatory and

an-oxidave stress

eects, and protecve

eect of pregnancy

Toxoplasma gondii-induced

adverse pregnancy

in female mouse

ED∼100, 200, and

400 mg/kg BW/day

(oral administraon)

Reduced the aboron rate; inhibited the

decreases of serum progesterone and estriol

levels and the increase of MDA level; increased

the acvies of SOD and GSH in blood and/or

placenta; Inhibited the producon of TNF-α,

IL-6, IFN-γ, IL-1β and IL-17A; and promoted

the producon of an-inammatory cytokine

IL-10 and TGF-β in placenta; Up-regulated

the expression of Fox-p3, whereas down-

regulated the expressions of ROR-γt, STAT-3

and TLR-4, and inhibited the phosphorylaons

of NF-κB and IκBα in placental ssues

Xu et al.

(2020)

Puried polysaccharide

(42 kDa)

An-inammatory, an-

oxidave stress, and

hepatoprotecve eect

Toxoplasma gondii-induced

mouse liver injury

ED∼100, 200, and

400 mg/kg BW/day

(oral administraon)

Decreased the liver coecient, the levels of

ALT, AST, MDA, and NO; increased the contents

of SOD and GSH in liver/serum; Decreased

the expression of serum TNF-α, IL-6, IL-1β,

IFN-γ and IL-4; down-regulated TLR2, TLR4,

phosphorylaon of NF-κB and IκBα; up-

regulated the expressions of Nrf2 and HO-1

Xu et al.

(2019a)

Low-molecular-

weight polysaccharide

(10–100 kDa)

Renal protecve eect HFD/STZ-Induced

diabec nephropathy

in C57BL/6 male mice

ED∼300 and 1,000

mg/kg BW/day (oral

administraon)

Restored the integrity of the glomerular

capsules and increased the numbers of

glomerular mesangial cells; alleviated the

glucotoxicity in renal tubular cells;

Chou et al.

(2016)

An-hyperglycemic

eect

Decreased insulin tolerance,

triglyceride levels, urinary albumin/

creanine rao and LDL/HDL rao

An-inammatory eect Decreased NF-κB and TGF-β expression;

decreased expression of TGF-β on renal cortex

Puried polysaccharide

(32.5 kDa)

An-inammatory

and an-oxidave

stress eects

DDC-induced chronic

pancreas mice

ED∼100, 200 and

400 mg/kg BW/day

(oral administraon)

Alleviated pancreac acinar atrophy and weight

loss; increased SOD and MDA level in pancreac

ssue; decreased LDH, hydroxyproline,

AMS, IFN-γ, and IL-1 levels in serum

Hu et al.

(2016)

Unknown polysaccharide An-inammatory eect DSS-induced colis mice ED∼100–300 mg/

kg BW/day (oral

administraon)

Reduced the losses of ght juncon proteins

Occludin and ZO-1 in colon ssues; regulated

imbalanced Th1/Th2 and Th17/Treg in

colon ssues, mesenteric lymph nodes and

spleen; upregulated p-STAT1 and p-STAT3;

down-regulated expression of p-STAT6

Chen et al.

(2019b)

Crude polysaccharide An-inammatory

acvity

LPS-induced RAW 264.7

murine macrophage cells

ED∼50–500 μg/ml Down-regulated IL-6 and TNF-α levels; no

eect on IL-1β; reduced NO producon

Van et al.

(2009)

Table 7. Bioacvies of polysaccharides and other compounds puried from chaga - (connued)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Compounds Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

Crude endo-

polysaccharide from

submerged cultures

An-inammatory

acvity

LPS-induced RAW 264.7

murine macrophage cell

ED∼1–10 μg/ml Up-regulated the mRNA expression of the

INOS and inammatory eector cytokines

(IL-1β, IL-6 and TNF-α); Increased total

nitrite-producing acvity of macrophages

Kim et al.

(2005)

Crude endo-

polysaccharide from

submerged cultures

Immunomodulatory

acvity

Fraconated fresh

B and T cells

ED∼1–100 μg/ml Smulated proliferaon and dierenaon

of B cells into anbody-producing plasma

cells; smulated IgM anbody yield

Kim et al.

(2005)

Crude polysaccharide Immunomodulatory

acvity

Macrophage and

splenocytes

ED∼20 and

100 μg/ml

Promoted cell proliferaon and

producon of IL-2 and GM-CSF

Lee et al.

(2017b)

Puried polysaccharide

(40 kDa)

Immunomodulatory

acvity

RAW 264.7 murine

macrophage cell

ED∼50–500 μg/ml Smulated NO producon Liu et al.

(2019)

Puried α-linked

fucoglucomannan

(∼1,000 kDa)

Immunomodulatory

acvity

RAW 264.7 murine

macrophage cell

ED∼1–100 μg/ml Smulated proliferaon and NO producon Kim et al.

(2006)

Puried proteoglycan

(40 kDa)

Immunomodulatory

acvity

LPS-induced RAW 264.7

murine macrophage cell

ED∼50–500 µg/ml Increased the release of NO Liu et al.

(2019)

Puried polysaccharides

(32–119 kDa)

Immunomodulatory

acvity

Human peripheral blood

mononuclear cells

ED∼15–150 µg/ml Smulated cell proliferaon and secreon

of TNF-α, IFN-γ, IL-1β, and IL-2

Xu et al.

(2014b)

Alkaline (>450 kDa) and

acidic polysaccharides

(10–31 kDa)

Immunomodulatory

acvity

J774.A1 murine

macrophage cell

and D2SC/1 murine

dendric cell

ED∼100 µg/ml Increased NO producon Wold et

al. (2018)

Neutral polysaccharides

(60–73 kDa)

ED∼10 µg/ml

Crude protein-

polysaccharide complex

An-proliferaon

acvity

Unclear cellular model –Inhibited the acvity of cdc25 and cdc2/cyclin B; Mizuno et

al. (1999)

Puried α-linked

fucoglucomannan

(∼1,000 k Da)

An-proliferaon

acvity

MCF-7, Hur7 cells ED∼10 and 50 μg/ml –Kim et al.

(2006)

An-tumor eect B16F10 melanoma cells-

implanted (SPF) BDF1 mice

ED∼30 mg/kg BW/

day (intraperitoneal

administraon) or

300 mg/kg BW/day

(oral administraon)

Enhanced survival rate; decreased

tumor incidence

Puried polysaccharides

(48.82 kDa)

An-tumor eect Jurkat cells implanted

Kunming mice

ED∼20–80 mg/

kg BW/day (oral

administraon)

Increase Bax expression and

inhibit Bcl-2 expression

Chen et al.

(2015)

Immunomodulatory

eect

Smulated proliferaon splenocyte

and lymphocyte; promoted cytokine

secreon (IL-2, IL-6, IL-12 and TNF-) and

macrophage phagocytosis in mice;

Table 7. Bioacvies of polysaccharides and other compounds puried from chaga - (connued)

Journal of Food Bioactives | www.isn-jfb.com 61

Peng et al. Bioactive compounds and bioactive properties of chaga

Compounds Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

Crude polysaccharide An-tumor eect B16-F10 melanoma

cells implanted female

C57BL/6 mice

ED∼200 mg/

kg BW/day (oral

administraon)

Inhibited the growth of the

peritoneal tumor mass

Won et al.

