Anti-inflammatory potential of mushroom extracts and isolated metabolites

Abstract Background In the recent years natural resources are being in focus due to their great potential to be exploited in the discovery/development of novel bioactive compounds and, among them, mushrooms can be highlighted as alternative sources of anti-inflammatory agents. Scope and approach The present review reports the anti-inflammatory activity of mushroom extracts and of their bioactive metabolites involved in this bioactive action. Additionally the most common assays used to evaluate mushrooms anti-inflammatory activity were also reviewed, including in vitro studies in cell lines, as well as in animal models in vivo . Key findings and conclusions The anti-inflammatory compounds identified in mushrooms include polysaccharides, terpenes, phenolic acids, steroids, fatty acids and other metabolites. Among them, polysaccharides, terpenoids and phenolic compounds seem to be the most important contributors to the anti-inflammatory activity of mushrooms as demonstrated by numerous studies. However, clinical trials need to be conducted in order to confirm the effectiveness of some of these mushroom compounds namely, inhibitors of NF-κB pathway and of cyclooxygenase related with the expression of many inflammatory mediators.


Introduction
Inflammation is a physiological response to injury, characterised by loss of function and pain, heat, redness and swelling. It is usually associated with the pathogenesis of diseases such as diabetes, arthritis, obesity, metabolic syndrome, cancer and several cardiovascular diseases (Bellik et al., 2012;Moro et al., 2012;Ma, Chen, Dong, & Lu, 2013).
Natural products are good resources for development of therapeutic compounds with antiinflammatory potential and without or lower toxic effects (Yuan, Wahlqvist, He, & Yang, 2006). Several bioactive compounds from plants (Wang et al., 2013c), rhizomes (Debnath et al., 2013) and marine algae (Kim et al., 2014) have been isolated and their anti-inflammatory effect studied by various mechanisms.
Mushrooms are nutritionally functional foods that have been an integral part of our diet for years. They have not just been consumed for their culinary importance because of their unique taste and flavour (Kalač, 2013), but also because of their potential therapeutic properties which dates back to over 2000 years ago and are recognized as effective to treat and prevent varieties of disorders (Lim et al., 2007;Moro et al., 2012;Silveira et al., 2014).
Most research studies conducted on the pharmacological potential of mushrooms are mainly focused on crude extracts. Nevertheless, it is also important to identify the bioactive compounds responsible for each one of the ascribed bioactivities. In this context, the antiinflammatory activity of several mushroom species has been reported as well as of their bioactive metabolites. It has been related with a reduction in the production of nitric oxide (NO) and other inflammatory mediators such as interleukins (IL 1β, IL-6, IL-8), tumor necrosis factor (TNF-α) and prostaglandin E2 (PGE2), causing reduction of inflammation (Jedinak, Dudhgaonkar, Wu, Simon, & Sliva, 2011;Moro et al., 2012;Fangkrathok, Junlatat, NF-κB is a transcription factor that regulates the expression of several pro-inflammatory cytokines and enzymes such as IL-1β, TNF-α, iNOS, and COX-2 that play vital roles in apoptosis, in the immune system, as well as in the inflammation (Hseu, Huang, & Hsiang, 2010). When there is an immune stimulant such as lipopolysaccharide, viral proteins or cytokines, the NF-κB becomes activated (Kim et al., 2003). Toll like receptors (TLRs) and tumor necrosis factor receptor (TNFr) localised in the macrophages membrane have the ability to detect these pathogen-associated microbial patterns (PAMPs) necessary for activation of several signalling cascade (Figure 1). After ligand binding, these receptors activate the myeloid differentiation protein 88 (MyD88) responsible for activation of mitogen activated protein kinase (MAPKs). This MAPKs further activate the IKK kinases (IKKα, IKKβ, IKKγ) leading to phosphorylation of IkB proteins complex (Hasnat, Pervin, Cha, Kim, & Lim, 2015). Cytosolic IKB forms a complex with NF-κB and the IkB proteins becomes degraded allowing NF-κB to translocate to the nucleus where it triggers the transcription of several chemokine and cytokine genes involved in the innate and adaptive immune response (Kim et al., 2003). Some polyphenols have been known to inhibit specific steps in the pathway leading to NF-κB release (Ruiz & Haller, 2006). These authors investigated the antiinflammatory mechanisms of flavonoids that were able to inhibit the phosphorylation of IkB preventing translocation of NF-kB to the nucleus. Hence, finding natural inhibitors of NF-kB for treatment and prevention of various inflammatory diseases have been the target of several scientists (Kim et al., 2003).

