Secondary Metabolites from Hericium erinaceus and Their Anti-Inflammatory Activities

Hericium erinaceus, a culinary and medicinal mushroom, is widely consumed in Asian countries. Chemical investigation on the fruiting bodies of Hericium erinaceus led to the isolation of one new ergostane-type sterol fatty acid ester, erinarol K (1); and eleven known compounds: 5α,8α -epidioxyergosta-6,22-dien-3β-yl linoleate (2); ethyl linoleate (3); linoleic acid (4); hericene A (5); hericene D (6); hericene E (7); ergosta-4,6,8(14),22-tetraen-3-one (8); hericenone F (9); ergosterol (10); ergosterol peroxide (11); 3β,5α,6α,22E-ergosta-7,22-diene-3,5,6-triol 6-oleate (12). The chemical structures of the compounds were determined by 1D and 2D NMR (nuclear magnetic resonance) spectroscopy, mass spectra, etc. Anti-inflammatory effects of the isolated aromatic compounds (5–7, 9) were evaluated in terms of inhibition of pro-inflammatory mediator (TNF-α, IL-6 and NO) production in lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophage cells. The results showed that compounds 5 and 9 exhibited moderate activity against TNF-α (IC50: 78.50 μM and 62.46 μM), IL-6 (IC50: 56.33 μM and 48.50 μM) and NO (IC50: 87.31 μM and 76.16 μM) secretion. These results supply new information about the secondary metabolites of Hericium erinaceus and their anti-inflammatory effects.


Introduction
Mushrooms are familiar food ingredients and frequently appear on the daily dining table. Their wide consumption is not only due to their unique flavor and texture as an attractive food, but also to their beneficial effects on human health. Hericium erinaceus (Bull.) Pers. (family Hericiaceae), also known as Houtougu (monkey head) in Chinese, Lion's Mane in English and Yamabushitake in Japanese after its shape, is a popular edible and medicinal mushroom widely consumed in Asian countries (China, Japan and Korea, etc.) [1]. H. erinaceus grows on old or dead trunks of hard woods and its fruiting bodies have been used in traditional Chinese medicine for treatment of gastritis for more than 1000 years [2]. Recently, the beneficial effects of the fruiting bodies of H. erinaceus on depression, anxiety and cognitive impairment were also reported [3,4]. Previous chemical investigations on H. erinaceus have established the presence of an exceptionally large amount of structurally different bioactive and potential bioactive components, such as diterpenoids (erinacines) [5], aromatic compounds (hericerins, erinacerins and erinaceolactones) [6][7][8][9][10], sterols [11,12], polysaccharides and glycoproteins [13][14][15]. These isolated components of H. erinaceus were reported to possess various bioactivities, such as cytotoxicity [9,10], immunomodulation [16,17], nerve growth factor (NGF) promotion [18,19], and antidiabetic [7,8] properties.
In our continuing investigation on edible and medicinal mushrooms [20][21][22][23], one new (1) and eleven known compounds (2-12, Figure 1) were isolated from the fruiting bodies of bodies of H. erinaceus. Here, we report the structural elucidation of the isolated comp nents and the anti-inflammatory effects of the isolated aromatic compounds.

Results and Discussion
Compound 1 was isolated as a colorless, oily solid. Its molecular formula was esta lished as C64H106O5 using HRESIMS (
TNF-α, IL-6 and NO, the major pro-inflammatory mediators, are able to induce inflammation due to overproduction in abnormal situations [33][34][35], and the inhibition effects on their secretion are often used in evaluating the potential anti-inflammatory activities of the isolated natural products [12,36]. Bacterial lipopolysaccharide (LPS) is the best characterized stimulus for the induction of inflammatory mediators in macrophage RAW 264.7 [37]. On the basis of the traditional use in treating gastritis by H. erinaceus [2], we evaluated the potential anti-inflammatory activity of hericene A, D and E (5-7), hericenone F (9), one type of characteristic aromatic compound only isolated from H. erinaceus, using LPS-stimulated RAW 264.7 mouse cells as the cell model.
First, cell viability was evaluated using the CCK-8 assay. The results showed that compounds 5-7 and 9 did not affect cell viability at the tested concentrations. As shown in Figure 4, the secretion of TNF-α was significantly inhibited by compounds 5-7 and 9 in a dose-dependent manner, and compounds 5 and 9 showed the most potent inhibitory activities on the production of inflammatory factor TNF-α, with IC 50 values of 78.50 and 62.46 µM, respectively, compared with the positive control (Aspirin, IC 50 27.08 µM) ( Table 2). We, therefore, further evaluated compounds 5 and 9 for their inhibition on the secretion of IL-6 and NO, another two pro-inflammatory mediators, in LPS-stimulated RAW 264.7 mouse cells. As shown in Figure 5 and Table 2

