Inonotus obliquus – from folk medicine to clinical use

Abstract

The Inonotus obliquus (I. obliquus) mushroom was traditionally used to treat various gastrointestinal diseases. For many years, mounting evidence has indicated the potential of I. obliquus extracts for treatment of viral and parasitic infections. Furthermore, substances from I. obiquus have been shown to stimulate the immune system. The most promising finding was the demonstration that I. obliquus has hypoglycemic and insulin sensitivity potential. This review summarizes the therapeutic potential of I. obliquus extracts in counteracting the progression of cancers and diabetes mellitus as well as their antiviral and antiparasitic activities and antioxidant role. As shown by literature data, various authors have tried to determine the molecular mechanism of action of I. obliquus extracts. Two mechanisms of action of I. obliquus extracts are currently emerging. The first is associated with the broad-sense impact on antioxidant enzymes and the level of reactive oxygen species (ROS). The other is related to peroxisome proliferator-activated receptor gamma (PPARγ) effects. This receptor may be a key factor in the anti-inflammatory, antioxidant, and anti-cancer activity of I. obliquus extracts. It can be concluded that I. obliquus fits the definition of functional food and has a potentially positive effect on health beyond basic nutrition; however, studies that meet the evidence-based medicine (EBM) criteria are needed.

Graphical abstract

Highlights

• •Extracts or polysaccharides from I. obliquus exhibit an anti-cancer potential in vitro.
• •Extracts or polysaccharides from I. obliquus exhibit anti-inflammation potential.
• •Extracts or polysaccharides from I. obliquus exhibit hypoglycemic and insulin sensitivity potential.

Abbreviations

ABTS

2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)

ALT

alanine aminotransferase

AST

aspartate aminotransferase

Bax

Bcl-2-associated X protein B-cell

Bcl-2

B- cell lymphoma 2

CAT

catalase

COX-2

cyclooxygenase-2

DPPH•

2,2-diphenyl-1-picrylhydrazyl

GPx

glutathione peroxidase

H₂DCFDA

2′,7′-dichlorodihydrofluorescein diacetate

HCV

Hepatitis C Virus

HO-1

heme oxygenase-1

IFNγ

interferon gamma

IL-12

interleukin 12

IL-1β

interleukin 1 beta

IL-2

interleukin 2

IL-4

interleukin 4

IL-6

interleukin 6

IL-10

interleukin 10

iNOS

inducible nitric oxide synthase

IκBα

inhibitor kappa B alpha

LPS

lipopolysaccharides

MDA

malondialdehyde

MMPs

matrix metalloproteinases

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

NO

nitric oxide

Nrf2

nuclear factor erythroid 2-related factor 2

p-AKT

phospho-protein kinase B

PGE₂

prostaglandin E₂

PI3k

phosphatidylinositol 3-kinase

p-mTOR

phospho-mammalian target of rapamycin

PPARγ

peroxisome proliferator-activated receptor gamma

ROS

reactive oxygen species

SOD

superoxide dismutase

STZ

streptozotocin

T2DM

type 2 diabetes mellitus

TGF-β

transforming growth factor beta

TIMPs

tissue metallopeptidase inhibitors

TLR2

toll-like receptor 2

TLR4

toll-like receptor 4

TNFα

tumor necrosis factor alpha

XOD

xanthine oxidase

1. Introduction

Inonotus obliquus (I. obliquus) (Ach. ex Pers.) Pilát, belonging to the family Hymenochaetaceae, is a black-brown plant parasitic fungus.^1,2 This mushroom has also at least a few common and regional names such as [Pol.] czerniak brzozy, czyreń, czernidło, czarcie oko, or czanga, which most likely has been coined from the Russian word "chaga".³ The genus Inonotus is widespread in North America, Asia, and Europe and includes approximately 100 species.^3,4 In Europe, this genus is represented only by 4 species, with I. obliquus as one of them.⁴ Although it is widely distributed in North America, Asia, and Europe, I. obliquus is on the list of partially protected species of mushrooms in Poland.⁵

I. obliquus is a primary tree parasite causing decomposition of live trunks.⁶ It has been seen on many trees species such as alder, beech, maple, rowan, hornbeam, poplar, oak, ash, willow, plane-tree, chestnut, and walnut, but the main hosts of I. obliquus are various species of birch.^3,7 It should be noted that the reports on the occurrence of I. obliquus on different species of deciduous trees are not reliable, because other fungi of the genus Inonotus are very often confused with the analyzed species.^3,7