NSAIDs and their mechanism of action
The nonsteroidal anti-inflammatory drugs (NSAIDs) are a group of medications commonly administered to manage pain and inflammation (Moro et al., 2012). Most of them are available over the counter in the United States while the rest needs prescription (Meek, van de Laar, & Vonkeman, 2010). Several side effects have been associated with frequent administration of NSAIDs particularly in the gastrointestinal (GI) tract where they cause bleeding, intestinal perforation and peptic ulcer (Dugowson & Gnanashanmugam, 2006).
Prostaglandins (PG) are hormone-like chemicals in the body that perform ''housekeeping'' functions required for normal physiological activities. They are structurally related and have regulatory roles as well as pathological implication (Silveira et al., 2014). Cyclooxygenase enzymes catalyzes the conversion of arachidonic acid to PGH 2 , which is converted to other prostanoid species including PGD 2 , PGE 2 , prostacyclin (PGI 2 ), and thromboxane (TXA 2 ) by the action of specific synthases (Figure 2) (Joo & Sadikot, 2012). COX-1 is primarily involved in the regulation of homeostatic functions and is constitutively expressed in a wide variety of cells, promoting physiological functions, such as gastric mucosal protection, control of renal blood flow, hemostasis, autoimmune responses, lungs, central nervous system, cardiovascular system and reproductive functions (Grosser, Fries, & Fitzgerald, 2006).
On the other hand, COX-2 is an inducible isoform of prostaglandin synthase in activated macrophages, fibroblasts, and endothelial cells that are responsible for inflammation. They are expressed significantly due to stimuli such as cytokines, endotoxins, viral proteins and growth factors. COX-2 originates inducing prostaglandins, which contributes to the development of the four cardinal signs of inflammation: pain, heat, redness and swelling, thus being considered as the main target for the anti-inflammatory action (Fitzgerald, 2004).
The search for selective inhibitors of COX-2 is considered important, on the basis of the theory that the side effects, such as gastric lesions, that occurred from inhibition of COX-1 activity, were observed with some non-selective nonsteroidal anti-inflammatory drugs (NSAIDs) (Supplementary material S1) such as dexamethasone, diclofenac and indomethacin . Until now, very few compounds of natural origin have been reported to possess COX-2 inhibitory effects. Yoshikawa et al. (2005) were the first to report the potential of lanostane triterpenoids and their glycosides as selective inhibitors of COX-2 enzyme.
Usually, NSAIDs inhibit both isoforms of the cyclooxygenase enzyme but the recently discovered selective COX-2 inhibitors (Supplementary material S2) are specific for the COX-2 isoform, thus exerting the anti-inflammatory property of COX-2 inhibition while theoretically evading the adverse effect associated with COX-1 isoform inhibition (Nowak, 2012). Considerable resources have thus been invested in the pharmaceutical industries for development and design of drugs that act through selective inhibition of COX-2 to control inflammation with improved tolerability, less adverse effects, and without affecting normal metabolic processes (Shukla, Bafna, Sundar, & Thorat, 2014 Several in vitro measurement of NO production in LPS stimulated RAW 264.7 cells have been reported by several authors in the past, and this is one of the possible ways to screen various extracts and bioactive compounds with potential anti-inflammatory properties. RAW 264.7 cells are seeded in 96-well plates, they are then treated with different concentrations of the sample to be studied followed by stimulation with LPS. The cell culture supernatant is then transferred to a new plate followed by addition of sulphanilamide and NED solutions. The NO produced is determined by measuring the absorbance at 540 nm. This assay is one of the most common and widely used for evaluation of anti-inflammatory activity as reported by different authors (Moro et al., 2012;Taofiq et al., 2015).

