Extracts and Steroids from the Edible Mushroom Hypholoma lateritium Exhibit Anti‐Inflammatory Properties by Inhibition of COX‐2 and Activation of Nrf2

Hypholoma lateritium is an edible macrofungus with a common distribution in Europe, North America, and the Far East. The aim of this study was to investigate the potential anti‐inflammatory effects of H. lateritium extracts and its isolated steroids: fasciculic acid B, fasciculol E, fasciculol C, lanosta‐7,9(11)‐diene‐12β,21α‐epoxy‐2α,3β,24β,25‐tetraol, fasciculol F, and demethylincisterol A2. Organic (hexane, chloroform and 50 % methanol) and water extracts of H. lateritium were subjected to in vitro assays to determine pro‐inflammatory protein levels, such as cyclooxygenase‐2 (COX‐2), cytosolic prostaglandin E2 synthase (cPGES), and antioxidant nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2). Fungal extracts demonstrated significant activities on pro‐inflammatory protein levels with minor differences among the activities of the fractions of different polarities. All the compounds proved to exert notable inhibitory properties on COX‐2 and were capable to stimulate the Nrf2 pathway. Fungal extracts and the compounds exerted no cytotoxic activities on RAW 264.7 cells.


Introduction
The genus Hypholoma, which means 'mushrooms with threads', belongs to the family Strophariaceae and includes mushroom species possessing characteristic well-pigmented pileus and variably developed threadlike veil, which does not form a membranous annulus on the stipe. [1] The genus consists of about 30 species worldwide, occurring in temperate to tropical regions, growing on decomposing wood, living trees, or soil. [2] Hypholoma species are recognized as active wood and litter decomposers and play a significant role in forest ecosystems, being used not only in bioconversion of cellulose, fabric and dye industrial residues, [3,4] but also in biological control of phytopathogenic fungi. [5,6] Hypholoma fasciculare is the most widespread and investigated member of the genus, which is known for its antioxidant and antimicrobial activities, [7] producing different types of fungal metabolites, e. g., styrylpyrone-type compounds (hypholomins, fasciculins), [8] steroids (fasciculic acids, fasciculols), [9,10] and sesquiterpenoids (fascicularones). [11,12] Apart from H. fasciculare, there is H. lateritium (brick cap mushroom), a less known related species, but still with a quite common distribution in Europe, North-America and the Far East. It is a saprobic macrofungus, occurring regularly in small tufts or sometimes singly on hardwood stumps and exposed roots of dead hardwood trees. H. lateritium was reported to contain steroid compounds, e. g., fasciculols, fasciculic acids and sublateriols [13,14] as well as sesquiterpenes, e. g., naematolin, a caryophyllane derivative with antiproliferative property. [15] We have recently explored the chemistry of this species and identified a series of steroids with remarkable structural diversity including ergostane and lanostane derivatives, along with highly degraded sterols with ion channel modulating properties. [16,17] As regard the pharmacology of H. lateritium, previous investigations revealed that this species possesses considerable biological properties; however, these experiments were performed with crude extracts without identifying the major fungal metabolites responsible for the observed biological activity. Lee at al. demonstrated that the extract of this species decreases TNF-α-induced inflammation in human umbilical vein endothelial cells. The butanol fraction of H. lateritium inhibited TNF-α-induced monocyte adhesion to endothelial cells; moreover, it dose-dependently decreased the expression of inducible nitrogen oxygen synthase and cyclooxygenase-2. [18] In another article, Lee et al. investigated the inhibitory effect of H. lateritium extract on highly invasive and metastatic tumor cells. The hexane fraction of brick cap significantly inhibited the invasion and migration of MDA-MB-231 breast cancer cells in the Matrigel invasion assay and wound-healing investigations, respectively. The results obtained suggested that hexane extract of H. lateritium inhibits the metastatic potential of MDA-MB-231 cells by inhibiting the phosphorylation of JNK/ p38 and reducing AP-1 and NF-кB DNA-binding activities. [19] Despite of its wide geographical distribution and richness in various fungal metabolites with pharmacological potential, the ethnomycological profile of H. lateritium is rather unexplored. Nonetheless, this mushroom was used in Swedish folk medicine as an anti-inflammatory agent in alleviating symptoms of rheumatic disorder. [20] Therefore, we conducted a research to explore the anti-inflammatory properties of H. lateritium extracts and its characteristic constituents, fasciculic acid B (1), fasciculol E (2), fasciculol C (3), lanosta-7,9(11)-diene-12β,21α-epoxy-2α,3β,24β,25tetraol (4), fasciculol F (5), and demethylincisterol A2 (6) for the purpose of confirming the traditional use of this species.