I. obliquus infects approximately 30–50-year-old trees through wounds in the bark and can grow on the trunk for another 30–80 years.^3,5 A few years after penetrating the trunk of live trees, it produces sclerotia (vegetative or asexual fruiting bodies) with a lumpy irregular shape, cracked surface, and black-brown color. The interior of the sclerotium itself is made of rust-brown, yellow-veined, very dense mycelium.³ The sclerotia grow very slowly, reaching a diameter of more than 10 cm after 10–15 years.⁵ On old trees, the growths can exceed 50 cm in diameter.³ After many years, the host tree dies and annual fruiting bodies of the sexual stage appear. Fruiting bodies of this stage develop in the warm season of the year in places with the most advanced rot. They very rarely grow on live trees.^3,8 This type of fruiting bodies may have considerable sizes (up to 3–4 m in length and up to 50 cm in width) and are eaten very quickly by insects, which are assumed to be the main (besides the wind) spore-spreading vector of I. obliquus.³

Numerous scientific studies have shown that the some assumptions of folk medicine beliefs are reasonable. Mexican and European folk medicine plants have been tested on in vivo or in vitro models, e.g. Jasminum fruticans, Mentha longifolia, and Artemisia absinthium extracts, which show anthelmintic or antibacterial effects.^9,10 Given these examples, the search for new potentially active, natural compounds in folk medicine extends the present knowledge and help to find a promising cure to such prevalent diseases as cancers, parasitic infestations, or bacterial infections. Therefore, the usefulness of I. obliquus extract in medicine, e.g. in the treatment of diabetes and parasitic and viral infections, was evaluated. However, there has been no work so far, presenting the current scientific findings on I. obliquus in a concise and substantive form. Therefore, the aim of this work is to present the current state of knowledge of the biological properties of the I. obliquus mushroom and the potential possibilities of its medical use.

2. Application of I. obliquus in folk medicine

In folk medicine, rational premises are intertwined with elements of magic. The rational premises are based on observation of the natural environment and finding substances that alleviate disease symptoms. In many countries, this is the only way in which people can improve their health due to poverty in society or difficult access to scientific medicine. I. obliquus has been used in folk medicine since ancient times. One of the oldest documents confirming the use of its conks for medicinal purposes is the work by Hippocrates "Corpus Hippocraticum".³ The father of medicine used infusions of this mushroom externally to wash wounds.

In Eastern Europe, the Chaga mushroom has been used since the 12th century. Historical sources describe healing of a lip tumor in a Kiev Kniaz.¹¹ I. obliquus was used in traditional medicine for many indications by the people of Siberia.¹² The fungus was applied due to its antiparasitic, anti-tuberculosis, anti-inflammatory, and gastrointestinal properties.¹³ It was also recommended for heart and liver diseases. Most often, it was used in the form of infusions, inhalations, or aqueous macerates.¹³ Also antiseptic soaps containing I. obliquus were prepared for external use. In the middle of the 20th century, Chaga infusions were used as a substitute for tea in Siberia.¹⁴ Relatively early attention was paid to the potential I. obliquus antitumor or supportive effect in cancer treatment, which was particularly important before the era of scientific oncology. Such descriptions can be found in popular literature, e.g. in Aleksander Solzhenitsyn’s Cancer Ward.¹⁴

In Asia (China, Japan, Korea), Russia, and the Baltic countries, extracts of Chaga mushroom were used due to their beneficial effects on the plasma lipid system and heart function as well as antibacterial, anti-inflammatory, and anti-cancer activity.¹⁵ Furthermore, the antioxidant activity of I. obliquus may be important for prevention of free radical-related civilization diseases (atherosclerosis, cancer, diabetes, accelerated aging, and degenerative diseases of the central nervous system).¹⁶ Chaga extracts have been shown to inhibit the reproduction of hepatitis C virus (HCV) and human immunodeficiency viruses (HIV).¹⁷

The only objective method to assess the effectiveness of any therapy in scientific medicine is to perform research that meets the criteria of evidence-based medicine (EBM). Unfortunately, no observations from folk medicine or folk tales meet these criteria. Therefore, while drawing information from these sources, it is necessary to conduct scientific research that will either refute or confirm the folk premises. Importantly, the highest EBM standards should be maintained during the research, giving grounds for verifying folk knowledge.