(2011)

Immunomodulatory

eect

Female C57BL/6 mice ED∼300 and 500 μg/

mice (intraperitoneal

administraon)

Promoted phagocytosis, NO/ROS producon,

and TNF-α secreon of peritoneal macrophages

Fraconated fresh

mouse splenocyte

ED∼10–1,000 μg/ml Promoted cell proliferaon, comitogenic

eect and IFN-γ/IL-4 secreon

RAW 264.7 murine

macrophage cell

ED∼100, 300 and

500 μg/ml

Induced NO/ROS producon and TNF-α

secreon; induced the phosphorylaon

of three MAPKs (ERK, JNK and p38) and

nuclear translocaon of NF-κB; secreon of

TNF-α were inhibited by an-TLR2 mAb

Puried polysaccharide

(93k Da)

An-tumor eect SGC7901 cells

implanted nude mice

ED∼50, 75 and 100

mg/kg BW/day

–Fan et al.

(2012)

Immunomodulatory

eect

Spleen lymphocyte

and Macrophage

ED∼25–400 g/mL –

Puried polysaccharide

(111.9 kDa)

An-oxidave

stress acvity,

L-glutamic acid-damaged

HT22 hippocampal

neuronal cells

ED∼5 or 10 μg/mL Reduced the release of lactate dehydrogenase;

restored the dissipated mitochondrial

membrane potenal; enhanced levels of

Bcl-2, Nrf2, HO-1, SOD-1, and cysteine

ligase catalyc subunit and suppressed the

excess accumulaon of intracellular ROS

Han et al.

(2019)

An-apoptoc acvity Inhibited cellular apoptosis and caspase-3

acvity; reduced levels of Bax and Keap1

An-Alzheimer’s

disease eect

APP/PS1 transgenic mice ED∼25 or 50 mg/

kg BW/day (oral)

Improved the pathological behaviors related to

memory and cognion; reduced the deposion

of β-amyloid pepdes and neuronal ber tangles

induced by enhanced phosphor-Tau in the brain;

modulated the levels of an- and prooxidave

stress enzymes; Enhanced the expression levels

of Nrf2 and its downstream proteins, including

HO-1 and SOD-1, in the brains of APP/PS1 mice

Puried polysaccharide

(45 kDa)

An-proliferaon

acvity

LLC1 Lewis lung cancer cell ED∼0.1 or 1 mg/mL Acvated AMPK via phosphorylaon of

threonine 172 by LKB1; downregulates

Bcl-2, upregulates Bax; enhances

cleavage of Caspase-3 and PARP

Jiang et al.

(2019)

An-tumor eect LLC1 cells implanted

C57BL/6J mice

ED∼50 mg/kg BW/

day (intraperitoneal

injecon)

Inhibited allogra tumor growth

Table 7. Bioacvies of polysaccharides and other compounds puried from chaga - (connued)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Compounds Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

Crude polysaccharide An-proliferaon

acvity

A549 human non-small

cell lung cancer cell

ED∼50 and

100 µg/ml

Suppressed the migraon and invasive ability

of A549 cells throughout reducing MMP

expression and inhibing NF-κB nuclear

translocaon and phosphorylaon of JNK/AKT

Lee et al.

(2017a)

Crude polysaccharide An-proliferaon

acvity

B16-F10 mouse

melanoma cell

ED∼50 and

100 µg/ml

No eects on migraon of B16-F10

cells; inhibited the invasion of B16-F10

cells and suppressed the expression of

MMPs (2/7/9); inhibited NF-κB nuclear

translocaon; inhibited the phosphorylaon

of c-Jun N-terminal kinases and AKT

Lee et al.

(2016)

Crude polysaccharide An-proliferaon

acvity

B16-F10 mouse

melanoma cell

ED∼25, 50 and

100 µg/ml

Suppressed the migraon and invasive ability

of B16-F10 cells and decreased the expression

levels and acvies of MMP-2 and MMP-

9; decreased the phosphorylaon levels of

MAPKs (ERK, JNK and p38); decreased the

expression level of COX-2, and inhibited

the nuclear translocaon of NF-κB;

Lee et al.

(2014b)

Crude polysaccharide An-proliferaon

acvity

A549 human non-small

cell lung cancer cell

ED∼25, 50 and

100 µg/ml

Suppressed the migraon and invasive ability

of A549 cells; decreased the expression levels

and acvity of MMP-2 and MMP-9; decreased

the phosphorylaon levels of MAPKs and PI3K/

(AKT) as well as the expression level of COX-2,

and inhibited the nuclear translocaon of NF-κB

Lee et al.

(2014c)

Crude polysaccharide An-proliferaon

acvity

U251 human

Neurogliocytoma Cells

ED∼25–500 µg/ml Decreased the expression of Bcl-2 and

increased the expression of caspase-3

Ning et al.

(2014)

Crude polysaccharide An-proliferaon

acvity

Human T lymphadenoma

jurkat cell and human B

lymphadenoma daudi cell

ED∼0.7–200 µg/ml –Chen et al.

(2010)

An-tumor eect Jurkat tumor cells-

implanted Balb/c-nu/

nu nude mice

ED∼50 and 100 mg/

kg BW/day (oral

administraon)

–

Crude protein-

polysaccharide complex

An-proliferaon

acvity

SMMC7721 hepatoma cell ED∼150 μg/ml –Mizuno et

al. (1999)

Crude polysaccharide Anhyperglycemic

acvity

α-Glucosidase

Inhibitory assay

IC50∼24.34–82.97

μg/ml

–Wang et

al. (2019)

Crude protein-

polysaccharide complex

An-hyperglycemic

eect

Type-1 diabec mice –Maintained hypoglycemic eect

for 3−48 h aer injecon

Mizuno et

al. (1999)

Table 7. Bioacvies of polysaccharides and other compounds puried from chaga - (connued)

Journal of Food Bioactives | www.isn-jfb.com 63

Peng et al. Bioactive compounds and bioactive properties of chaga

Compounds Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

Crude polysaccharide An-proliferaon

acvity

A549 human non-small

cell lung cancer cell

ED∼50 and

100 µg/ml

Suppressed the migraon and invasive ability

of A549 cells throughout reducing MMP

expression and inhibing NF-κB nuclear

translocaon and phosphorylaon of JNK/AKT

Lee et al.

(2017a)

Crude polysaccharide An-proliferaon

acvity

B16-F10 mouse

melanoma cell

ED∼50 and

100 µg/ml

No eects on migraon of B16-F10

cells; inhibited the invasion of B16-F10

cells and suppressed the expression of

MMPs (2/7/9); inhibited NF-κB nuclear

translocaon; inhibited the phosphorylaon

of c-Jun N-terminal kinases and AKT

Lee et al.

(2016)

Crude polysaccharide An-proliferaon

acvity

B16-F10 mouse

melanoma cell

ED∼25, 50 and

100 µg/ml

Suppressed the migraon and invasive ability

of B16-F10 cells and decreased the expression

levels and acvies of MMP-2 and MMP-

9; decreased the phosphorylaon levels of

MAPKs (ERK, JNK and p38); decreased the

expression level of COX-2, and inhibited

the nuclear translocaon of NF-κB;

Lee et al.