COX-1 and COX-2 catalyzed prostaglandin biosynthesis assay
The cyclooxygenase enzymes have been extensively used to study the anti-inflammatory potential of natural agents. This is not a very common method for anti-inflammatory activity assessment, but it has been reported in some publications (Noreen, Ringbom, Perera, Danielson, & Bohlin, 1998;Zhang et al., 2003;Yoshikawa et al., 2005;Stanikunaite, Khan, Trappe, & Ross, 2009

In vivo assays
A lot of studies have reported in vivo anti-inflammatory activity of natural products achieved by inducing inflammation in mouse and measuring the degree of swelling relative to a positive control. In these animal models there is capillary dilation, increase blood vessel permeability, and edema similar to the ones associated with human acute inflammation (Wang et al., 2013a). The carrageenan-induced hind paw edema model has been used in a lot of research studies (Jose, Ajith, & Jananrdhanan, 2004;Deng et al., 2013;. The methodology involves treating animals with extract at different concentration and also a control group with indomethacin, dexamethasone or any other non-steroidal antiinflammatory drug. This is followed by injection of hind paw with carrageenan in saline solution and by measuring the paw volume increment immediately and at different time intervals (Lu et al., 2008). The degree of swelling induced by the injection is evaluated and the result compared with the control. Inflammation can also be induced by topical application of xylene in the ear of the mouse. After few minutes, the difference in swelling (Lu et al., 2008) is estimated. Other in vivo anti-inflammatory assay induce inflammation, either by the croton oil-induced ear edema test (Kim et al., 2004;Dore et al., 2007) or by TPA, 12-O-tetradecanoylphorbol-13-acetate induced inflammation in mice, and sacrificed by cervical dislocation, to take punch biopsies to weight (Kamo, Asanoma, Shibata, & Hirota, 2003;Dore et al., 2007;Liu et al., 2007).

The anti-inflammatory potential of mushroom extracts
Numerous investigations have suggested that several mushroom species can exhibit antiinflammatory potential based on their ability to reduce the production of inflammatory mediators (Kim et al., 2003;Padilha et al., 2009;Wen et al., 2011;Elsayed, Hesham, Mohammad, & Aziz, 2014Taofiq et al., 2015. Their crude extracts ( Table 1) have been described to display activity, and attention is now being focused on efforts to discover bioactive compounds capable to suppress the production of inflammatory mediators through gene expression downregulation of different types of inflammatory mediators (Kim et al., 2006, Fangkrathok, Junlatat, & Sripanidkulchai, 2013. Previous research studies have been carried out on several mushroom species, mainly in methanolic (Kim et al., 2003;Wen et al., 2011;Moro et al., 2012) and ethanolic Kim et al., 2006;Ruthes et al., 2013b;Taofiq et al., 2015) extracts. Most studies have shown that these extracts display antiinflammatory activity, but it is also crucial to identify the metabolites responsible for this bioactivity.
Different compounds have been isolated from mushrooms and implicated as responsible for the anti-inflammatory activity, e.g. polysaccharides (Dore, et al., 2007;Lu, et al., 2008;Lavi, Levinson, Peri, Hadar, & Schwartz, 2010;Adebayo, Oloke, Majolagbe, Ajani, & Bora, 2012;Ruthes, et al., 2013aRuthes, et al., , 2013bRuthes, et al., , 2013cChang, Lur, Lu, & Cheng, 2013;Castro et al., 2014;Silveira et al., 2014;Silveira et al., 2015), terpenes (Kamo, Asanoma, Shibata, & Hirota, 2003;Yoshikawa et al., 2005;Dudhgaonkar, Thyagarajan, & Sliva, 2009;Ma, Chen, Dong, & Lu, 2013;Tung et al., 2013;Xu, Yan, Bi, Han, Chen, & Wu, 2013;Choi, et al., 2014a), phenolic compounds ( anti-inflammatory activity of extracts prepared from mushrooms after undergoing some food processing procedures. The results showed reduced activity compared to fresh samples, which implies that anti-inflammatory compounds present in these mushrooms were degraded, e.g due to suseptibility to heating. Ganoderma lucidum (Curtis) P. Karst., is a medicianal mushroom that has been used to reduce allergies, inflammation, has anti-tumor and anti-aging potential, as well as health promoting effects. Ethanolic extract of Ganoderma lucidum was studied for its antiinflammatory potential by stimulating murine BV2 cell line with LPS, and the amount of NO, PGE2 and Cytokine( IL-1β and TNF-α) in culture supernatants quantified as reported by Yoon et al. (2013). Treatment of cell line with extract up to 1 µg/ml significantly repressed the production of NO due to the inhibition of iNOS mRNA protein expression. The amount of cytokine release was measured by ELISA and a significant reduction in the level of cytokines after treatment with extract was observed. The anti-inflammatory ativity was further associated to the inhibition of the NF-κB signaling pathway by the ethanolic extract.
Methanolic extract of Ganoderma lucidum was also evaluated by Chu et al. (2015).

