Wild and Cultivated Mushrooms Exhibit Anti-Inflammatory Effects Including Inhibition of Platelet Aggregation and Interleukin-8 Expression

1. Introduction

Mushrooms have been used as herbal medicines in China, Japan, and other countries since ancient times [1,2]. In Japan, there are a number of commercially available medicines made from mushrooms; for example, krestin from Coriolus versicolor, lentinan from Lentinula edodes, and schizophyllan from Schizophyllum commune [3]. Currently, there are 2.2–3.8 million species of fungi on Earth. Of these, only 120,000 (3%–8%) species have been accepted in Species Fungorum [4], and very few have been studied for their pharmacological effects or edibility.

The potential of mushrooms with as-yet unknown medicinal properties as raw materials for drug discovery is widely accepted, and these putative properties are an important focus of current research. Poisonous mushrooms in particular are suspected to contain useful medicinal components, despite their toxicity.

There are approximately 130 reported medicinal effects of mushrooms and fungi; these include antitumor, immunomodulatory, antioxidant, radical scavenging, cardiovascular, antihypercholesterolemic, antiviral, antibacterial, antiparasitic, antifungal, detoxifying, hepatoprotective, and antidiabetic effects [5,6]. Among these, anti-inflammatory activity is a key functionality, which explains the diverse pharmacological effects of mushrooms. Thus, examining the anti-inflammatory effects of mushrooms could provide important insight for future drug-discovery research. Although many studies have compared the anti-inflammatory effects of a few mushroom species, few have examined the effects across wide range of species.

The preset study provides a comprehensive comparison of 66 species of wild and cultivated mushroom, focusing on their inhibition of platelet aggregation and interleukin (IL)-8 expression.

2. Materials and Methods

2.1. Obtaining and Storage of Fruiting Bodies

Table 1 presents the samples used in this study. Wild mushrooms were collected from mountains and fields in Japan—mainly in the eastern region from Hokkaido to northern Kanto—between 2010 and 2017. Artificially cultivated fruiting bodies were provided by Murata Shiitake Honpo, co. ltd., Miyazaki, Myogi Kinoko, Gunma, JA Nakano, Nagano and Takeuchi Kinoko, Nagano. The obtained fruiting bodies were using a mill, and stored at −25°C. Fungi were classified as edible, inedible, and poisonous based on a previous study [7]. Of the 66 mushroom species, there were 39 Agaricale, ten Polyporale, four Boletale/Pezizale, two Cantharellale/Russulale, and one Aphyllophorale/Auriculariale/Hymenochaetale/Thelephorale/Tremellale species. All samples were taken from mature fruiting bodies that had opened.

2.2. Preparation of Samples for Assay

Dried fruit bodies were ground using a Wonder Blender WB-1 (Osaka Chemical Co., Ltd.; Osaka, Japan), then sieved through a 1000-μm mesh. The resulting powder was used for analyses. To evaluate inhibition of platelet aggregation and IL-8 expression, the powder was extracted by incubating in 10 volumes of hot water (80°C) for 2 h, after which the hot-water extract was concentrated under reduced pressure after filtration using Advantec No. 2 filter paper (Toyo Roshi Kaisha, Ltd.; Tokyo, Japan).

2.3. Platelet Aggregation Assay

We evaluated the inhibition of platelet aggregation induced by sodium arachidonate (A-Na), platelet activating factor (PAF), and adenosine diphosphate (ADP). Human peripheral blood was collected from the median cubital vein of a medication-free healthy adult for at least 2 weeks. The blood was centrifuged (200 × g for 20 min at room temperature) and the upper layer collected to obtain platelet-rich plasma (PRP). The lower layer was then centrifuged (200 × g for 5 min at room temperature) and collected to obtain platelet-poor plasma (PPP).