Results and Discussion
Basidiomycota mushrooms are known to possess various beneficial pharmacological properties including anti-inflammatory activity. [21] Previous studies revealed that several extracts prepared from certain edible mushrooms have anti-inflammatory potential: Cantharellus cibarius, [22] Imleria badia, [23] and Agaricus bisporus. [24] In the current study, we examined the proor anti-inflammatory properties of H. lateritium extracts and identified specific fungal metabolites (1-6) which may contribute to the favorable biological activities of this fungal species. Accordingly, organic (hexane, chloroform and 50 % methanol) and H 2 O extracts of H. lateritium were prepared, and then, they were subjected to in vitro tests in order to determine the proinflammatory protein levels, such as COX-2, cPGES as well as Nrf2 using Western blot techniques. Regarding the cytotoxic effect, no such activities were observed in RAW 264.7 cells incubated with mushroom extracts and fungal metabolites 1-6. Cell viabilities were around 100 % after treatment. According to results, all fractions demonstrated significant biological activities in the assays performed, however, minor differences were observed among the activities of the fractions with different polarities (Figure 1).
In RAW 264.7 cells activated with LPS and incubated with mushroom extracts A -D, an increase of Nrf2 was observed. In the same way, higher levels of cPGES protein were detected in macrophages cotreated with LPS and extracts A -D, but the values obtained were significantly lower compared to those of the LPS-activated cells. The investigations revealed a decrease in COX-2-levels in RAW 264.7 cells cotreated with mushrooms extracts and LPS in comparison with the experiment of LPS-activated macrophages.
To identify the main constituents of H. lateritium responsible for the detected anti-inflammatory properties of the crude fungal extracts, we proposed to perform the pharmacological assay of characteristic compounds of H. lateritium. The fungal metabolites investigated in the current study belong to the vast class of steroids ( Figure 2).
Fasciculic acid B (1), fasciculol E (2), fasciculol C (3), and fasciculol F (5) represent a special group of compounds known as fasciculols which are specific to mushrooms of the Hypholoma genus, especially H. fasciculare and H. lateritium, lanosta-7,9(11)-diene-12β,21α-epoxy-2α,3β,24β,25-tetraol (4) is a related steroid recently identified in H. lateritium, while demethylincisterol A2 (6) is a highly degraded sterol  Chem. Biodiversity 2020, 17, e2000391 reported originally from a marine sponge of Homaxinella sp. [25] Previous investigations revealed that these compounds could have important pharmacological properties, including the calmodulin antagonistic activity of fasciculic acid B (1) and the cytotoxic property of demethylincisterol A2 (6). [10,25] Our experiments ( Figure 3) revealed that 1-6 activated cPGES, but levels of this protein were lower than those in LPSactivated RAW 264.7 cells. In cells activated with LPS and incubated with 1-6, we experienced an increase of Nrf2. Compounds 1 -6 in general proved to possess similar activities, however, fasciculol C (3) represents a particular case, because when cells were treated with 3 alone the amount of cPGES was the lowest, while the level of Nrf2 was the highest among the values obtained in all experiments.
Macrophages activated with LPS and incubated with fungal metabolites were characterized by decreased COX-2 levels when compared to LPS-activated macrophages.
Nrf2, or nuclear factor (erythroid-derived 2)-like 2, is an essential transcription factor that controls the expression of antioxidant proteins that protect against oxidative damage produced by injury and inflammation. It is a key participant of cellular defense mechanism; activation of Nrf2 leads to a subsequent production of proteins and antioxidant enzymes, providing the damaged cells and tissues with a complex antioxidant defense. Plenty of studies un-equivocally demonstrate that many plant metabolites from fruits and vegetables, e. g., curcumin, [26] resveratrol [27] and sulforaphane [28] are capable of regulating Nrf2. Although many plants produce a variety of compounds with Nrf2 activity, the potential of mushroom metabolites in this view is largely unexplored. However, extracts from Agaricus bisporus mycelia enriched in α-linolenic acid presented Nrf2 modulating activity. [24] Only a few fungal compounds are known to regulate the Nrf2 pathway, including the benzoid type antrolone and the ubiquinone derivative antroquinonol identified in Antrodia sp., and several steroids from the renowned Ganoderma lucidum. [29,30] The current study demonstrates that the examined fungal steroids could have several beneficial pharmacological properties providing multiple opportunities for the potential therapeutic application of these secondary metabolites.