(2014b)

Crude polysaccharide An-proliferaon

acvity

A549 human non-small

cell lung cancer cell

ED∼25, 50 and

100 µg/ml

Suppressed the migraon and invasive ability

of A549 cells; decreased the expression levels

and acvity of MMP-2 and MMP-9; decreased

the phosphorylaon levels of MAPKs and PI3K/

(AKT) as well as the expression level of COX-2,

and inhibited the nuclear translocaon of NF-κB

Lee et al.

(2014c)

Crude polysaccharide An-proliferaon

acvity

U251 human

Neurogliocytoma Cells

ED∼25–500 µg/ml Decreased the expression of Bcl-2 and

increased the expression of caspase-3

Ning et al.

(2014)

Crude polysaccharide An-proliferaon

acvity

Human T lymphadenoma

jurkat cell and human B

lymphadenoma daudi cell

ED∼0.7–200 µg/ml –Chen et al.

(2010)

An-tumor eect Jurkat tumor cells-

implanted Balb/c-nu/

nu nude mice

ED∼50 and 100 mg/

kg BW/day (oral

administraon)

–

Crude protein-

polysaccharide complex

An-proliferaon

acvity

SMMC7721 hepatoma cell ED∼150 μg/ml –Mizuno et

al. (1999)

Crude polysaccharide Anhyperglycemic

acvity

α-Glucosidase

Inhibitory assay

IC50∼24.34–82.97

μg/ml

–Wang et

al. (2019)

Crude protein-

polysaccharide complex

An-hyperglycemic

eect

Type-1 diabec mice –Maintained hypoglycemic eect

for 3−48 h aer injecon

Mizuno et

al. (1999)

Compounds Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

Crude polysaccharide Anhyperglycemic and

anhyperlipidemic

eects

STZ and high-fat-

diet-induced type-2

diabec mice

ED∼900 mg/

kg BW/day (oral

administraon)

Restored the body and fat mass weight,

reduced fasng blood glucose levels, improved

glucose tolerance ability, increased hepac

glycogen level and ameliorate insulin resistance;

Enhanced the cholesterol transportaon

in the liver; increased HDL-C levels and

decreased TC, TG and LDL-C levels; improved

the anoxidant acvies of liver and alleviate

the STZ-lesioned organ ssues (liver, kidney,

and pancreas); Up-regulated expressions

of PI3K-p85, p-Akt (ser473), GLUT4

Wang et al.

(2017c)

Crude polysaccharide

(46–41,508 kDa)

Anhyperglycemic, an-

inammatory and an-

oxidave stress eects

STZ-induced diabec mice ED∼50 mg/kg

BW/day (oral

administraon)

Increased the insulin and pyruvate kinase

levels in serum; improved the synthesis

of glycogen; restored the serum levels of

SOD, CAT, GPx, and MDA; down-regulated

IL-2R and MMP-9, and enhanced IL-2 level;

decreased the expression of phosphor-NF-

κB in the kidneys; repaired the damage on

kidney ssues, inhibited inammatory inltrate

and extracellular matrix deposit injuries

Wang et al.

(2017b)

Crude polysaccharide

of submerged cultures

Anhyperglycemic,

anhyperlipidemic, and

anoxidant eects

Alloxan-induced type-

1 diabec mice

ED∼150 and 300

mg/kg BW/day (oral

administraon)

Reduced blood glucose level; decreased serum

contents of free fay acid, TC, TG, and LDL-C;

increased HDL-C, insulin levels, and hepac

glycogen contents in the liver; increased CAT,

SOD, and GPx acvies and decreased MDA

level; restored the damage of pancreac ssues

Xu et al.

(2010b)

Crude polysaccharide Anhyperglycemic

eects

STZ-induced diabec mice ED∼10–30 mg/

kg BW/day (oral

administraon)

Restored the altered in vivo glycoprotein

components; diminished the focal necrosis,

congeson in central vein; protect β-

cells from selecve destrucon

Diao et al.

(2014)

Polysaccharides-

Cr(III) complex

Anhyperglycemic and

anhyperlipidemic

eects

STZ and high-fat-

diet-induced type-2

diabec mice

ED∼300, 600, and

900 mg/kg BW/day

(oral administraon)

Improved the glucose tolerance capacity;

promoted the metabolism of glucose and

synthesis of glycogen; reduced TG, TC, LDL-C

levels; promoted the acvies of SOD, CAT,

GPx and reduced the MDA levels in liver;

ameliorated severe pathological kidney damages

including mesangial expansion, glomeruli

partly sclerosis and glomerular hypertrophy

Wang et al.

(2017a)

β-pyran-type puried

polysaccharide

fracons (200 kDa)

Anhyperglycemic

acvity

HepG2 Cell and insulin

resistant HepG2 Cell

ED∼10–40 μg/ml Increased the glucose consumpon in both

HepG2 Cell and insulin resistant HepG2 Cell

Xue et al.

(2018)

α-pyran-type puried

polysaccharide (20 kDa)

Table 7. Bioacvies of polysaccharides and other compounds puried from chaga - (connued)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Compounds Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

α/β-type puried

polysaccharide (13.6 kDa)

–Liu et al.

(2018)

β-type puried

polysaccharide (15.2 kDa)

–

α/β-type puried

polysaccharide (13.6 kDa)

Anhyperglycemic

eects

STZ-induced diabec mice ED∼4.5 mg/

kg BW/day (oral

administraon)

–Liu et al.

(2018)

α-Glucosidase

inhibitory acvies

α-Amylase inhibitory assay IC50∼7.875 μg/ml –

β-type puried

polysaccharide (15.2 kDa)

Anhyperglycemic

eects

STZ-induced diabec mice ED∼4.5 mg/

kg BW/day (oral

administraon)

–

α-Glucosidase

inhibitory acvies

α-Amylase inhibitory assay IC50∼3.841 μg/ml –

Puried polysaccharide

(105.02 kDa)

α-Amylase and

α-glucosidase

inhibitory ability

α-Amylase and

α-glucosidase

inhibitory assays

ED∼40–200 μg/ml –Wang et al.

(2018b)

Polysaccharides-

chromium (III)

complex (115 kDa)

α-Amylase and

α-glucosidase

inhibitory ability

α-Amylase and

α-glucosidase

inhibitory assays

ED∼3.0 mg/mL –Wang et al.

(2018a)

Puried polysaccharide

(97.12 kDa)

α-Amylase

inhibitory ability

α-Amylase inhibitory assay IC50∼482.49 μg/ml –Wang et al.