PRP and PPP (223 μL each) were preheated at 37°C. The 5% hot-water extract was dissolved in 2% dimethyl sulfoxide (DMSO) solution and 2 μL was added to PRP and PPP. The resulting mixtures were incubated for 3 min at 37°C, then 25 μL of an aqueous solution of either AA, PAF, ADP, or ion-exchanged water (control) was added to induce platelet aggregation. The concentrations of AA, PAF, and ADP were 500 nM. Aggregation was measured using an aggregometer (MCM Hema Tracer 313M; MC Medical Co., Ltd. Tokyo, Japan) and inhibitory effects evaluated by comparing the maximum aggregation rate (calculated as the maximum value of the aggregation curve for each sample by normalizing the value of the PPP sample to 100) with the control. Inhibition rates were normalized to the control to calculation of the efficacy of the test agents [8].

2.4. Inhibition of Interleukin-8 Expression

Normal human dermal fibroblasts were cultivated in Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum until the growth was confluent (6 cm in diameter). The 5% hot-water extract was dissolved in 2% DMSO solution and 2 μL was added to the dish (final concentration: 0.01% [dry mass]). For the positive control, 10⁻⁷ M hydrocortisone was added in place of the hot-water extract. Next, 1 ng/mL of tumor necrosis factor alpha (TNF-α, which promotes chemokine gene expression) was added, and samples were incubated for 6 h at 37°C. A sample-free control was prepared without TNF-α, and incubated under identical conditions.

Total RNA was isolated using the ISOGEN reagent (Nippon Gene Co., Ltd.; Tokyo, Japan) according to the manufacturer’s instructions. Total RNA (1 μg) was reverse transcribed to cDNA using M-MLV reverse transcriptase (Life Technologies Co. Ltd.; Maryland, USA) according to the manufacturer’s instructions. Expression of IL-8 was measured using quantitative real-time polymerase chain reaction. cDNA was prepared using TaqMan reverse transcription reagent, and samples were quantified using TaqMan universal PCR master mix and the ABI Prism 7,700 sequence detection system (Applied Biosystems; Foster City, CA, USA). Nucleotide sequences for PCR primers and probes were as follows: IL-8 forward primer, 5′-TCAGAGACAGCAGAGCACACA-3′; reverse primer, 5′-CTTGGCAGCCTTCCTGATT-3′; probe, 5’-AACATGACTTCCAAGCTGGCCA-3’; GAPDH forward primer, 5’-GAAGGTGAAGGTCGGAGTC-3’; reverse primer, 5’-GAAGATGGTGATGGGATTTC-3’; probe, 5’-AGGCTGAGAACGGGAAGCTTG-3’. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal standard gene. Inhibition rates were normalized to that of TNF-α for calculation of the efficacy of samples [9].

2.5. Statistical Analyses

Data are expressed as means ± standard deviation of at least three replicates for each sample. Statistical analyses were performed using Microsoft 365 Excel with Statcel4 add-in software (OMS; Tokyo, Japan). Data were compared using one-way analysis of variance followed by the Tukey–Kramer post-hoc test for multiple comparisons. P values of <0.05 were considered statistically significant.

3. Results

3.1. Platelet Aggregation Assay and Inhibition of Interleukin-8 Expression

Table 2 shows the rates of inhibition of platelet aggregation and IL-8 expression. All samples exhibited inhibitory effects. Regarding platelet aggregation inhibition, the highest mean value for A-Na was 98.1% ± 0.21%, whereas Helvella crispa had the lowest of 38.3% ± 0.47%. In terms of PAF, the highest mean value was 96.8% ± 0.74%, whereas Mycena haematopoda had the lowest of 37.3% ± 1.25%. In terms of ADP, the highest mean value was 96.6% ± 0.36%, whereas Helvella crispa had the lowest of 41.0% ± 3.74%. In terms of the IL-8 gene, the highest mean value was 97.0% ± 1.41%, whereas Agaricus bisporus var. albidus had the lowest of 17.0% ± 2.45%. Gyromitra esculenta exhibited the highest inhibition rate among all assays. There were no significant differences in the IL-8 expression among all poisonous mushrooms, including Gyromitra esculenta, Amanita muscaria, Amanita pantherina, Pleurocybella porrigens, Tricholoma flavovirens, Amanita virosa, Psilocybe argentipes, and Chlorophyllum molybdites. The nonedible mushrooms; Trametes versicolor, Ganoderma lucidum, Wolfiporia cocos, and Polyporus umbellatus did not statistically differ. In the edible mushrooms; Tricholoma matsutake, Pleurotus ostreatus, Lyophyllum decastes, Hericium erinaceum, and Sparassis crispa, the inhibition rates were not statistically significant.