General Experimental Procedures
Optical rotation was measured using a Rudolph Research Analytical APVI/6W automatic polarimeter (Hackettstown, NJ, USA). The FT-IR spectrum was recorded on a Ther-moFisher Nicolet 6700 FT-IR spectrometer (Waltham, MA, USA). The NMR spectra were recorded using Bruker AV II 600 and 400 (Billerica, MA, USA), with tetramethylsilane as an internal standard. The high-resolution electrospray ionization mass spectra

Fungal Material
Mature fruiting bodies of H. erinaceus were collected from a planting base in Jintang District, Chengdu, China, in September 2018 and identified by one of the authors (L.X.). A voucher specimen (HE-201809) was deposited at the Sichuan Institute of Edible Fungi, Sichuan Academy of Agricultural Sciences.

Extraction and Isolation
Oven-dried fruiting bodies (10 kg) of H. erinaceus were extracted with 95% EtOH (45 L × 3) under room temperature (7d each time). The EtOH extract was concentrated in vacuo to yield a residue (2.1 L), which was further suspended in water and partitioned with EtOAc (6 L × 3), yielding EtOAc fractions (125 g).

Cell Viability
Cell viability was evaluated by the CCK-8 (Cell Counting Kit-8) method. Compounds in different concentration were added to the cells and incubated for 2 h, and then CCK-8 solution (10 µL, Beyotime, Shanghai, China) was added. The cells were further incubated for 4 h and then the absorbance was measured at 450 nm.

Pro-Inflammatory Cytokines (TNF-α and IL-6) Assay
The production of TNF-a and IL-6 was measured according to the literature with minor modification [38]. RAW 264.7 cells were cultured at a density of 1 × 10 5 cells/well in RPMI 1640. Cells were pretreated with different concentrations of compounds for 2 h before LPS stimulation. Twenty-four hours after LPS (200 ng/mL) stimulation, TNF-α (TNF-α Elisa kit, Boster Biological Technology Co. Ltd., Wuhan, China) and IL-6 (Mouse IL-6 Elisa kit, Beyotime, Shanghai, China) levels in the supernatant were measured by the ELISA test according to the manufacturer's instructions.

Nitric Oxide (NO) Assay
The production of NO was measured using the Griess method as previously reported with minor modification [39]. Briefly, the RAW 264.7 cells were pretreated with different concentrations of compounds for 2 h before LPS stimulation. Twenty-four hours after LPS (200 ng/mL) stimulation, 50 µL Griess reagent I and 50 µL Griess reagent II (Beyotime, Shanghai, China) were added into the 50 µL supernatant, respectively. This mixture was incubated for 10 min at room temperature, and the absorbance was measured at 540 nm using a microplate reader (LB 941, Berthold Technologies, Bad Wildbad, Germany). The amount of nitrite in the samples was obtained by a calibration curve using NaNO 2 as the standard.

Statistical Analysis
GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA) was used for data processing and analysis. The data obtained are presented as the means ± SD of five independent experiments. A one-way analysis of variance (ANOVA) followed by Tukey's test was used to determine significant differences between each treated group and the LPS group. Values of p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) were considered to indicate statistical significance.

Conclusions
In this study, the chemical constitution of the fruiting bodies of H. erincacus was studied, and twelve compounds, including one new compound and eleven known compounds were isolated. The four typical aromatic compounds were evaluated for their inhibition effects on the secretion of TNF-α, IL-6 and NO, three major pro-inflammatory mediators, in the macrophage RAW 264.7 model. Two of them showed moderate inhibitory effects indicating their potential anti-inflammatory activity, which may provide the basis for the traditional medical use of H. erincacus.