3. Chemical composition and approaches to extraction of substances from I. obliquus

Only young and fresh sclerotia growing on birches, harvested throughout the year, are used in medicine.³ Sclerotia should be harvested in uncontaminated areas distant from sources of pollution, which may accumulate in the fungus.¹⁸ In the natural environment, I. obliquus grows in a cool climate with high seasonal temperature fluctuations, freezing, UV radiation, as well as occurrence of bacterial and viral infections.^19,20 In response to numerous stressors, I. obliquus has developed complex defense mechanisms. These mechanisms include the production of various bioactive substances: antioxidants, triterpenoids, ergosterol and its peroxide, sesquiterpenes, benzoic acid derivatives, hispidin analogues, and melanins. In addition, high expression of antioxidant enzymes has been detected in I. obliquus.21, 22, 23, 24, 25, 26 Moreover, many reports have highlighted that the polysaccharide fraction present in I. obliquus extracts is the largest group of active compounds, besides phenols. Therefore, many investigations have been performed to evaluate the polysaccharide content in this mushroom extracts, also in various conditions of extraction such as freeze, hot air, or vacuum drying methods.²⁷ As reported by Ma et al. I. obliquus extracts contain many low molecular polysaccharides, which are considered antioxidant agents responsible for this activity of these mentioned extracts.²⁷ This antiradical property has been repeatedly proved in the literature inter alia by Ma et al. or Cui et al. who tested the antioxidant properties of I. obliquus saccharides in vitro or in vivo on rat livers, respectively.^1,27 The water extract contained 19.76–26.51% of neutral sugars, in which the sugar composition varied depending on the drying method, but rhamnose, galactose, and glucose were the dominant sugars, whereas arabinose or mannose were the least abundant components.²⁷ The chemical structure of I. obliquus polysaccharides was also strictly related to the method applied, indicating that the saccharides were characterized by a dense spherical, branching, and elongated rod structure.²⁷

Currently, many approaches for isolation of substances from I. obliquus have been described; they are summarized in Table 1. They can be divided into techniques yielding the whole of isolated substances, individual fractions, or even single active substances from I. obliquus.^1,28,29 The use of total aqueous or ethanol extracts is the most similar approach to the methods used in folk medicine.^28,30 These types of extracts are suitable for direct consumption. Unfortunately, the application of other methods of total extraction using organic solvents such as methanol, ethyl acetate, or cyclohexane prevents the use of such a product.30, 31, 32 From a practical point of view, the use of a whole extract in scientific research is very important for practical natural or herbal medicine. In addition, many substances present in the whole extract can abolish or enhance each other’s activity. On the other hand, it is not possible to investigate the exact mechanism of specific substances in a non-fractionated extract. Another approach is to use individual fractions obtained from I. obliquus. In the literature, there are descriptions of research on various substances isolated from I. obliquus, including melanin, inodothiol, flavans, triterpenoids, steroids, polyphenols, or polysaccharides.^1,24,33, 34, 35, 36 This type of approach makes it possible to determine precisely which substance has therapeutic effects. Currently, the most frequently performed research is focused on the polysaccharide fraction (summarized in Table 1). As shown in the literature, this is a very promising group of substances associated with anti-cancer activity and insulin-improving sensitivity.^37,38 It should be noted that proteins contained in extracts are not studied. The extraction methods denature the protein at an early stage of isolation. In the case of aqueous extracts, the isolation of the substance is carried out at a temperature between 80 and 100 °C.^37,39,40 Similarly, various organic solvents (ethanol, methanol, cyclohexane, etc.) as well as multi-stage extraction methods can destroy the bioactive active ingredients contained in I. obliquus.

Table 1.

Approaches to isolation and administration of extracts or various substances isolated from Inonotus obliquus.

TYPES OF EXTRACTS FROM Inonotus obliquus TESTED	REFERENCES
ENTIRE WATER EXTRACTS	28,30,31,39,41, 42, 43, 44, 45
ENTIRE ETHANOL EXTRACTS	1,30,36,41,42,46, 47, 48
ENTIRE METHANOL EXTRACTS	30,32,41,49
ENTIRE SODIUM HYDROXIDE EXTRACTS	41
ENTIRE CYCLOHEXANE EXTRACTS	31
ENTIRE ETHYL ACETATE EXTRACTS	31
MELANIN FRACTION	24,29,50
INODOTIOL FRACTION	33,51
FLAVAN FRACTION	34
TRITERPENOIDS AND STEROIDS FRACTIONS	1,35,52, 53, 54
POLYSACCHARIDE FRACTIONS	1,37,62, 63, 64, 65, 66, 67, 68, 69,38,55, 56, 57, 58, 59, 60, 61
POLYPHENOLIC FRACTION	1,32,36,70

4. Biological properties of Inonotus obliquus extracts

Many different properties of extracts or substances derived from I. obliquus have been described to date. These include antiviral, antidiabetic, antioxidant, antiparasitic, immunomodulatory, anti-inflammatory, neuroprotective, anticancer properties in vitro, and recently antifatigue effects.^{1,30,36,37,39,65,71} Relevant literature data are summarized in Table 2.

