Chemical Constituents with Inhibitory Activity of NO Production from a Wild Edible Mushroom, Russula vinosa Lindbl, May Be Its Nutritional Ingredients

Russula vinosa Lindbl is a wild edible mushroom that is usually used for original material of food and soup and has rich nutritional value. What are the nutritional ingredients? In order to answer this question, we investigated the chemical constituents of this wild functional food. Six new compounds (1–6), together with nine known ones (7–15), were isolated from R. vinosa. The six new compounds were named as vinosane (1), rulepidadione C (2), (24E)-3,4-seco-cucurbita-4,24-diene-26,29-dioic acid-3-methyl ester (3), (24E)-3,4-seco-cucurbita-4,24-diene-26-oic acid-3-ethyl ester (4), (24E)-3β-hydroxycucurbita-5,24-diene-26,29-dioic acid (5), and (2S,3S,4R,2′R)-2-(2′-hydroxydocosanoylamino)eicosane-1,3,4-triol (6). Their structures were determined based on spectroscopic methods including HR-ESI-MS, 1D, and 2D NMR. Moreover, a cell counting kit-8 (CCK-8 kit) was used to screen for the cytotoxicity of compounds 1–5 and 7–13 on mouse macrophage RAW 264.7 cells. The results showed that compounds 1–5 and 7–13 had no obvious cytotoxicity. In addition, the inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated mouse macrophage RAW 264.7 cells were evaluated. Compounds 1, 3, 4, 7, 12, and 13 showed moderate inhibitory activity on NO production.


Introduction
Russula vinosa Lindbl belongs to the genus of Russula, a multifunctional edible fungus, which is distributed mainly in Northwest Fujian province and Jiangxi province in China. Studies have shown that R. vinosa has good antitumor and anti-inflammatory activities. Related research has revealed that the water-soluble components of R. vinosa are mainly polysaccharides, and the fat-soluble components are sesquiterpenes, triterpenes, steroids, ceramides, fatty acids, and other compounds [1][2][3]. Research progress of fat-soluble components in edible mushroom has shown that they have several bioactivities, such as anti-tumor, anti-inflammation, anti-oxidant, and antibacterial effects [4][5][6][7]. Consequently, the chemical analysis of the Russula edible fungus was performed by the natural product chemistry and food chemistry scientists. The chemical constituents of several other mushrooms have been investigated, such as R. japonica, R. subnigricans, R. lepida, R. cyanoxantha, etc. [8][9][10][11]. R. vinosa has been used as a multifunctional edible food in our life and although there are some studies on the water-soluble polysaccharides, few systematic investigations on the fat-soluble components have been performed. In order to systematically investigate the fat-soluble components of R. vinosa, we separated and identified the fat-soluble components of R. vinosa by silica gel column, ODS column, gel column, and preparative high-performance liquid chromatography (Pr-HPLC), and finally 15 compounds ( Figure 1) were determined according to their physicochemical data and spectral data such as 1D, 2D-NMR, and MS. Interestingly, six new compounds were found in this fungus, and also the inhibitory effect of those compounds on nitric oxide (NO) production in lipopolysaccharide-activated macrophage RAW 264.7 cells was evaluated. As a result, compounds 1, 3, 4, 7, 12, and 13 showed moderate inhibitory activity on NO production at higher concentrations. several bioactivities, such as anti-tumor, anti-inflammation, anti-oxidant, and antibacterial effects [4][5][6][7]. Consequently, the chemical analysis of the Russula edible fungus was performed by the natural product chemistry and food chemistry scientists. The chemical constituents of several other mushrooms have been investigated, such as R. japonica, R. subnigricans, R. lepida, R. cyanoxantha, etc. [8][9][10][11]. R. vinosa has been used as a multifunctional edible food in our life and although there are some studies on the water-soluble polysaccharides, few systematic investigations on the fat-soluble components have been performed. In order to systematically investigate the fat-soluble components of R. vinosa, we separated and identified the fat-soluble components of R. vinosa by silica gel column, ODS column, gel column, and preparative high-performance liquid chromatography (Pr-HPLC), and finally 15 compounds ( Figure 1) were determined according to their physicochemical data and spectral data such as 1D, 2D-NMR, and MS. Interestingly, six new compounds were found in this fungus, and also the inhibitory effect of those compounds on nitric oxide (NO) production in lipopolysaccharide-activated macrophage RAW 264.7 cells was evaluated. As a result, compounds 1, 3, 4, 7, 12, and 13 showed moderate inhibitory activity on NO production at higher concentrations.

Marked Peaks of Isolated Compounds
After the isolation, 15 isolates were marked on the UPLC-Q/TOF-MS chromatograms in Figure 3. The identification of these 15 compounds could be seen in Tables S1 and S2.