(2018c)

α-Glucosidase

inhibitory ability

α-Glucosidase

inhibitory assay

IC50∼51.47 μg/ml –

H2O2-induced hemolysis

inhibitory assay

Hemolysis inhibitory assay IC50∼47.63 μg/ml –

Puried polysaccharide

(114.30 kDa)

α-Amylase

inhibitory ability

α-Amylase inhibitory assay IC50∼2.83 mg/ml –

α-Glucosidase

inhibitory ability

α-Glucosidase

inhibitory assay

IC50∼159.73 μg/ml –

H2O2-induced hemolysis

inhibitory assay

Hemolysis inhibitory assay IC50∼58.53 μg/ml –

Puried polysaccharide

(75.94 kDa)

α-Glucosidase

inhibitory ability

α-Glucosidase

inhibitory assay

IC50∼55.20 μg/ml –

H2O2-induced hemolysis

inhibitory assay

Hemolysis inhibitory assay IC50∼51.53 μg/ml –

Table 7. Bioacvies of polysaccharides and other compounds puried from chaga - (connued)

Journal of Food Bioactives | www.isn-jfb.com 65

Peng et al. Bioactive compounds and bioactive properties of chaga

Compounds Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

Crude polysaccharide An-obesity and

probioc eects

High-fat diet fed

C57BL6/J mice

ED∼1,000 mg/

kg BW per day

Improved the obesity of mice, including the

adjustment of body weight gain, energy intake,

energy eciency, liver glucose metabolism and

triglyceride metabolism, tricarboxylic acid (TCA)

cycle, and degradaon of three major nutrients

(carbohydrate, lipid, and protein); Improved the

level of cecal butyrate by Lactobacillus and the

Bacteroidales S24-7 group, resulng in increased

energy consumpon, and fat degradaon

by regulang the TCA cycle of the host

Yu et al.

(2020)

Puried polysaccharide

(32.5 kDa)

An-inammaon,

anoxidave stress

and probioc eects

DDC-induced chronic

pancreas in mice

ED∼100, 200, 400

mg/kg BW/day (oral

administraon)

Increased GPx and TAOC levels in pancreas,

and decreased TNF-α, TGF-β, lipase and trypsin

levels in serum; Increased the proporon of

Bacteroidetes and decreased that of Firmicutes

at phylum level; maintained the microbiota

structure and richness to normal level

Hu et al.

(2017b)

Puried polysaccharide An-fague eect Forced sports test of

male Kunming mice

ED∼50 mg/kg

BW/day (oral

administraon)

Increase the climbing duraon and swimming

me as well as reduced the immobility me;

Decreased the level of blood lacc acid, urea

nitrogen and lacc dehydrogenase; Decreased

the 5-HT concentraons in the mice brain

Zhang et

al. (2020)

Crude polysaccharide An-fague eect Forced sports test of

male Kunming mice

ED∼100, 200, 300

mg/kg BW/day (oral

administraon)

Extended the swimming me; Enhanced liver

and muscle glycogen content; Decreased the

level of blood lacc acid and urea nitrogen

Zhong et

al. (2015)

Puried polysaccharide

(32.5 kDa)

An-virus FHV-infected CRFK cells IC50∼18.15 μg/ml Shoed a low cytotoxicity to CRFK and

MDCK cells and broad-spectrum anviral

acvity against feline calicivirus

Tian et al.

(2017)

FPV-infected CRFK cells IC50∼45.33 μg/ml

FIPV-infected CRFK cells IC50∼22.87 μg/ml

H5N6-infected MDCK cells IC50∼68.47 μg/ml

H3N2-infected MDCK cells IC50∼48.51 μg/ml

Other compounds

Pepde

Trp-Gly-Cys Platelet aggregaon

inhibitory acvity

83.3% Platelet

aggregaon inhibitory

acvity in collagen/

epinephrine-induced

thromboc ICR mice,

– – Hyun et

al. (2006)

Melanin

Table 7. Bioacvies of polysaccharides and other compounds puried from chaga - (connued)

Journal of Food Bioactives | www.isn-jfb.com

Bioactive compounds and bioactive properties of chaga Peng et al.

Compounds Bioacvity Model IC50 value or experi-

mental dosage (ED) Mechanism or manifestaon Reference

Puried melanin-

polysaccharide

complex (<10 kDa)

An-hemolysis acvity Sheep erythrocytes IC50∼4.9–8.4 µg/ml –Wold et

al. (2020)

An-inammatory

acvity

LPS + IFNγ-acvated

C57BL/6 primary

macrophages

IC50∼24.1 ±

7.9 µg/ml

Reduced NO producon

Anoxidant acvity DPPH radical

scavenging assay

IC50∼61.4 μg/ml –

An-proliferaon

acvity

CI-H460 and HT29-MTX IC50 > 50 μg/ml –

Puried melanin

(2–20 kDa or 90–100

kDa or more)

Anoxidant acvity Total anoxidant assay;

DPPH, ABTS and hydroxyl

radical assays; FRAP and

Fe2+ chelaon assays;

β-carotene bleaching assay

– – Olennikov

et al. (2012)

Crude melanin Probioc acvity Bidobacterium bidum

1 and Bidobacterium

animalis subsp. lacs

ED∼10−10, 10−7,

10−2 mg/cm3

–Burmasova

et al. (2019)

Anoxidant acvity Total anoxidant assay

(phosphomolybdate

method)

– –

Crude melanin Anoxidant acvity Total anoxidant assay

(phosphomolybdate

method) and Ferric

Ions reducon assay

(phenanthroline method)

– – Parfenov et

al. (2019)

Hepatoprotecve

acvity

D-Galactosamine-

treated normal human

(Chang) Liver cell

– –

Hepatoprotecve eect Tetrachloromethane-

treated Sprague

Dawley rats

ED∼100 mg/

kg BW/day

Decreased steatosis, necrosis, fat accumulaon,

and normalized various indicators including

the total and unconjugated bilirubin,

total protein, serum cholinesterase, and

γ-glutamyl transpepdase levels

HFD: high-fat diet; STZ: streptozotocin; MDA: maleic dialdehyde; TC: total cholesterol; TG: triglyceride; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; CAT: catalase; SOD: superoxide

dismutase; GPx: glutathione peroxidase; GSH: glutathione; TBARS: thiobarbituric acid-reacve substances; ALT: alanine aminotransferase; AST: aspartate aminotransferase; BW: body weight; CYP: cyclophosphamide; DDC:

diethyldithiocarbamate; TAOC: total anoxidant capacity; AMS: amylase; MMP: matrix metalloproteinase; MSPKs: mitogen-acvated protein kinases; PI3K: phosphoinoside 3-kinase; AKT: protein kinase B; ERK: extracellular

signalregulated protein kinase; JNK: c-Jun N-terminal kinase; P38: Cytokinin Specic Binding Protein (CSBP); MAPKs: mitogen-acvated protein kinases; NF-κB: nuclear factor κB p65; COX: cyclooxygenase, IL-2R: interleukin-2

receptor; Bax: Bcl-2 associated X protein; Keap1: Kelch-like ECH-associated protein 1; Bcl-2: B-cell lymphoma-2; Nrf2: NF-E2p45-related factor 2; HO-1: heme oxygenase-1; APP/PS1: amyloid precursor protein/presenilin 1 ; NO:

nitric oxide; IL-6: interleukin-6; IL-1β: interleukin-1β; INF-γ: interferon-γ; IL-4: interluekin-4; TLR2: toll-like receptor 2; TLR4: toll-like receptor 4; IκBα: inhibitor kappaBα of NF-κB, or nuclear factor of kappa light polypepde gene

enhancer in B-cells inhibitor alpha; TGF-β: transforming growth factor; Fox-p3: forkhead box; ROR-γt renoic acid-related orphan receptor; STAT-3: signal transducer and acvator of transcripon; STAR: steroidogenic acute

regulatory protein; NQO-1: NADPH quinoneoxidoreductase-1; p-AKT: phospho-protein kinase B; p-mTOR: phospho-mammalian target of rapamycin; Nrf2: erythroid 2-related factor 2; GM-CSF: granulocyte macrophage-colony

smulang factor.