Conclusions
Organic and water extracts of H. lateritium and compounds 1 -6 proved to demonstrate not only considerable inhibitory properties on COX-2, but they are also capable to stimulate the Nrf2 pathway. Our results provide experimental evidence that extracts of Hypholoma lateritium and characteristic compounds of the hexane (6), chloroform (1-5) and more polar (1, 3)

Mushroom Material
Sporocarps of Hypholoma lateritium (Schaeff.) P. Kumm (Strophariaceae family) were gathered in September 2015 in the vicinity of Bakonybél, Hungary. Fungal identification was made by Attila Sándor (Hungarian Mycological Society). A voucher specimen (No. H018) has been deposited at the Department of Pharmacognosy, University of Szeged, Szeged, Hungary.

Sample Preparation
Sporocarps of H. lateritium were lyophilized and ground with a grinder, then, a 10 g sample was extracted with 3 × 100 mL methanol for 3 × 15 min using ultrasonic bath. Following filtration, the extracts were combined and concentrated in vacuum. The residue was dissolved in 50 mL of 50 % aqueous MeOH and was subjected to liquidÀ liquid partition between hexane (4 × 25 mL) (extract A) and CHCl 3 (4 × 25 mL) (extract B) and the remaining material provided extract C. After extraction with MeOH, the residual fungal material was dried and extracted with 50 mL of boiling H 2 O for 15 min. The filtered extract was lyophilized to give extract D.

Cell Proliferation XTT Assay
Cell proliferation was evaluated using a sodium 2,3bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium) inner salt (XTT) with N-methyldibenzopyrazine methyl sulfate) working as the intermediate electron carrier (PMS). RAW 264.7 cells were seeded in 96-well plates (2.5 × 10 3 cells/well) and incubated for 24 h. The medium was then removed and 0.5; 1; 2.5; 5; 10; 50 and 100 μg of mushroom extracts A -D as well as compounds 1-6 were added to FCS-free medium and incubated for the next 24 h. Then, XTT solution (50 μL) was added to each well and incubated for 4 h at 37°C according to the manufacturer instruction (SigmaÀ Aldrich). The absorbance was measured at 475 nm and 630 nm in a Omega plate reader (BMG LABTECH, San Diego, CA, USA). The specific absorbance of the sample was calculated as follows: Specific Absorbance = A 475nm (sample)À A 475nm (blank)À A 660nm (sample). Cell viability was expressed as the percentage of control.

Statistical Analysis
All the results are presented as means � standard deviation (SD). The statistical analysis was carried out using the one-way ANOVA; p < 0.05 was considered to be significant.