Constituents from the Fruiting Bodies of Trametes cubensis and Trametes suaveolens in Vietnam and Their Anti-Inflammatory Bioactivity

It is reported that various fungi have been used for medicine and edible foods. The tropical Trametes genus is popular and well-known in Vietnam for its health effects and bioactivities. In this study, the fruiting bodies of the edible fungi T. cubensis and T. suaveolens were collected in Vietnam. The preliminary bioactivity screening data indicated that the methanol extracts of the fruiting bodies of T. cubensis and T. suaveolens displayed significant inhibition of superoxide anion generation and elastase release in human neutrophils. Therefore, the isolation and characterization were performed on these two species by a combination of chromatographic methods and spectrometric analysis. In total, twenty-four compounds were identified, and among these (1–3) were characterized by 1D-, 2D-NMR, and HRMS analytical data. In addition, the anti-inflammatory potentials of some purified compounds were examined by the cellular model for the inhibition of superoxide anion generation and elastase release in human neutrophils. Among the isolated compounds, (5,14), and (19) displayed significant anti-inflammatory potential. As the results suggest, the extracts and isolated compounds from T. cubensis and T. suaveolens are potential candidates for the further development of new anti-inflammatory lead drugs or natural healthy foods.


Introduction
Once the human body is stimulated by bacteria, viruses, wounds, or other environment factors, the immune system will respond by inflammation to resist the infection and irritation. When organs and tissues are in inflammation, neutrophils are usually the first lymphocytes to reach the infected region [1]. Various cytotoxins, such as superoxide anion and elastase can be secreted by neutrophils in response to activation of the immune system [2]. Neutrophil overexpression has been demonstrated to be related to many human diseases in recent years [3][4][5][6][7]. Coussens et al. showed the relationship between inflammation and cancer, and they also indicated that the formation of cancer cells is attributed effective system for the screening of anti-inflammatory drugs. Chinese herbal medicine has been used for thousands of years, and it is believed to display relatively lower side effects on the human body. Thus, the application of Chinese herbal medicine to the development of anti-inflammatory lead drugs for the treatment of immune diseases and cancer has become popular in recent years.
Trametes cubensis (Mont.) Sacc. and T. suaveolens ( Figure 1) are commonly used edible fungi in Vietnam and belong to the Trametes genus. They mainly grow on living or dead trees. The fruiting bodies of these fungi display various shapes, white color, and are types of saprophytic fungi. The research reports of the Trametes genus mostly focus on T. versicolor but other species are relatively few. Walder et al. reported the water extracts of T. cubensis exhibited strong anti-HIV-1 activity [26]. In addition, T. suaveolens demonstrated antioxidant [27], anti-complementary [28], and anti-HIV-1 [29] bioactivities. In preliminary examination, the methanol extracts of fruiting bodies of T. cubensis and T. suaveolens were subjected to evaluation for their anti-inflammatory bioactivities. The results that T. cubensis promoted superoxide anion generation and elastase release, indicated the immunostimulatory effect (Table 1). Therefore, in the present study the bioactive constituents of fruiting bodies of T. cubensis and T. suaveolens were investigated and the purified compounds were evaluated for their anti-inflammatory bioactivity in a cellular neutrophil model. Hopefully, the extracts and isolated compounds from the fruiting bodies of T. cubensis and T. suaveolens can be developed as lead compounds in anti-inflammatory drugs as well as natural ingredients of healthy foods.    50 ). e A phosphatidylinositol-3-kinase inhibitor was used as a positive control for superoxide anion generation and elastase release.