Table 7. Bioacvies of polysaccharides and other compounds puried from chaga - (connued)

Journal of Food Bioactives | www.isn-jfb.com 67

Peng et al. Bioactive compounds and bioactive properties of chaga

well as the physiochemical properties of non-nitrogenous melanin

are somewhat similar to those of lignin (Babitskaya et al., 2000;

Kukulyanskaya et al., 2002; Solano, 2014; Varga et al., 2016).

Kukulyanskaya et al. (2002) suggested that the melanin in wild

chaga was allomelanin while in cultured ones was eumelanin

according to the dierence in their mole ratio of C/N. In fungi,

the allomelanin is believed to be mainly composed of 1,8-dihy-

droxynaphthalene (DHN)/tetrahydroxynaphthalene, while for eu-

melanin was L-3,4-dihyroxyphenylalanine (L-DOPA) (Eisenman

and Casadevall, 2012; Plonka and Grabacka, 2006). One review

mentioned around 17 amino acids hydrolyzed from eumelanin of

cultured chaga, but reliability of this data needs to be conrmed

(Balandaykin and Zmitrovich, 2015). The allomelanin of wild

chaga is known to be heterogeneous and contains aromatic meth-

oxy group, carboxyl group, pyrocatechol, along with the phenolic

hydroxyl group (Olennikov et al., 2012; Wold et al., 2020). How-

ever, no original study has successfully claried the exact units

and linkages of chaga allomelanin. In fungi, melanin granules are

localized in the cell wall, where they are likely cross-linked to

polysaccharides, protein, or lignin. It contributes to the strength-

ening of the fungus cell wall and their defence mechanisms

against harsh environmental conditions, such as ultraviolet radia-

tion, extreme temperatures, free radicals, toxic heavy metal, and

enzymatic degradation (Eisenman and Casadevall, 2012; Gómez

and Nosanchuk, 2003; Varga et al., 2016). In addition, the fun-

gus melanin could promote the penetration and invasion ability

against the plant host by providing mechanical strength to the ap-

pressoria (Eisenman and Casadevall, 2012). So far, various MWs

of dierent melanin fractions have been reported. Babitskaya et

al. (2000) reported that the MWs of melanin from cultured and

wild chaga mainly ranged from 50 to 60 kDa, and the MWs of

a minor amount of the other melanin fractions went up to 100 or

even several hundred daltons. Similarly, Olennikov et al. (2012)

isolated dozens of fractions of puried chaga melanin, the MWs

of which mainly ranged from 2 to 20 kDa, and the rest were more

than 100 kDa. The melanin fraction in the study of Wold et al.

(2018) had a MW range of 10–31 kDa because it was detected

in a polysaccharide fraction of similar MW range. Meanwhile,

this study suggested that melanin was not covalently bound to

the polysaccharides (Wold et al., 2018). More recently, Wold et

al. (2020) specically measured the approximate polymer size of

the melanin fraction to be less than 10 kDa. Furthermore, their

study strengthened the hypothesis that melanin from wild chaga

was allomelanin according to the analysis of the combustion and

chemical degradation constituents, though still none of these deg-

radation products were isolated and identied. Meanwhile, GC

analysis of melanin hydrolysis of chaga showed that around 5%

polysaccharides existed in the melanin after repeated sedimenta-

tion purication, which demonstrated that the sugars were cova-

lently bound to the melanin polymer (Wold et al., 2020). The mel-

anin is also considered a main bioactive compound in the water

extract of chaga. Besides the antioxidant nature of chaga melanin,

its hepatoprotective, probiotic, anti-hemolysis, anti-inammatory,

and anti-proliferation activities have also been studied (Table 7).

Table 6 presents more categories under “Other compounds”, in-

cluding various alkaloids, organic acids, organic acid esters, al-

kanes, alcohols, aldehydes, and amino acids. Chaga also contains

an abundance of mineral microelements. Chen et al. (2009) quan-

tied 12 microelements (in μg/g) in chaga including 22.41 boron,

726.00 calcium, 0.21 cobalt, 0.58 chromium, 5.55 copper, 213.33

iron, 1,127.80 magnesium, 117.84 manganese, 0.88 nickel, 0.18

selenium, 12.90 strontium, and 88.13 zinc, which indicated the

possibility that chaga and its products might act as a candidates

for mineral supplementation.

5. Conclusion

Chaga is recorded with numerous historical applications and anec-

dotal evidence of medicinal properties worldwide. The studies of

bioactivities of chaga along with the latest technologies/methodol-

ogies and prevalence of “open access” policy of scientic journal

may ourish even further. As summarized, the in vivo/in vitro bio-

active properties of chaga include anti-proliferation, anti-tumor,

immunomodulatory, anti-inammation, antioxidant, antimuta-

genic, analgesic, anti-virus, antibacterial, antifungal, antibacterial,

antihyperglycemic, anti-platelet-aggregation, anti-hypertension,

anti-hyperuricemia, anti-obesity, probiotic, hepatoprotective, and

enzyme inhibitory activities/eects. These bioactivity studies ex-

tend the understanding of pharmaceutical values of chaga and po-

tentiate its future application in modern medicine if more rigorous

biological/clinical studies could be conducted. In recent decades,

the investigations of the chemical diversity of chaga have also

achieved remarkable progress. The main secondary metabolites of

fungi such as terpenoids, phenolics, polysaccharides, and melanin

have been identied in various chaga extracts. They are considered

as the main contributors to their wide spectrum of bioactivities.

However, compared with small-molecule compounds, a further

characterization of specic structures of bioactive polymers in

chaga, such as polysaccharide, lignin, and melanin, is still needed.

Besides, to a great extent, the use of chaga has been guided by the

folk experience or obsolete data, and the reported cases showing

potential adverse health eects have provoked serious safety con-

cerns in administrating wild chaga and its products. On the other

hand, the compositional variation among chaga samples (wild/

wild; wild/cultivated; cultivated/cultivated) are inuencing judge-

ment of both their safety and eectiveness. The standardized qual-

ity control based on fast detection technologies, and the dosage

guideline under the promise of sucient preclinical/clinical data

of its acute and chronic toxicity are most urgently needed.

Funding

This work was supported by the NSERC (Natural Sciences and

Engineering Research Council) and CSC (China Scholarship

Council).

Conict of interest

There is no conict interest.