Bioactivity Evaluation
In this study, the inhibition of NO production by LPS-induced RAW 264.7 mouse macrophages was measured. Twelve components were used to screen the potential inhibitory activity on NO production.

Bioactivity Evaluation
In this study, the inhibition of NO production by LPS-induced RAW 264.7 mouse macrophages was measured. Twelve components were used to screen the potential inhibitory activity on NO production.

Cytotoxic Activity Assay
The results (Figure 4) showed that there was no significant difference in cell viability between LPS, dexamethasone sodium phosphate (Dex), the monomer groups (50, 25, 12.5 µg/mL), and the control group.

Bioactivity Evaluation
In this study, the inhibition of NO production by LPS-induced RAW 264.7 mouse macrophages was measured. Twelve components were used to screen the potential inhibitory activity on NO production.

Cytotoxic Activity Assay
The results (Figure 4) showed that there was no significant difference in cell viability between LPS, dexamethasone sodium phosphate (Dex), the monomer groups (50, 25, 12.5 μg/mL), and the control group.

Inhibitory Activity on NO Production Assay
In the results ( Figure 5), compared with the control group, the release of NO in RAW264.7 cells was significantly increased after LPS stimulation, and the monomers inhibited the release of NO in different degrees. Compounds 1, 3, 4, 7, 12, and 13 showed better activity at higher concentrations. Through this experiment, we screened some compounds with inhibitory activity on NO production.

Inhibitory Activity on NO Production Assay
In the results ( Figure 5), compared with the control group, the release of NO in RAW264.7 cells was significantly increased after LPS stimulation, and the monomers inhibited the release of NO in different degrees. Compounds 1, 3, 4, 7, 12, and 13 showed better activity at higher concentrations. Through this experiment, we screened some compounds with inhibitory activity on NO production.

Bioactivity Evaluation
In this study, the inhibition of NO production by LPS-induced RAW 264.7 mouse macrophages was measured. Twelve components were used to screen the potential inhibitory activity on NO production.

Cytotoxic Activity Assay
The results (Figure 4) showed that there was no significant difference in cell viability between LPS, dexamethasone sodium phosphate (Dex), the monomer groups (50, 25, 12.5 μg/mL), and the control group.

Inhibitory Activity on NO Production Assay
In the results ( Figure 5), compared with the control group, the release of NO in RAW264.7 cells was significantly increased after LPS stimulation, and the monomers inhibited the release of NO in different degrees. Compounds 1, 3, 4, 7, 12, and 13 showed better activity at higher concentrations. Through this experiment, we screened some compounds with inhibitory activity on NO production.

General
HPLC was run on Agilent 1260 HPLC (Agilent, Palo Alto, CA, USA). Semi-preparative HPLC was performed on Waters 2489 equipped with a diode array detector and a C 18 column (250 mm × 10 mm, 5 µm, Waters, Maple St. Milford, MA, USA). NMR spectra was measured on an AV-400 spectrometer (Bruker Corporation, Faellanden, Switzerland). Thin-layer chromatography (TLC) was performed on glass precoated silica gel GF 254 plates (Qingdao Haiyang Chemical Co., Ltd, Qingdao, China), detection under UV light or by heating after spraying with 10% sulfuric acid (H 2 SO 4 ) in 90% ethanol (EtOH). Column chromatography was performed on silica gel (200-300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), and Sephadex LH-20 (Amersham Pharmacia Biotech, Uppsala, Sweden) were used for the chromatography column. Other chemicals and reagents of analytical grade were from Tianjin Concord Technology (Tianjin, China).

UPLC-QTOF-MS/MS Conditions
The chromatography was performed with a Waters Acquity UPLC BEH C 18 column (2.1 × 100 mm, 1.7 µm; Waters, Milford, MA, USA) and the column temperature was maintained at 50 • C. One µL of sample was used for separation. The parameters of the mass spectrometer were set as follows: capillary voltage, 3kV in negative ion mode and positive ion mode; cone voltage, 40 V; ion source temperature, 120 • C; desolvation temperature, 450 • C; desolvation gas (N 2 ) flow rate, 750 L/h; the first range scan, m/z 100-1600 Da; collision gas, Argon. During low energy scanning, trap collision energy was 4 eV, transfer collision energy was 6 eV, during high energy scanning, trap collision energy was 15 eV, transfer collision energy was 30-50 eV. The mass range was from m/z 50 to 1500. Leucine-enkephalin (m/z 556.2771(+)/554.2615(−)) was selected as the lock mass at a concentration of 400 µg/L and flow rate of 5 µL/min.

Plant Materials
The

Conflicts of Interest:
No conflict of interest was reported by the authors.