Structural Elucidation of Compounds 1-3
Compound 1 was isolated as optically active white powder, and the molecular formula was assigned as C30H46O6 by HR-ESI-MS analysis ([M − H] − , m/z 501.3216, calculated for C30H45O6, 501.3216, Figure S1). The IR spectrum indicated the presence of an hydroxyl (3406 cm −1 ) and a conjugated carbonyl group (1674 cm −1 ). There were six methyl singlets in 1 H-NMR spectrum ( Figure S2 (Table 2). According to its 13 C-NMR, DEPT, and HSQC spectra ( Figures S3 and S4), there were two oxygenated methines at δ 74.3 (C-15) and 79.7 (C-3), two quaternary carbons which are tetrasubstituted double bond at δ 135.6 (C-9) and 136.2 (C-8), one set of trisubstituted double bond at δ 134.7 (C-25) and 137.2 (C-24), and one carboxylic acid group at δ 178.1 (C-26). The 2 J-and 3 J-HMBC correlations ( Figure S5) from H-3 to CH3-28 and CH3-29; from H-12 to C-9 and CH3-19; from H-17 to C-20; from CH3-18 to C-1, C-5, C-9, and C-10; from H-24 to C-23, C-26, C-27; and from CH3-30 to C-8, C-13, C-14, and C-15, constructed the planar structure of 1 ( Figure 2). The configuration of OH-3 was determined as β according to the coupling constant of H-3 (7.2 Hz). Comparing the NOE correlations ( Figure S6      Trametin B (2) was isolated as a white solid and its molecular formula was also proposed as C 30 H 46 O 6 based on a deprotonated molecular ion peak at m/z 501 in the ESI-MS analysis ( Figure S7), which was the same as that of 1 and suggested 2 was an isomer of 1. The absorptions in the IR spectrum (3434 and 1629 cm −1 ) implied the hydroxy and conjugated carbonyl moieties, respectively. The 1 H-NMR spectrum of 2 ( Figure S8) revealed almost identical spectral characteristics of a lanostanoid triterpene to those of 1 ( Table 2). The significant HMBC correlations ( Figure S11) from H-3 to CH 3 -28 and CH 3 -29; from H-5 to C-4, C-6, C-9, and C-10; from H-15 to CH 3 -30; from CH 3 -18 to C-5, C-9, and C-10; from CH 3 -19 to C-12, C-13, and C-14; from H-24 to C-22 and C-27; and from CH 3 -30 to C-8, C-13, and C-14, established the planar structure of 2 as the same as that of 1 (Figure 2 (Figure 3), which was 20S rather than the common 20R. In addition, the NOE correlation between H-23 and CH 3 -27 also supported the E configuration at C-24/C-25. Conclusively, the structure of 2 was established as a stereoisomer of 1 based on the spectral data and its structure was provided ( Figure 2).
Compound 3 was obtained as a white powder and its molecular formula was assigned as C 30 Figure S13). The IR absorption peaks at 3368 and 1658 cm −1 indicated the hydroxy and carbonyl functionalities. The 1 H-NMR data of 3 ( Figure S14) showed much similarity with those of 1, except for the loss of a carboxyl group and the presence of an aldehyde moiety in 3 (δ H 9.36 and δ C 197.3) ( Table 2). All the 1 H-and 13 C-NMR data were coincided well with those reported for hexagonin F [30]. However, the clear HMBC spectral analytical data revealed the different substituted pattern as compared with the previous paper. The 2 Jand 3 J-correlation peaks ( Figure S17) from H-3 to CH 3 -28 and CH 3 -29; from H-5 to C-4, C-6, C-7, and C-9; from H-15 to CH 3 -30; from CH 3 -18 to C-5 and C-10; from CH 3 -19 to C-12, C-13, and C-14; from H-26 to C-24, C-25, and CH 3 -27; from H-24 to C-26; from CH 3 -27 to C-25; from CH 3 -30 to C-13 and C-14, were observed in its HMBC spectrum (Figure 2), which suggested it was 3,11-dihydroxylanosta-8,24-dien-26-al-21-oic acid. In the NOESY spectrum ( Figure S18), the correlations of H-3/H-5, H-15/CH 3 -19 and H-20, H-17/CH 3 -30, and CH 3 -18/CH 3 -28, established its configuration as the same as that of 1. According to the present spectral data, the chemical structure of 3 should be revised as shown (Figure 2).

Anti-Inflammatory Activity
The isolated compounds which were found to be more abundant in the present study were subjected to evaluation of their inhibitory activity against superoxide anion generation and elastase release by human neutrophils in response to fMLF/CB (Table 3) Table 3). The anti-inflammatory data revealed multiple anti-inflammatory bioactivities in active components which were purified from the fruiting bodies of T. cubensis and T. suaveolens. In previous reports, 5 showed growth inhibition on several tumor cell lines (IC 50 10-25 µg/mL) [32]. Compound 14 was reported to attenuate the growth and triggered an apoptotic process in prostate cancer cells [55]. Inhibition of various tumor cell lines by compound 19 was also determined [46]. Based on our anti-inflammatory examination results and reported bioactivities in the literature, it can be concluded that the fruiting bodies of T. cubensis and T. suaveolens could be promising as anti-inflammatory lead drugs or natural, healthy ingredients.

In Silico Study of the Potential Compounds
According to the bioactivity examination results, compounds 5, 14, and 19 exhibited significant inhibition of elastase release. Thus, the three compounds were subjected to computing to evaluate their binding affinity with human neutrophil elastase, which was a trypsin-like serine protease, and played an important role in inflammation [56]. The simulation of elastase and small molecules has been reported [57,58]. The best poses were decided by the lowest binding energy of each ligand. LY294002 was used as a positive control. After simulation, binding energy of LY294002 was determined as −6.0 kcal/mol (Table 4). LY294002 was linked to Arg147 by a hydrogen bond, and to Cys220, Phe192, and Val216 by different effects ( Figure 5A). Compounds 5, 14, and 19 showed even lower binding energy (Table 4), and this meant that these three compounds could connect to elastase easier than LY294002. Compound 5 and elastase formed a stable complex through alkyl and π-alkyl interactions with Cys42, His57, Leu99B, Phe41, Phe192, and Val216 ( Figure 5B). For 14, the hydrogen bond with Ser195, and other interactions with Arg217A, His57, Leu99B, and Phe215 established the affinity with elastase ( Figure 5C). Compound 19 was bound with Arg217A, Lue99B, Phe215, and Val216 via alkyl and π-alkyl interactions ( Figure 5D). All these in silico computing results coincided well with those afforded from the biological activity experiments.