Table 2.

Properties of different extracts from Inonotus obliquus. In vitro means a cell culture model or chemical analysis (cell-free model) in vivo means experiments performed on animals. To date, no human experiments have been performed.

PROPERTIES OF DIFFERENT EXTRACTS FROM Inonotus obliquus	TYPE OF BIOLOGICAL MODEL USED	REFERENCES
ANTIVIRAL	in vivo	17,39,72, 73, 74
ANTIDIABETIC	in vivo/in vitro	29,37,55,56,75,76
ANTIOXIDANT/ROS SCAVENGER	in vivo/in vitro	1,30,59,60,70,75,77, 78, 79, 80, 81, 82,36,83, 84, 85, 86, 87,37,42,50,55, 56, 57, 58
ANTIFATIGUE	in vivo	71
ANTIPARASITIC	in vivo	63, 64, 65
IMMUNOMODULATORY	in vivo/in vitro	33,48,61,62,64,88, 89, 90
ANTI-INFLAMMATORY	in vivo/in vitro	33,36,41,57,86,91
NEUROPROTECTIVE	in vivo/in vitro	34,66,82,92
ANTI-CANCER POTENTIAL	in vitro	28,30,49,51,63,67,75,93, 94, 95,31,32,38,43, 44, 45, 46, 47

4.1. Antioxidant properties

The antioxidant properties of extracts and individual fractions of substances obtained from I. obliquus are the most frequently studied parameter in scientific publications. To determine the antioxidant potential, methods based on the stable 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) are used.^{1,57,61,77,85,}96, 97, 98 Less often, another stable 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical is used.^70,79 The DPPH• and ABTS methods are based only on the antioxidant properties of chemical substances and do not fully reflect the antioxidative potential of a given extract or substance.^99,100 Methods based on the impact on the activity or expression of antioxidant enzymes as well as the formation of reactive oxygen species (or scavenging) in living cells reflect better the actual state in the organism. To date, the effects of I. obliquus extracts on the activity and/or expression of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), xanthine oxidase (XOD) in cell cultures and mouse tissues have been described.^{30,37,55,57,59,66,78} In addition, extracts from I. obliquus affected reactive oxygen species (ROS) production measured with the malonic dialdehyde (MDA) and 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) methods.^58,63,92 Interestingly, the effects were different depending on the type of cell. Szychowski et al. showed that extracts from I. obliquus reduced the amount of ROS in normal cells^30,58; in turn, numerous research teams described an increase in ROS in cancer cell lines.^30,67

Flavan derivatives, polysaccharides, and 3,4-dihydroxybenzalacetone isolated from I. obliquus have also been shown to have neuroprotective effects in neurodegenerative disease models such as Alzheimer and Parkinson diseases and the SH-SY5Y cell line.^34,66,92 These substances were found to protect against oxidative stress or increase SOD expression. Similarly, in other cells considered normal, such as pancreatic (RINm5F cell line) and hepatic (L02 cell line) cells, I. obliquus polysaccharides protected against H₂O₂-induced damage.^42,58,77