RAW264.7 monocytic cells were stimulated with LPS and treated with extract at different
concentrations. From the result, 100 µg/ml of extract significantly inhibited NO production in the culture medium up to 85%. Cordyceps, a genus of mushroom known to grow on insects and have been reported to strengthen the immune system . Rao, Fang, Wu, & Tzeng (2007), studied anti-inflammatory activity using methanolic extracts from the fruiting body of Cordyceps sinensis (Berk.) Sacc., and by stimulating macrophages cells with LPS and NO production later quantified. The amount of TNF-α level and IL-12 were quantified by the ELISA test. production by using ELISA kit as well as NO production in LPS stimulated RAW264.7 macrophages. The extracts were known to suppress gene expression of IL-1β, TNF-α, inducible nitric oxide synthase, and cyclooxygenase-2 enzyme. This is due to the inhibition of nuclear transcription factor NF-κB activation.

Polysaccharides
Mushrooms have been known as valuable sources of bioactive carbohydrates, namely polysaccharides which represent the main group with various health promoting properties (Villares, 2013). They include several different β-glucans (Supplementary material S3), fucomannogalactans, xylomannans and mannogalactans, known to play different biological roles such as the ones of antioxidation, anti-inflammatory, antitumour, antimicrobial, antiaging, neuroprotective and immunomodulatory (Li et al., 2013).
Studies on the anti-inflammatory properties of carbohydrates have led to positive results as shown in Table 2. Several types of polysaccharides have been obtained from mushroom dry fruiting bodies or mycelia and tested for anti-inflammatory activity either in vivo (Lu et al., 2008;Ruthes et al., 2013c;Silveira et al., 2015), following a typical model similar to human acute inflammation, or in vitro for inhibition of cytokine or NO production (Dore et al., 2007;Chang et al., 2013;Castro et al., 2014).
Several pharmacological properties have been reported on the extracts and bioactive compounds isolated from Cordyceps militaris, namely the anti-inflammatory activity (Rao, et al., 2010). D-Glucose, D-mannitol and 3,4-O-isopropylidene-D-mannitol were isolated from its fruiting body and the anti-inflammatory potential evaluated in mouse peritoneal macrophages regarding the inhibition of NO production and cytokine release. Among the three cited compounds, D-Glucose showed the highest NO inhibition potential with an IC 50 value of 11.3 µg/mL, followed by D-mannitol (14.2 µg/mL) and 3, 4-O-isopropylidene-Dmannitol (17.2 µg/mL). They also inhibit significantly cytokines (TNF-α and IL-12) production, indicating that they may be useful compounds for the design of antiinflammatory agents.
Mushroom polysaccharides vary in structure and sometimes they exhibit different biological effects. Silveira et al. (2014) isolated a (1→3)-β-D-glucan from the fruiting body of Pleurotus sajor-caju (Fr.) Singer and tested its anti-inflammatory effect in a monocytic cell line THP-1 after LPS induction, for inhibition of pro-inflammatory genes production.
Monocyte cells showed a significant decrease in TNF-α expression (61.8% inhibition) while IL-1β and COX-2 mRNAs were also significantly inhibited (37.0% and 63.6%, respectively). Silveira et al. (2015) isolated a mannogalactan from P. sajor-caju by submerged fermentation. The purified polysaccharide was chemically characterized and its antiinflammatory potential evaluated in vivo for reduction of carrageenan-induced paw edema in mice. The group treated with purified mannogalactan was able to reduce edema after 5-6h of exposure to 1% carrageenan, with 69-71% edema reduction observed, and was quite as effective as dexamethasone used as control. Therefore, mushroom polysaccharides have shown to be lead compounds for development of anti-inflammatory agents.
Agaricus bisporus is one of the most commonly consumed mushrooms in the world. Ruthes