In Silico Study of the Potential Compounds
According to the bioactivity examination results, compounds 5, 14, and 19 exhibited significant inhibition of elastase release. Thus, the three compounds were subjected to computing to evaluate their binding affinity with human neutrophil elastase, which was a trypsin-like serine protease, and played an important role in inflammation [56]. The simulation of elastase and small molecules has been reported [57,58]. The best poses were decided by the lowest binding energy of each ligand. LY294002 was used as a positive control. After simulation, binding energy of LY294002 was determined as -6.0 kcal/mol (Table 4). LY294002 was linked to Arg147 by a hydrogen bond, and to Cys220, Phe192, and Val216 by different effects (Figure 5A). Compounds 5, 14, and 19 showed even lower binding energy (Table 4), and this meant that these three compounds could connect to  alkyl and π-alkyl interactions with Cys42, His57, Leu99B, Phe41, Phe192, and Val216 (Figure 5B). For 14, the hydrogen bond with Ser195, and other interactions with Arg217A, His57, Leu99B, and Phe215 established the affinity with elastase ( Figure 5C). Compound 19 was bound with Arg217A, Lue99B, Phe215, and Val216 via alkyl and π-alkyl interactions ( Figure 5D). All these in silico computing results coincided well with those afforded from the biological activity experiments.

General Experimental Procedures
A WRX-4 melting-point apparatus was utilized to record the melting points without

General Experimental Procedures
A WRX-4 melting-point apparatus was utilized to record the melting points without correction. The Jasco P-2000 digital polarimeter was used to obtain the optical rotations. The ultraviolet (UV) and infrared (IR) spectra were examined by an Hitachi U-2001 UV/Vis spectrometer and a PerkinElmer FT-IR Spectrum RX1 spectrophotometer, respectively. 1 H-, 13 C-, and 2D nuclear magnetic resonance (NMR) spectra were recorded on the Bruker AV-400 NMR spectrometer. Chemical shifts are expressed in δ values (ppm) using tetramethylsilane as an internal standard. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) were examined on a JEOL JMS-700 spectrometer that the experimental data were afforded in the negative-ion mode.

Fungi Material
The fruiting bodies of T. cubensis and T. suaveolens were collected and identified by Ngo Anh (Department of Biology, Hue University) at the Pumat National Park of Nghean Province, Vietnam, in August 2018. The voucher specimen (Vinh 2018A001 and 2018A002) was deposited at the School of Chemistry, Biology and Enviroment, Vinh University, Vinh City, Vietnam.

Extraction and Isolation
The fruiting bodies of T. cubensis (dried weight 1.0 kg) were powdered and extracted with methanol under reflux, the combined extracts then concentrated in vacuo to obtain a brownish syrup (120 g). The methanol extract was partitioned between dichloromethane and water to yield a dichloromethane layer (40 g) and a water layer (80 g).
The powdered fruiting bodies of T. suaveolens (dried weight 3.0 kg) were extracted by methanol and fractionated with dichloromethane and water to obtain a dichloromethane layer (132 g), a water layer (172 g), and an insoluble layer (36 g).

Anti-Inflammatory Bioactivity Examination
The present study of human neutrophils was approved by the Chang Gung Memorial Hospital Institutional Review Board (No. 1612200032, Taoyuan, Taiwan) and was conducted according to the Declaration of Helsinki (2013). The examination for the superoxide anion and elastase release inhibition was based on the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c and degranulation of azurophilic granules as reported [59]. Detailed procedures are provided in the Supplementary Materials S1.

Molecular Docking Study
The AutoDock Vina program was performed for in silico calculation [60]. The crystal structure of human neutrophil elastase has been clarified [61], the PDB file was obtained from the Protein Databank (PDB ID: 1H1B). The B chain of protein was deleted, as well as the water molecules (without hydrogen bonds). The three-dimensional structures of ligands were established by Chem3D program. The AutoDockTools (ADT ver. 1.5.

Conclusions
Sixteen ingredients, including two new compounds and a revised structure constituent, were isolated from the methanol extracts of fruiting bodies of T. cubensis. In addition, eleven constituents were characterized from the fruiting bodies of T. suaveolens. Their chemical structures were characterized via spectroscopic and spectrometric analyses. In total, eleven purified compounds were evaluated for their anti-inflammatory activity by inhibition of superoxide anion generation and elastase release on a neutrophil model. The examined data demonstrated that 5, 14, and 19 display significant anti-inflammatory bioactivities. Therefore, the fruiting bodies of T. cubensis and T. suaveolens have the potential to be developed as new anti-inflammatory lead drugs or food products.