Inhibition rates of 80% or more are considered empirically applicable for the purposes of drug discovery. Twenty-nine (43.9%) of the 66 species that we tested achieved this rate for aggregation induced by A-Na, while 31 (47.0%) and 31 (47.0%) species achieved high rates for inhibition of aggregation induced by PAF and ADP, respectively. In inhibition of IL-8 gene expression, 33 (50.0%) species achieved high rates.

Of the samples that exhibited “low” inhibition rates (50% or lower), four (6.1%), five (7.6%), and seven (10.6%) showed low inhibition of A-Na-, PAF-, and ADP-induced platelet aggregation, respectively; while nine (13.6%) exhibited low inhibition of IL-8 expression (Table 3). 87.5%–100% of poisonous mushrooms exhibited high inhibition rates of 80% or more through four assays.

3.2. Taxonomical Classification

Our examination of the taxonomic commonality of species revealed the variance of specimens in each order to be large, ranging from 23.3 for Cantharellale to 760.7 for Pezizale, and no trends were identified (Table 4).

4. Discussion

Inhibition of platelet aggregation is an important aspect of anti-inflammatory activity. Platelets aggregate at the site of vascular injury to stop bleeding, as well as to induce an inflammatory response through the release of inflammatory mediators including prostaglandin E2 (PGE2), thromboxane A2 (TXA2), histamine, serotonin, and PAF. Anti-inflammatory drugs suppress inflammatory responses by inhibiting platelet aggregation; for example, nonsteroidal anti-inflammatory drugs inhibit cyclooxygenase and suppress the production of PGE2 and TXA2. In contrast, inflammatory mediators released from platelets induce the expression of inflammatory chemokine genes such as IL-8, promoting inflammation [10]. Inhibition of platelet aggregation and chemokine expression can have effects both upstream and downstream of the inflammatory response. The present study supports previous reports of the anti-inflammatory effects of fungi [11,12,13]. Inhibition of cyclooxygenase 2 has been confirmed in several fungi [14,15]. Although COX inhibition was not examined in this study, we expect a COX2 inhibitory effect, and we would like to consider this as a topic for future study.

Our results suggest that many species of fungi have anti-inflammatory properties and that poisonous species in particular have great potential value as raw materials for drug discovery. Our findings highlight the high number of poisonous species with medicinal properties.

Isolation and/or identification of specific bioactive compounds responsible for the observed activities of the tested species was beyond the scope of the present study; however, polysaccharides, proteins, fatty acids [16,17,18], sterols and polyisoprene polyols [19,20], and carbohydrate-protein complexes [21] have been suggested to be involved in the bioactivity.

We investigated the inflammatory effects using hot-water extracts, which may lead to different results compared with other extraction methods. Further detailed studies are required to identify the active compounds. Furthermore, pharmacological effects cannot be determined on the basis of in-vitro tests alone; thus, further in vivo tests and human clinical trials are required to draw firm conclusions from the present findings, as well as identify the absence or involvement of cytotoxicity etc.

The commonality between genera could not be examined due to the small sample number, but we speculate that it may be due to the characteristics of each species. The pharmacological effects of mushrooms have been reported to be influenced by the composition of the medium [22], as well as by the strain and cultivation stage [23]. This suggests that the characteristics of fungi species may vary depending on the growth environment and stage.

5. Conclusions

The present examination of the anti-inflammatory effects of 66 wild and cultivated fungi species revealed approximately half of the mushrooms to have high rates of inhibition of inflammation. Our findings imply that the presence of substances with anti-inflammatory effects may be a universal property of fungi. Mushrooms have been reported to exhibit various pharmacological activities, and their anti-inflammatory effects are speculated to be involved in the underlying mechanisms. The results presented here highlight the potential of fungi as promising raw materials for drug discovery and open up new avenues for detailed research to identify the active pharmacological components in these species.