4.2. Anti-inflammatory and immunomodulatory effects

So far, few research teams have tried to determine the impact of extracts obtained from I. obliquus on the immune system.^33,88,89 Fan et al. have shown that water-soluble polysaccharides obtained from I. obliquus increased the proliferation of murine peritoneal lymphocytes.³⁸ Polysaccharides from I. obliquus also stimulated the production of tumor necrosis factor-alpha (TNFα) by mouse macrophages. TNFα production increased in direct proportion to the increasing concentration of the tested polysaccharides.³⁸ Similarly, Chen et al.¹⁰¹ observed that polysaccharides from I. obliquus increased the proliferation of splenocytes and lymphocytes. In addition, I. obliquus increased the secretion of cytokines such as interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-12 (IL-12), and TNFα and intensified the phagocytic processes. I. obliquus also significantly increased Bax expression and inhibition of Bcl-2 ¹⁰¹. In turn, the latest research has shown that I. obliquus extracts very strongly reduced TNFα secretion in the RAW 264.7 cell line.⁴¹ Similarly, Chen et al. showed that polysaccharides from I. obliquus affected the Th1/Th2 lymphocyte ratio and Th17/Treg in the colon, mesenteric lymph nodes, and spleen of male BALB/c mice.⁸⁹ Using a mouse model, the authors showed that the studied polysaccharides from I. obliquus could be used to treat inflammatory bowel disease. Other studies performed on BALB/c mice showed that extracts from I. obliquus suppressed Th2 and Th17 immune response.³³ It is currently thought that I. obliquus extracts or their polysaccharide fraction inhibit inflammatory reactions.^41,57 Most studies show a reduction in the production and/or secretion of proinflammatory cytokines such as interleukin-1 beta (IL-1β), interferon gamma (IFNγ), and TNFα.^41,57,89 In vivo studies showed that an I. obliquus extract administered to Sprague-Dawley rats for 7 days protected the animals against the development of induced inflammation. The same studies showed that the I. obliquus extract exhibited analgesic properties as well.¹⁶ Park et al. tried to elucidate the mechanism of the anti-inflammatory and analgesic effects of I. obliquus extracts on the RAW 264.7 cell line.¹⁶ It was demonstrated that the extract caused a decrease in the production of nitric oxide (NO) and prostaglandin E₂ (PGE₂).¹⁶ In addition, LPS-stimulated macrophages produced nitric oxide synthase (iNOS), which was inhibited by the I. obliquus extract. Furthermore, the tested extract inhibited lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2) production.¹⁶ The I. obliquus extract also reduced mRNA production and expression of TNFα and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) induced by LPS.¹⁶ Recent studies have shown that individual fractions from I. obliquus can act antagonistically. Wold et al. proved that some polysaccharide fractions increased NO production by murine macrophage and dendritic cell lines J774. A1 and D2SC/1 ⁶². It cannot be excluded that, depending on the type of cells tested, the extracts or individual substances from I. obliquus may increase or inhibit NO production.

4.3. Antiviral, antibacterial, and antiparasitic properties of I. obliquus

Data on the antiviral activity of I. obliquus extracts are very limited. To date, it has been shown that polysaccharides derived from the aqueous fraction of I. obliquus inhibit the protease from HIV type 1 (HIV-1), and thus impede the entry of virions into cells.³⁹ In other studies, Shibnev et al. showed that the I. obliquus water extract was active against the HCV.¹⁷ At 48 h after addition of the I. obliquus aqueous extract to the HCV-infected embryonic porcine kidney epithelial inoculated line (SPEV), the amount of the virus in the cells was inversely proportional to the concentration of the added extract. At the highest concentrations, the extract completely stopped or significantly inhibited the reproduction of HCV. Moreover, it was shown that the addition of the extract to the cells 24 h before their exposure to the virus protected the SPEV cells from infection.¹⁷ In another experiment, an aqueous extract of I. obliquus showed an effect against the herpes virus - Herpes simplex type 1 (HSV-1) in normal kidney cells (Vero) of infected Cercopithecus aethiops.¹⁰² The presented studies confirm the antiviral effect of I. obliquus and indicate its potential use in the treatment of diseases associated with viral infections. In addition, the polysaccharide fraction of I. obliquus exhibited a broad-spectrum antiviral activity against feline herpesvirus 1, feline influenza virus H3N2 and H5N6, feline panleukopenia virus, and feline infectious peritonitis virus, which cause respiratory and gastrointestinal diseases in cats.⁷³ Similarly, Seo and Choi showed that ethanolic extracts from I. obliquus inhibited the entry of murine norovirus (MNV) and feline calicivirus (FCV) into RAW264.7 cells ⁷².

In the available literature, there are only two reports suggesting the antibacterial or probiotic effect of I. obliquus extracts. Niu et al. showed that various fractions derived from I. obliquus stimulated NO production and additionally increased phagocytosis in RAW 264.7 cells.⁶¹ As demonstrated by Hu et al. I. obliquus polysaccharide was found to regulate the gut microbiota in chronic pancreatitis in mice.¹⁰³ Moreover, the authors suggest that administration of I. obliquus polysaccharides could regulate the gut microbiota composition and diversity to a healthy profile in mice with chronic pancreatitis.