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Lignin-Based Hydrogels for the Delivery of Bioactive Chaga Mushroom Extract

Article

Full-text available

Mar 2024

Lignin-poly(ethylene)glycol diglycidyl ether hydrogels were synthesized from lignin fractions readily extracted during the hot-water treatment of angiosperms: hardwoods, sugar maple and energy-crop willow, monocotyledons, grasses, miscanthus and agriculture residues, and wheat straw. These lignins represent a broad range of chemical structures and properties as a comparative analysis of their suitability to produce the hydrogels as a novel carrier of chaga–silver nanoparticles. The formation of hydrogels was assessed via attenuated total reflectance Fourier-transformed infrared spectroscopy. Then, the hydrogels were observed via scanning electron microscopy and evaluated for their free-absorbency capacity and moduli of compression. Furthermore, a hydrogel produced from kraft lignin and two commercial hydrogels was evaluated to benchmark the effectiveness of our hydrogels. Chaga extracts were prepared via the hot-water extraction of chaga mushroom, a method selected for its relatively higher yields and preserved antioxidizing activities. Hydrogels synthesized with lignins of monocotyledons, wheat straw, and miscanthus were found to be suitable carriers for chaga–silver nanoparticles due to their favorable absorption and release behaviors.

Cloning and functional characterization of sesquiterpene synthase genes from Inonotus obliquus using a Saccharomyces cerevisiae expression system

Article

Full-text available

Jan 2025
WORLD J MICROB BIOT

Inonotus obliquus (Chaga mushroom) is a large medicinal and edible fungus that contains a wealth of bioactive terpenoids. However, the detection of certain low-abundance sesquiterpenoids remains a challenge due to limitations in extraction and analytical techniques. Furthermore, the synthase genes responsible for the biosynthesis of the identified terpenoids have not yet been clearly elucidated. To address this, our study combined transcriptome mining with yeast heterologous expression to investigate the synthase genes involved in sesquiterpenoid production in I. obliquus. We successfully identified eight sesquiterpene synthase genes and one farnesyltransferase. Among these, only cis-β-farnesene, synthesized by IoTPS2, had been previously detected before in the sclerotium of I. obliquus, while the other nine sesquiterpenoids—including neoisolongifolene-8-ol, β-longipinene, vetiselinenol, isolongifolene, 7,8-dehydro-8a-hydroxy-, 4a,8b,10b,11a-tetramethylbicyclo[6.3.0]undec-1-en-5-one, 6,11-oxido-acor-4-ene, β-maaliene, neointermedeol, and longifolenaldehyde—were discovered for the first time. This research provides a critical scientific foundation for expanding the known repertoire of sesquiterpenoids and their corresponding synthase genes in I. obliquus.

A heart-rot fungus, Inonotus obliquus (chaga), mediates microhabitat creation in birch snags and contributes to forest fungal diversity

Article

Dec 2024

Bioactivity, pharmacological research, and application of birch fungus

Article

Nov 2024

Birch fungus, known as birch brown porcupine fungus (Inonotus obliquus), is a rare medicinal fungus that grows in the Frigid Zone. This study reviews the bioactive components of birch mushrooms, including polysaccharides, triterpenoids, polyphenols, flavonoids, etc., and discusses in detail their pharmacological effects in antitumor, hypoglycemic, anti-inflammatory, antioxidant and immunomodulatory aspects. This paper also outlines the application prospects of birch mushrooms in the fields of medicinal nutraceuticals and functional foods, aiming to provide reference for the in-depth research and development of birch mushrooms.

Protoilludene and Alkenoic Acid Derivatives from the European Polypore Fomitiporia hartigii

Article

Jul 2024

Chemical investigation of the solid-state rice culture of the endangered European polypore Fomitiporia hartigii (Hymenochaetaceae) afforded a previously undescribed protoilludene derivative (1) in addition to six known compounds (2−7). Chemical structures of the isolated compounds were established based on HR-ESI-MS, comprehensive 1D/2D NMR spectroscopic analyses, and comparisons with the literature. All isolated compounds were assessed for their cytotoxic and antimicrobial activities. Among the tested compounds, hymeglusin (3) revealed potent cytotoxic activity against all tested cell lines with IC 50 values between 0.3 and 6.8 μM. Compound 3 and fusaridioic acid A (4) revealed weak to moderate antimicrobial activities with its most potent effect against Candida albicans (minimum inhibitory concentration of 4.2 μg/mL).

Therapeutic Potential of Fungal Terpenes and Terpenoids: Application in Skin Diseases

Article

Full-text available

Mar 2024
MOLECULES

Terpenes and their derivatives comprise a diverse group of natural compounds with versatile medicinal properties. This article elucidates the general characteristics of fungal terpenes and terpenoids, encompassing their structure and biogenesis. The focal point of this work involves a comprehensive overview of these compounds, highlighting their therapeutic properties, mechanisms of action, and potential applications in treating specific skin conditions. Numerous isolated terpenes and terpenoids have demonstrated noteworthy anti-inflammatory and anti-microbial effects, rivalling or surpassing the efficacy of currently employed treatments for inflammation or skin infections. Due to their well-documented antioxidant and anti-cancer attributes, these compounds exhibit promise in both preventing and treating skin cancer. Terpenes and terpenoids sourced from fungi display the capability to inhibit tyrosinase, suggesting potential applications in addressing skin pigmentation disorders and cancers linked to melanogenesis dysfunctions. This paper further disseminates the findings of clinical and in vivo research on fungal terpenes and terpenoids conducted thus far.

Discovery and Relevance of Novel Pharmacological Substances from Beneficial Mushrooms

Chapter

Mar 2025

Chestnut flower extract as a natural inhibitor of Fusarium graminearum: antifungal activity and mechanisms

Article

Full-text available

Feb 2025
PEST MANAG SCI

BACKGROUND Plant pathogenic fungi are a major contributor to reductions in crop yield and quality, posing significant challenges to global food security. The extensive application of chemical fungicides has led to the development of resistance in pathogenic fungi and the accumulation of harmful residues, which threaten environmental sustainability and human health. Plant‐derived fungicides, with low toxicity and broad‐spectrum activity, offer an eco‐friendly alternative to synthetic chemicals. RESULTS This study evaluated the antifungal activity of chestnut flower extract (CFE) against Fusarium graminearum (FG) and investigated its underlying mechanisms through cellular and transcriptomic analyses. Liquid chromatography‐mass spectrometry (LC–MS) identified phenolic acids and flavonoids as the primary active constituents of CFE. CFE inhibited mycelial growth and spore germination with median effective concentration (EC50) values of 2.51 and 0.39 mg mL⁻¹, respectively. It disrupted biofilm integrity and membrane permeability by reducing ergosterol content, increasing extracellular conductivity, and affecting malondialdehyde (MDA) levels. Protein and nucleic acid leakage were observed. Additionally, CFE inhibited energy metabolism by reducing adenosine triphosphate (ATP) levels and suppressing the activities of key respiratory enzymes, including succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and nicotinamide adenine dinucleotide (NADH) dehydrogenase. Transcriptomic analysis further revealed that CFE affected multiple biological processes in FG, including cell structure, protein synthesis, ion transport and mitochondrial function. CONCLUSION The chestnut flower contains active antibacterial ingredients that exhibit targeted inhibition of FG, thereby providing theoretical and technical support for the development of natural antibacterial agents with specific targeting capabilities. © 2025 Society of Chemical Industry.