Polyphenols
Phenolic compounds are characterized by at least one aromatic ring (C6) and one or more hydroxyl groups (Michalak, 2006).  Table 4).
Antrodia camphorata, very common in Taiwan

Steroids
Steroids are organic compounds with three hexagonal and one pentagonal carbon rings arranged in specific configuration with several functional groups found in plants, animals and fungi (Streck, 2009). Ergosterol is a precussor of vitamin D found in mushrooms membrane and known to vary among species depending on the physiological state of the mushroom (Chiocchio & Matković, 2011). Steroids in general have been reported to play several biological functions such as anti-tumor, anti-oxidant, immune function as well as prevention of common diseases (Phillips et al., 2011). Antrodia camphorata is a medicinal mushroom with reported anti-inflammatory activity (Hseu et al., 2005;Liu et al., 2007;Hsieh et al., 2010;Hseu, Huang, & Hsiang, 2010;Lee et al., 2011b;Liao, Kuo, Liang, Shen, & Wu, 2012;Deng et al., 2013;Chen et al., 2013).
Ergostatrien-3β-ol was isolated from the fruiting body of A. camphorata by submerged fermentation (Huang et al., 2010). The anti-inflammatory activity was evaluated in vivo for reduction of NO production as well as for serum levels of TNF-α using a commercial ELISA kit. This compound significantly inhibited NO and TNF-α levels after Carrageenan injection and inhibited iNOS and COX-2 protein expression in the animal model. These results suggest that the compound may be useful to develop new anti-inflammatory agents, and the mechanism of action may be related to inhibition of iNOS expression. inhibited NO production in a dose dependent manner, very similar to control. They also significantly inhibited TNF-α, IL-6, and PGE2 release from the cell culture supernatant. The anti-inflammatory activity was reported to be due to down regulation of iNOS and COX-2 mRNA protein expression.

Other metabolites
Beside the major compounds with reported anti-inflammatory activity, other bioactive metabolites (Supplementary material S7) such as fatty acids, succinic and maleic derivatives, adenosine, cordycepin and glycopeptides have been studied ( Table 6) and known to inhibit the production of inflammatory mediators as well as to suppress the induced inflammation in vivo.
Fomes fomentarius is an inedible mushroom specie common in Europe, Asia and North America, known for its large fruiting body and decomposing property. Its fruiting body was extracted with methanol and methyl 9-oxo-(10E, 12E)-octadecadienoate isolated (Choe, Yi, Lee, Seo, Yun, & Lee, 2015). The anti-inflammatory activity was evaluated in peritoneal macrophages for NO, PGE2 production, TNF-α and IL-6 release. At 20 µg/mL, the compound significantly inhibited NO and PGE2 by 65% and 50%, respectively, while TNF-α and IL-6 levels were inhibited up to 35% and 13%, respectively. Finally, the mechanism of action of the compound was found to be due to inhibition of iNOS and COX-2 protein expression and also due to a slight inhibition of phosphorylation of ERK1/2 kinase.
Polysaccharide protein complexes obtained from mushroom have been known to play several biological functions such as immunomodulatory, antitumour and antioxidant (Wu, Chen, & Siu, 2014). The anti-inflammatory activity of several glycoproteins have been reported by some authors (Chen, De Mejia, & Wu, 2011;Lau, Abdullah, Aminudin, Lee, & Tan, 2015).
The anti-inflammatory activity of a glycopeptide from Ganoderma capense (Lloyd) Teng was evaluated in RAW264.7 cells for NO production and for iNOS enzyme activity (Zhou, Chen, Ding, Yao, & Gao, 2014). It was found that LPS-induced NO production and iNOS expression were significantly inhibited in a dose-dependent manner.