To date, only one team has studied the antiparasitic aspects of I. obliquus extracts and polysaccharides ^63–65. Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan that causes toxoplasmosis in humans and many warm-blooded animals. Approximately 30%–50% of the population is infected with T. gondii worldwide.¹⁰⁴ Xu et al. described that I. obliquus polysaccharides significantly reduced the abortion rate, inhibited the decreases in progesterone (P) and estriol (E3) levels and the increase in the MDA level, and increased the activities of SOD and GSH.⁶⁴ Furthermore, I. obliquus polysaccharides inhibited the production of inflammatory cytokines, such as TNFα, IL-6, IFNγ, IL-1β, and IL-17A, and promoted the production of the anti-inflammatory cytokine interleukin-10 (IL-10) and transforming growth factor (TGF)-β in T. gondii-infected pregnant mice.⁶⁴ Similarly, Xu et al. described that I. obliquus polysaccharides significantly decreased the liver coefficient and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), MDA, and nitric oxide NO. In turn, the polysaccharides increased the levels of antioxidant enzymes SOD and GSH.⁶³ As mentioned previously, I. obliquus polysaccharides also effectively decreased the expression of serum TNFα, IL-6, IL-1β, IFNγ, and interluekin-4 (IL-4) in T. gondii-infected mice. The authors found that I. obliquus polysaccharides down-regulated the levels of toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) and phosphorylation of NF-κB p65 and inhibitor kappaBα (IκBα), but up-regulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1).⁶³ Lastly, Ding et al. showed that I. obliquus polysaccharides significantly improved the spermatogenic capacity, ameliorated pathological damage to testis, and increased the levels of serum testosterone, luteinizing hormone, and follicular-stimulating hormone in T. gondii-infected male mice. I. obliquus polysaccharides effectively up-regulated the expression of testicular steroidogenic acute regulatory protein (StAR), P450scc, and 17β-HSD. These compounds further enhanced testicular phosphatidylinositol 3-kinase (PI3K), phospho-protein kinase B (p-AKT), and phospho-mammalian target of rapamycin (p-mTOR) expression levels.⁶⁵

4.4. Anti-cancer potential

In medical databases, there are many publications on the anti-cancer activity of I. obliquus extracts in vitro. The anti-cancer potential of the extracts or substances isolated from I. obliquus and tested on different tumor cell lines are described in Table 3. Unfortunately, studies on animals are very limited and there are no reliable publications on humans.^57,105 Most studies have been conducted on cell lines originating from the digestive system, such as AGS, HCT-116, HT-29, SW620, SGC-7901, DLD-1, and CACO-2 ^30,38,46,105. Extracts from I. obliquus have been shown to inhibit proliferation and/or are cytotoxic to these human gastrointestinal tumor cell lines. Similarly, their cytotoxic, anti-proliferative, or proapoptotic effects have been demonstrated in cell lines from many other tissues and systems, e.g. lung, cervix, mammary gland, ovary, liver, lymphoid cancers (A549, MCF-7, HepG2, HepG3B, HeLa, MCF-7, HL-60, PA-1, H1264, H1299) (described in detail in Table 3).^{28,32,43,49,94,95,106}

Table 3.

Anti-cancer potential of extracts or substances isolated from Inonotus obliquus tested on different tumor cell lines.

CANCER CELL LINE	ASSAYS	REFERENCES
A549 human lung adenocarcinoma	LDH release	28,95
	Caspase-3 activity	67,95
	DNA fragmentation
	Cell viability	28,32,49,67,68,95
	Cell migration	28
	Cell proliferation
	ROS production	67
	Cytotoxicity	31
HeLa human cervical cancer	Cell viability	49,51
	Caspase-3 activity	51
	Cell migration
4T1 mouse mammary carcinoma	Cell viability	47
MCF-7 human breast cancer	Cell viability	47,49,68
AGS human gastric cancer	Cell viability	49
HEK-293 human embryonic kidney	Cell viability	49
PA-1 human ovarian teratocarcinoma	Cell viability	32
U937 human myeloid leukemia cell	Cell viability	32
HL-60 human myeloid leukemia	Cell viability	32
CACO-2 human colon carcinoma	Cell viability	30
	LDH release
	ROS production
	Cell proliferation
HT-29 human colorectal adenocarcinoma	Cell viability	28,46
	DNA synthesis
	Cell proliferation
	LDH release	28
	Cell migration
H1264 lung adenocarcinoma	LDH release	95
	Caspase-3 activity
	DNA fragmentation
	Cell viability
HCT-116 human colon carcinoma	Cell viability	45
	LDH release
	Caspase-3 activity
H1299 human non-small cell lung cancer	LDH release	95
	Caspase-3 activity
	DNA fragmentation
	Cell viability
B16–F10 murine skin melanoma	Cell viability	44,68,93
	Caspase-3 activity	44
	Cell cycle inhibition
	Cell migration	93
Calu-6 human pulmonary adenocarcinoma	LDH release	95
	Caspase-3 activity
	DNA fragmentation
	Cell viability
HEC-1B human endometrial adenocarcinoma	Cell viability	68
P388 mouse leukemia	Caspase-3 activity	94
	Cell viability
	DNA fragmentation
Fao rat hepatoma	Cell viability	28
	Cell proliferation
	LDH release
	Cell migration
P19 mouse embryo teratocarcinoma	Cell viability	28
	Cell proliferation
	LDH release
	Cell migration
C6 rat glioma	Cell viability	28
	Cell proliferation
	LDH release
	Cell migration
Hep3B human hepatoma	Cell viability	43,107
	Cell cycle inhibition	43
HepG2 human hepatoma	Cell viability	43,107
	Cell cycle inhibition	43
	ROS production	108
Hur7 human hepatoma	Cell viability	68
KATO-III human stomach carcinoma	Cell viability	68
DLD-1 human colon carcinoma	DNA fragmentation	40
SGC-7901 human gastric carcinoma	Cell viability	38
SK-OV3 human ovary adenocarcinoma	Cell viability	68
SW156 kidney adenocarcinoma	Cell viability	68
SW620 human colorectal adenocarcinoma	Cell proliferation	105