The anti-diabetic potential of mushrooms: a review The Anti-Diabetic Potential Of Mushrooms: A Review

Article

Full-text available

Oct 2023

Diabetes is emerging as a pandemic, so it is essential to find new nutraceuticals or drugs to treat or prevent it. Some mushrooms appear to be effective in controlling blood glucose levels and correcting diabetes problems without any negative side effects. Mushrooms with immune-modulating polysaccharides are used in limited areas as palatable food or health-promoting dietary supplements or medicine. However, to date, scientific or clinical research on mushrooms has not been sufficient to allow them to be used as recognized medicines or nutraceuticals worldwide. These functional fungi may have a greater impact on the prevention and treatment of diabetes. Therefore, further studies are needed to identify their active compounds for improving diabetes drugs or nu-traceuticals. This review focuses on prospective mushrooms that have demonstrated anti-diabetic effects in clinical or experimental studies and prevent or slow the progression of diabetes mellitus

The Health and Clinical Benefits of Medicinal Fungi

Article

Jul 2023
ADV BIOCHEM ENG BIOT

Christopher Hobbs

The human uses of mushrooms and cultured mycelium products for nutrition and medicine are detailed and supported by available human studies, which in many cases are clinical trials published in peer-reviewed journals. The major medically active immunomodulating compounds in the cell walls-chitin, beta-glucans, and glycoproteins, as well as lower weight molecules-nitrogen-containing compounds, phenolics, and terpenes-are discussed in relation to their current clinical uses. The nutritional content and foods derived from mushrooms, particularly related to their medical benefits, are discussed. High-quality major nutrients such as the high amounts of complete protein and prebiotic fibers found in edible and medicinal fungi and their products are presented. Mushrooms contain the highest amount of valuable medicinal fiber, while dried fruiting bodies of some fungi have up to 80% prebiotic fiber. These fibers are particularly complex and are not broken down in the upper gut, so they can diversify the microbiome and increase the most beneficial species, leading to better immune regulation and increasing normalizing levels of crucial neurotransmitters like serotonin and dopamine. Since the growth of medicinal mushroom products is expanding rapidly worldwide, attention is placed on reviewing important aspects of mushroom and mycelium cultivation and quality issues relating to adulteration, substitution, and purity and for maximizing medicinal potency. Common questions surrounding medicinal mushroom products in the marketplace, particularly the healing potential of fungal mycelium compared with fruiting bodies, extraction methods, and the use of fillers in products, are all explored, and many points are supported by the literature.

Cecal Butyrate (Not Propionate) Was Connected with Metabolism-Related Chemicals of Mice, Based on the Different Effects of the Two Inonotus obliquus Extracts on Obesity and Their Mechanisms

Article

Full-text available

Jun 2020

Obesity is a metabolic disease and causes significant changes in host and gut microbial metabolite levels. However, little research has been done on the relationship between host and gut microbial metabolites. Thus, this study investigated the connection of the chemicals, based on the different effects of two Inonotus obliquus extracts on high-fat-diet-induced mice and their mechanisms. In this study, C57BL6/J mice fed with a high-fat diet were given I. obliquus ethanol extract (IOE) and polysaccharide (IOP). ¹H NMR-based metabolomics, 16S rRNA sequencing, and real-time reverse transcription polymerase chain reaction (RT-PCR) were used to detect metabolites, cecal microbes, and expressions of genes in liver. IOE and IOP effectively improved the obesity of mice, including the adjustment of body weight gain, energy intake, energy efficiency, liver glucose metabolism and triglyceride metabolism, tricarboxylic acid (TCA) cycle, and degradation of three major nutrients (carbohydrate, lipid, and protein). IOE significantly increased cecal propionate based on Bacteroides and Akkermansia, thereby inhibiting energy intake and fat accumulation in mice. IOP remarkably improved the level of cecal butyrate by Lactobacillus and the Bacteroidales S24-7 group, resulting in increased energy consumption, and fat degradation by regulating the TCA cycle of the host. Two extracts containing different bioactive substances of I. obliquus improved obesity in mice through different effects on production of cecal microbial metabolites. Moreover, cecal butyrate (not propionate) was connected with chemicals of mice, including four metabolites of the TCA cycle and other metabolism-related chemicals.

Genoprotective effects of Chaga mushroom (Inonotus obliquus) polysaccharides in UVB-exposed embryonic zebrafish (Danio rerio) through coordinated expression of DNA repair genes

Preprint

Full-text available

Jun 2020

Background Chaga mushroom (Inonotus obliquus) is one of the most promising antioxidants with incredible health-promoting effects. Chaga polysaccharides (IOP) have been reported to enhance immune response and alleviate oxidative stress during development. However, the effects of IOP on the genotoxicity in model organisms are yet to be clarified. Methods Zebrafish embryos (12 hours post fertilization, hpf) were exposed to transient UVB (12 J/m ² /s, 310 nm) for 10 secs using a UV hybridisation chamber, followed by IOP treatment (2.5 mg/mL) at 24 hpf for up to 7 days post fertilization (dpf). The genotoxic effects were assessed using acridine orange staining, alkaline comet assay, and qRT-PCR for screening DNA repair genes. Results We found significant reduction in DNA damage and amelioration of the deformed structures in the IOP-treated zebrafish exposed to UVB (p < 0.05) at 5 dpf and thereafter. In addition, the relative mRNA expressions of XRCC-5, XRCC-6, RAD51, P53, and GADD45 were significantly upregulated in the IOP-treated UVB-exposed zebrafish. Pathway analysis demonstrated coordinated regulation of DNA repair genes, suggesting collective response during UVB exposure. Conclusions Overall, IOP treatment ameliorated the genotoxic effects in UVB-exposed zebrafish embryos, which eventually assisted in normal development. The study suggested the efficacy of Chaga mushroom polysaccharides in mitigating UV-induced DNA damage.

Bioactive triterpenoids and water-soluble melanin from Inonotus obliquus (Chaga) with immunomodulatory activity

Article

Full-text available

Aug 2020

The fungus Inonotus obliquus has historically been used in traditional medicine in Europe and Asia. A melanin fraction and six triterpenoids were obtained from I. obliquus sclerotia, and evaluated in various bioassays including for immunomodulatory, cytotoxicity and enzyme-interacting properties. The water-soluble, nitrogen-free melanin fraction and the triterpenoids 3β-hydroxy-8,24-dien-21-al (1) and inotodiol (2) displayed potent activity in a human complement assay. The melanin fraction inhibited the complement cascade, whereas 1 and 2 activated the same cascade. Compound 2, as well as betulinic acid (3) and betulin (4) had anti-proliferative properties against the colon adenocarcinoma cell line HT29-MTX. Further, the melanin fraction and betulin-3-O-caffeate (6) reduced nitric oxide production in primary murine macrophages. Furthermore, the metabolites were nontoxic against the common gut bacteria E. coli and B. subtilis. The results demonstrate the anti-inflammatory and immunomodulatory effects of I. obliquus melanin and triterpenoids, which could potentially justify the consumption of this increasingly popular “edible” fungus.