Commercial and synthesised compounds
Researchers have attempted to synthesize compounds with improved properties as drug candidates with the potential to inhibit production of NO and other inflammatory mediators such as interleukins (IL 1β, IL-6, IL-8), TNF-α and PGE2. Some synthesised and commercial compounds with positive anti-inflammatory potential have been reported (Table 7). Taofiq et al. (2015)  including the anti-inflammatory activity (Kim et al., 2003;Rao, Fang, & Tzeng, 2007;Rao, Fang, Wu, & Tzeng, 2010;Han, Oh, & Park, 2011). This mushroom contains a lot of bioactive compounds and "Cordycepin" an adenosine analogue is the most important one. Jeong et al. (2010) studied the anti-inflammatory activity of commercial cordycepin in LPS stimulated murine BV2 microglia cells for inhibition of NO production as well as PGE2, and pro-inflammatory cytokine release. Cordycepin at 7.5 µg/mL, decreased levels of NO production up to 65% while the PGE2 concentration measured using ELISA kit was also repressed up to 60%. This anti-inflammatory mechanism was found to be due the inhibition of iNOS and COX-2 protein expression. Choi et al. (2014b) also studied the antiinflammatory potential of cordycepin in LPS stimulated RAW 264.7 macrophage cell line for NO production, cytokine (TNF-α and IL-1β) levels and PGE2 production. At 30 µg/mL exposure of induced cells to cordycepin, there was significant decrease in NO, TNF-α, IL-1β and PGE2 levels. The mechanism of inhibition was further confirmed by decreased levels of LPS-induced NF-κB/p65 levels in the nucleus and inhibition of phosphorylation of IkB-α complex.

Concluding remarks
The present review focuses on the anti-inflammatory activity of some important worldwide edible, wild and medicinal mushrooms as well as on the bioactive metabolites they contain, Also the IC 50 values, concentration responsible for 50% inhibition of inflammatory mediators production, even for the same mushroom species using the same anti-inflammatory assay, might be different as the bioactive compounds are released depending upon the type of cultivation environment, solvent and extraction procedure, extraction time and mushroom maturation..
Among the several bioactive compounds isolated from mushrooms and studied for antiinflammatory activity, polysaccharides, terpenes and phenolic derivatives are the most implicated and known to show positive bioactivity. Only few research studies reported the bioactivity of ergosterol, fatty acids, glycopeptides, and nucleic acid analogues as well as of other metabolites.
Hence, mushrooms have valuable therapeutic compounds whose mechanism of action need to be fully elucidated and corroborated by conducting clinical trials in order to confirm the effectiveness of some of these mushroom-based inhibitors of NF-κB pathway as well as inhibition of the cyclooxygenase enzyme responsible for expression of many inflammatory mediators. This will consolidate the basis for the development of mushroom-based nutraceuticals or drugs effective against inflammation.
Akihisa, T., Nakamura, Y., Tagata  Schematic diagram of nuclear factor-κB (NF-κB) pathway. Macrophage cells express membrane receptors such as toll-like receptors (TLRs) and tumor necrosis factor receptors (TNFR). These receptors recognize pro-inflammatory stimuli such as lipopolysaccharides and viral proteins. Attachment of these pathogen-associated molecular patterns (PAMPs) to membrane receptors activates the myeloid differentiation protein 88 (MyD88). MyD88 activates specific protein kinases that are responsible for activation of IKK kinase (IKKα, IKKβ, IKKγ). This kinase further phosphorylates IkB-α complex leading to dissociation of the complex, and its proteasomal degradation, allowing NF-kB to translocate to the nucleus, where it binds to specific DNA sequences encoding the pro inflammatory cytokines (e.g., IL-1, IL-2, IL-6, TNF-α as well as Cyclooxygenase-2 (Cox-2) and inducible nitric oxide synthase (iNOS).