Unfortunately, many of these studies lack in-depth experiments. In some publications, the authors have limited themselves to reporting whether a given extract is toxic to cells without elucidation of the molecular mechanism of action, using only one parameter. These include investigations of 4T1, MCF-7, AGS, HEK-293, PA-1, U937, HL-60, P388, Hur7, HEC-1B, KATO-III, SG-7901, SK-OV3, and SW156 cell lines.^{32,38,47,49,68} Interestingly, the majority of studies were conducted on the A549 line, which is a human lung adenocarcinoma line with the best documented anti-cancer potential of I. obliquus extracts or fractions of substances derived from the extract.^{28,31,67,68,95} In publications elucidating the molecular mechanism of extracts, the authors consistently emphasize the role of ROS in the mechanism of action of substances derived from I. obliquus.^28,30 The authors also agree that extracts from I. obliquus intensify the process of apoptosis measured by capase-3 activity or the level of DNA fragmentation and stop the cell cycle in the G0 phase.^44,95,109 Very important in migration/metastasis of tumors are matrix metalloproteinases (MMPs).¹¹⁰ Interestingly, it has been shown that, although the I. obliquus extract was not toxic to the A549 cell line at low concentrations, it statistically significantly reduced cell migration.¹¹¹ The authors attributed this effect to the declining MMP-2 and MMP-9 expression and the increasing expression of tissue metallopeptidase inhibitor-2 (TIMP-2) as well as the reduced expression of NF-κB.¹¹¹ Similarly, a decrease in NF-κB expression in A549 induced by I. obliquus extracts has been described.¹⁰⁹ Moreover, it has been reported that I. obliquus polysaccharides inhibit the expression of MMP-2, MMP-7, and MMP-9 in B16–F10 mouse cells.⁹³ Recently, Zhang et al. described that inotodiol from I. obliquus decreases the expression of MMP-2 and MMP-9 in HeLa human cells and reduces cell invasiveness.⁵¹ Unfortunately, the tests performed in vivo are very limited. To date, it has been shown that tumors of the 3LL mouse lung cancer line implanted in C57BL/6 mice developed significantly more slowly in animals treated with an I. obliquus aqueous extract. In the experiment, it was shown that the tumor in mice receiving the extract was by 60% smaller and the number of metastases decreased by 25%. Interestingly, ingestion of I. obliquus reduced the body weight of the mice and increased their body temperature, which may have contributed to the protection against cancer, as suggested by the researchers.¹¹² Furthermore, I. obliquus extracts have been shown to inhibit the development of two tumor lines: melanoma B16–F10 and sarcoma-180 after implantation into Balbc/c strain mice.^44,49 It has also been shown that ergosterol isolated from I. obliquus inhibited the development of human colorectal cancer in the C57BL/6 mouse strain.¹⁰⁵ It is currently believed that the high content of polysaccharides is responsible for the anti-cancer potential of I. obliquus.^67,111 However, the anti-cancer potential have been found in all types of extracts used.¹¹³