Molecular insights and cell cycle assessment upon exposure to Chaga (Inonotus obliquus) mushroom polysaccharides in zebrafish (Danio rerio)

Article

Full-text available

May 2020

chaga (Inonotus obliquus) mushroom is considered as one of the most powerful antioxidants across the world. Though the therapeutic effects of Chaga components are well characterized in vitro, the in vivo developmental effects are not elucidated in detail. In this study, we assessed the in vivo developmental effects of Chaga polysaccharides in zebrafish, along with revealing the effects on cell cycle and apoptosis. chaga mushroom polysaccharides comprised xylulose, rhamnose, mannose, glucose, inositol, and galactose, in addition to phenolic compounds; zebrafish embryos exhibited normal embryonic development upon transient exposure to Chaga extract (24 hours). Most embryos (>90%) were found to be healthy even at high concentrations (5 mg/mL). In addition, staining with the DnA binding dye, acridine orange showed that chaga polysaccharides alleviated oxidative stress. flow cytometric analysis using H2DCFDA that specifically binds to cells with fragmented DNA showed significantly reduced levels of intracellular reactive oxygen species (ROS) (p < 0.05), which in turn reduced apoptosis in the developing embryos. cell cycle analysis by measuring the DnA content using flow cytometry revealed that Chaga polysaccharides moderately arrested the cells at G1 stage, thereby inhibiting cell proliferation that can be further explored in cancer studies. overall, transient exposure of chaga polysaccharide extract reduced intracellular RoS and assisted in the normal development of zebrafish.

Development of End Stage Renal Disease after Long-Term Ingestion of Chaga Mushroom: Case Report and Review of Literature

Article

Full-text available

May 2020

Chaga mushrooms are widely used in folk remedies and in alternative medicine. Contrary to many beneficial effects, its adverse effect is rarely reported. We here report a case of end-stage renal disease after long-term taking Chaga mushroom. A 49-year-old Korean man with end stage renal disease (ESRD) was transferred to our hospital. Review of kidney biopsy finding was consistent with chronic tubulointerstitial nephritis with oxalate crystal deposits and drug history revealed long-term exposure to Chaga mushroom powder due to intractable atopic dermatitis. We suspected the association between Chaga mushroom and oxalate nephropathy, and measured the oxalate content of remained Chaga mushroom. The Chaga mushroom had extremely high oxalate content (14.2/100 g). Estimated daily oxalate intake of our case was 2 times for four years and 5 times for one year higher than that of usual diet. Chaga mushroom is a potential risk factor of chronic kidney disease considering high oxalate content. Nephrologist should consider oxalate nephropathy in ESRD patients exposed to Chaga mushrooms.

Effect of Colloidal Metal Nanoparticles on Biomass, Polysaccharides, Flavonoids, and Melanin Accumulation in Medicinal Mushroom Inonotus obliquus (Ach.:Pers.) Pilát

Article

Full-text available

Jul 2020
APPL BIOCHEM BIOTECH

The article explores effect of colloidal nanoparticles (NPs) of Ag, Fe, and Mg metals on the growth activity of the medicinal mushroom Inonotus obliquus (Ach.:Pers.) Pilát and the synthesis of biologically active compounds (polysaccharides, flavonoids, and melanins). It was found that all the studied NPs stimulated growth activity. AgNPs inhibited polysaccharide and flavonoid synthesis, and stimulated melanin synthesis by 140%. Using MgNPs was effective to increase the level of accumulation of endopolysaccharides, flavonoids, and melanin pigments. FeNPs significantly increased the yield of endopolysaccharides. This effect should be used for biosynthesis stimulation for polysaccharides, flavonoids, and melanins obtaining from I. obliquus cultivated in vitro. The results demonstrate the potential of the use of metal colloidal solutions NPs for the development of environmentally friendly and effective biotechnology to produce biologically active compounds by medicinal macromycete I. obliquus.

The Influence of Submerged Fermentation of Inonotus obliquus with Control Atmosphere Treatment on Enhancing Bioactive Ingredient Contents

Article

Full-text available

May 2020
APPL BIOCHEM BIOTECH

This study led in the pioneering technique incubated in a bioreactor with the forced air injection system. The purpose of this study was to establish the optimal incubation conditions for this technique. The results showed that the speed at which Inonotus obliquus was incubated with the forced air injection system was superior to that with a normal bioreactor. A nitrogen to oxygen ratio of 50:50 provided the best results with the forced air injection system, including in terms of the achievement of biomass, total triterpenes, betulinic acid content, and the scavenging activities of DPPH radicals, which reached up to 21.3 g/1000 mL, 2.1 g/1000 mL, 1.9 g/1000 mL, and 87.3%, respectively. The results showed that the bioreactor with the forced air injection system could more effectively incubate I. obliquus by using less vapor while still utilizing a model close to that of a traditional bioreactor. The innovative bioreactor fermentation model was thus more economical than the traditional bioreactor model.

18. Bioactive foods and herbs in prevention and treatment of cardiovascular disease

Chapter

Sep 2017

T. Koyama

Inonotus taiwanensis sp. nov. (Basidiomycota) from Taiwan

Article

Sep 2018

Inonotus taiwanensis (Hymenochaetales) is described as a new species collected from southern Taiwan, and all specimens grew on the trunk of Trema tomentosa. This new species is characterized by having resupinate, effuse-reflexed to pileate basidiocarps, bright yellow context, long setal hyphae in context and trama, setae in hymenium and trama, and fairly small basidiospores. Maximum likelihood and Bayesian inference phylogenies inferred from both sequences of 28S and internal transcribed spacer (ITS) region of rDNA indicated that I. taiwanensis forms a distinct clade within Inonotus, and the species is sister to I. tricolor. The comparison of morphological differences among I. taiwanensis and some closely related Inonotus spp. is presented.

Inonotus obliquus polysaccharide ameliorates impaired reproductive function caused by Toxoplasma gondii infection in male mice via regulating Nrf2-PI3K/AKT pathway

Article

Feb 2020
INT J BIOL MACROMOL

This study was carried out to investigate the effects of Inonotus obliquus polysaccharide (IOP) on impaired reproductive function and its mechanisms in Toxoplasma gondii (T. gondii)-infected male mice. Results showed that IOP significantly improved the spermatogenic capacity and ameliorated pathological damage of testis, increased serum testosterone (T), luteinizing hormone (LH) and follicular-stimulating hormone (FSH) levels in T. gondii-infected male mice. IOP effectively up-regulated testicular steroidogenic acute regulatory protein (StAR), P450scc and 17β-HSD expressions. IOP also significantly decreased the levels of malondialdehyde (MDA) and nitric oxide (NO), but increased the activities of antioxidant enzyme superoxide dismutase (SOD) and glutathione (GSH). Furthermore, IOP up-regulated the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and NADPH quinoneoxidoreductase-1 (NQO-1), and suppressed the apoptosis of testicular cells by decreasing Bcl-2 associated x protein (Bax) and cleaved caspase-3 expressions. IOP further enhanced testicular phosphatidylinositol 3-kinase (PI3K), phospho-protein kinase B (p-AKT) and phospho-mammalian target of rapamycin (p-mTOR) expression levels. It demonstrates the beneficial effects of IOP on impaired reproductive function in T. gondii-infected male mice due to its anti-oxidative stress and anti-apoptosis via regulating Nrf2-PI3K/AKT signaling pathway.