4.5. Hypoglycemic and insulin sensitivity action

ROS play a key role in many signaling pathways. Moreover, the relationship between oxidative stress and metabolic disorders such as type 2 diabetes (T2DM), cancer, obesity, and cardiovascular disease is well described.¹¹⁴ To date, it has been shown that I. obliquus polysaccharides have hypoglycemic activity.^115,116 Unfortunately, the exact mechanism of action has not been elucidated. It has been described that I. obliquus polysaccharides in streptozotocin (STZ)-induced diabetic Wistar rats reduced blood glucose levels and restored the structure of β-cells after diabetes-induced cellular damage.¹¹⁷ I. obliquus polysaccharides lowered the level of lipid peroxidation products (such as low-density lipoprotein), whereas the high-density lipoprotein cholesterol level was enhanced.¹¹⁷ Moreover, Wang et al. reported that I. obliquus polysaccharides enhanced the serum levels of insulin and alleviated the metabolic derangement of glucose enzymes in STZ-induced diabetic mice.⁵⁶ In the same experimental model, I. obliquus polysaccharides with the chromium (III) complex significantly decreased fasting blood glucose levels, plasma insulin levels, and body weight in mice.⁵⁵ This is consistent with the previously described studies of total extracts. Cha et al. demonstrated that I. obliquus dietary treatment lowered serum glucose and leptin levels and alleviated obesity-related complications in T2DM OLETF rats.¹¹⁸ Similarly, Sun et al. described that feeding the experimental mice with I. obliquus improved serum insulin levels, moderately expanded the pancreatic islets, and reduced pancreatic injuries in alloxan-induced diabetic mice.¹¹⁹ In turn, I. obliquus polysaccharides elevated insulin levels in C57BL/6 mice with diabetic nephropathy; however, the cholesterol and triglyceride levels remained unaffected.¹²⁰ Recently, I. obliquus polysaccharide has been reported to help to alleviate pancreatic acinar atrophy and weight loss in chronic pancreatitis mice induced by diethyldithiocarbamate. Hu et al. postulated that I. obliquus polysaccharides possessed strong antioxidant activity for scavenging free radicals in vitro and in vivo, which could be beneficial for chronic pancreatitis therapy in mice.⁵⁷

Peroxisome proliferator-activated receptor gamma (PPARγ) is a key receptor involved in insulin resistance and cell sensitivity to this hormone.¹²¹ Moreover, PPARγ is also involved in anti-inflammatory action, by controlling activation and expression of NF-κB and pro-inflammatory cytokines.¹²² However, only one paper showed that I. obliquus extract activated adipogenesis of 3T3-L1 preadipocytes, enhanced the expression PPARγ target genes, and increased triacylglycerol accumulation.¹²³

5. Conclusions and perspectives

For decades, extracts of I. obliquus have been used as a remedy for numerous diseases in folk medicine. Recent discoveries have provided evidence of the appropriateness of using these drugs in vitro. Extracts from I. obliquus prepared in various solvents as well as individual fractions of substances derived from the fungus show antiviral, antibacterial, immunostimulating, and anti-tumor activity in vitro. To date, various studies have suggested significant therapeutic potential of substances derived from I. obliquus. Based on the literature data, it can be concluded that the effectiveness of extracts and/or substances derived from I. obliquus is based on two main mechanisms. The first one is the effect of I. obliquus on the production of ROS and/or on mechanisms of ROS scavenging, which varies depending on the cell type. The proposed scheme of the effect is described in Fig. 1.

Fig. 1.

Proapoptotic and antioxidant action of Inonotus obliquus extracts in normal and neoplastic cells described in literature.

The other mechanism is based on the action via the PPARγ receptor. Experimental evidence suggests that extracts and/or substances obtained from I. obliquus may affect the level of expression or activity of the PPARγ receptor.¹²³ In this way, extracts from I. obliquus can reduce insulin resistance. However, it cannot be excluded that the anti-cancer mechanism of action of I. obliquus extracts also involves the PPARγ receptor. The proposed scheme of the effects is described in Fig. 2. Unfortunately, due to the lack of data on the involvement of the PPARγ receptor in the anti-tumor activity of I. obliquus extracts, further research is required. It can be concluded that I. obliquus fits the definition of functional food and has a potentially positive effect on health beyond basic nutrition; however, studies that meet the EBM criteria are needed. Taking the above into account, we suppose that the I. obliquus extracts can be included in cancer therapies in the future; however, more specialistic analysis should be performed, especially on in vivo models.

Fig. 2.

Antidiabetic and anti-inflammatory action of Inonotus obliquus extracts with key involvement of the PPARγ receptor described in literature.

CRediT authorship contribution statement

Konrad A. Szychowski: Data curation, Formal analysis, Writing - original draft. Bartosz Skóra: Writing - original draft. Tadeusz Pomianek: Writing - original draft. Jan Gmiński: Writing - original draft.

Declaration of competing interest

The authors declare that there is no conflict of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: