Chemical Composition, Antioxidant and Anti-Inflammatory Activity of Shiitake Mushrooms (Lentinus edodes)

Abstract

Shiitake mushrooms (Lentinus edodes) are renowned as the “King of mountain treasures” in China due to their abundant nutritional and health-enhancing properties. Intensive chemical investigations of the fruiting bodies and mycelium of Shiitake mushrooms (Lentinus edodes) afforded five new compounds (1–5), named lentinmacrocycles A-C and lentincoumarins A-B, along with fifteen known compounds (6–20). Their structures and absolute configurations were elucidated by extensive spectroscopic analysis, including one-and two-dimensional (1D and 2D) NMR spectroscopy, circular dichroism (CD), and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The anti-inflammatory activity test showed that lentincoumarins A (4), (3S)-7-hydroxymellein (9), (3R)-6-hydroxymellein (11) and succinic acid (18) exhibited strong NO inhibitory effects (IC₅₀ < 35 μM), and that (3S)-5-hydroxymellein (10) and (3R)-6-hydroxymellein (11) exhibited potent TNF-α inhibitory effects (IC₅₀ < 80 μM) and were more potent than the positive control, Indomethacin (IC₅₀ = 88.5 ± 2.1 μM). The antioxidant activity test showed that (3R)-6-hydroxymellein (11) had better DPPH radical scavenging activity (IC₅₀ = 25.2 ± 0.5 μM).

1. Introduction

Most edible fungi are rich in bioactive substances, which not only have a high nutritional value but also have medicinal properties; therefore, they have attracted much attention [1]. At present, research on edible fungi has gradually shifted from traditional nutritional foods to health foods. Researchers have attached great importance to the development of edible fungi with good efficacy [2,3,4]. As a representative large edible fungus, shiitake mushrooms (Lentinula edodes) have a long history of consumption and are widely popular worldwide because of their attractive taste [5,6]. Bioactive compounds such as polysaccharides, purines, proteins (amino acids), fatty acids, polyphenols, and sterols [7,8] in shiitake mushrooms have been shown to have high nutritional value and enhance human health by promoting anti-inflammatory, antioxidant, antitumor, antiviral, antibacterial, and immunostimulatory effects [9,10,11,12,13].

At present, reports on the active substances in shiitake mushrooms have mostly focused on polysaccharides [14,15]. Other types of bioactive compounds in shiitake mushrooms have often been overlooked. The growth of Lentinus edodes can be divided into two stages: the vegetative phase (mycelium or mycelial growth) and the reproductive phase (fruiting bodies). After the scattered spores have invaded the substrate, the hyphae, which are only visible under a microscope, continually grow and branch to form mycelia, and the fruiting body grows out of the subterranean mycelium through a process called fructification. The fruiting bodies (at the bottom of the cap) are sporulation structures that are edible parts of edible fungi [16,17]. In recent years, research on medicinal fungi has expanded from the initial fruiting bodies to liquid fermentation [18], the number of functional secondary metabolites isolated has increased year by year, and their structures have become increasingly diversified [19].

In our study, we focused on the non-polysaccharide components of the fruiting bodies and mycelia of shiitake mushrooms. Ten compounds were obtained from the fruiting bodies and mycelia, and their anti-inflammatory and antioxidant properties were studied.

2. Materials and Methods

2.1. Instrument and Materials

UV data were collected on a Shimadzu UV-1700 spectrometer (Shimadzu, Kyoto, Japan). The IR spectra were measured on a Bruker IFS-55 (Bruker, Karlsruhe, Germany). The optical rotation values were obtained using a Perkinelmer 241 MC polarimeter (Perkinelmer, Waltham, MA, USA). The CD spectrum was measured by a Bio-Logic Science Instruments spectrometer (Bio-Logic Science Instruments, Seyssinet-Pariset, France). HRESIMS data were collected using a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific, Waltham, MA, USA). The NMR spectra were recorded using a Bruker AVANCEIII-600 and AVANCEIII-400 NMR spectrometer (Bruker Corporation, Karlsruhe, Germany). HPLC separation was carried out on a semi-preparative YMC-pack ODS-A column (250 × 10 mm, YMC-pack, Kyoto, Japan) equipped with a Shimadzu LC-8A detector (Shimadzu, Kyoto, Japan). Chromatographic silica gel (100–200 and 200–300 mesh, Yuwang Reagent Co., Ltd., Qingdao, China) and Sephadex LH-20 (18–110 lm; Pharmacia, Piscataway, NJ, USA), were used for column chromatography. The thermostatic shaker used for cultivating fungi uses an ZQPL-200 shaker (Tianjin Laiboteri Instrument Equipment Co., Ltd., Tianjin, China)

2.2. Reagents and Chemicals

The HPLC-grade solvents, such as methanol and acetonitrile (99.9%), and the analytical reagent solvents, such as petroleum ether, dichloromethane, ethyl acetate, and ethanol (99.5%), were purchased from Shandong Yuwang Reagent Co., Ltd. (Qingdao, China). The MTT used to detect the cells was obtained from Sigma Aldridge, St. Louis, MO, USA. The DPPH reagent used to detect the antioxidant activity of cells was derived from Qingbei Reagent Co., Ltd. Jinan 250100, China.

2.3. Fungal Material

Secondary strains of Lentinus edodes (with 78% wood chips, 20% bran, 1% sucrose, and 1% gypsum) were collected from the Edible Fungi Research Base of Shenyang Agricultural University and deposited at Shenyang Pharmaceutical University. After the colony grew, the strain was purified to a single colony, namely Lentinus edodes strain LE-M. Fresh shiitake mushrooms were obtained from Shenyang Farmers’ Market. Professor Pei of Shenyang Pharmaceutical University identified that the quality of this batch of shiitake mushrooms was excellent.

2.4. Large-Scale Fermentation of Strain LE-M

Preparation of the seed liquid: The purified mycelia of LE-M were inoculated in a 250 mL conical flask containing 150 mL of fungal No. 4 medium (1 L: 2% mannitol; 2% glucose; 0.5% yeast extract; 1% peptone; 0.05% KH₂PO₄; 0.03% MgSO₄·7H₂O; 0.1% corn steep liquor; water) and shaken at 25 °C for 10 days at 170 rpm. Afterward, the seed liquid (5 mL) was transferred to rice medium (100 g of rice and 100 mL of water, 220 bottles) and fermented at 25 °C statically for 45 days [20,21,22].

2.5. The Isolation of Chemical Components

Lentinus edodes mycelium rice fermentation was extracted with EtOAc 3 times at room temperature to obtain an EtOAc layer extract (72.5 g). The extract of the organic layer was subjected to column chromatography over silica gel (200–300 mesh, CH₂Cl₂-MeOH, step gradient elution 1:0, 50:1, 25:1, 10:1, 5:1, 2:1, 1:1 and 0:1) to obtain seven fractions (Fr. 1–Fr. 7). Fr. 1 (6.0 g) was separated on a silica gel column (200–300 mesh, PE-acetone, step gradient elution 10:1, 4:1, 2:1, and 1:1) to obtain six fractions (Fr. 11–Fr. 16). Then, Fr. 14 (2.6 g) was purified by Sephadex LH-20 (CH₃OH) to obtain compound 5 (12.5 mg). Fr. 15 (1.9 g) was purified by pre-HPLC (MeOH/H₂O = 35:65, 2.0 mL/min) to obtain compound 4 (4.3 mg, tR = 23.0 min).

The fruiting body of shiitake mushrooms was extracted with 95% ethanol 3 times to obtain a 25 L concentrate. The concentrate was extracted by EtOAc to yield a crude extract after evaporation under vacuum. The crude extract (19.6 g) of the organic layer was subjected to column chromatography over silica gel (200–300 mesh, CH₂Cl₂-MeOH, step gradient elution 1:0, 50:1, 25:1, 10:1, 5:1, 2:1, 1:1 and 0:1) to obtain eight subfractions (Fr. G1–Fr. G8). Fr. G5–2 (3.8 g) was purified by pre-HPLC (MeOH/H₂O = 60:40, 2.0 mL/min) to obtain compound 1 (24.1 mg, tR = 15.6 min), compound 2 (55.6 mg, tR = 17.8 min), and compound 3 (1.6 mg, tR = 19.7 min).

(3S,5S)-5-hydroxy-2,3,4,5-tetrahydrobenzo[b]oxepine-3,7-diyl (1): Yellow oil (MeOH); HR-ESI-MS: m/z 247.0941 [M + Na]⁺ (calcd for C₁₂H₁₆O₄Na, 247.0930); IR (KBr): 3396, 1634 cm⁻¹; [α${]}_{\mathrm{D}}^{25}$-6.0; UV: 254 nm; the ¹H NMR and ¹³C NMR measured using the reagent DMSO-d₆ are shown in

Table 1. The ¹H-NMR (600 MHz) and ¹³C-NMR (150 MHz) spectrum data of compounds 1–3 in DMSO-d₆.

NO	1		2		3
	δc	δH (m.; J in Hz)	δc	δH (m.; J in Hz)	δc	δH (m.; J in Hz)
1	-	-	-	-	-	-
2	75.0	3.17 (1H, d, 11.0) 4.31 (1H, dd, 11.0, 3.8)	80.7	4.17 (1H, d, 12.0) 3.84 (1H, d, 12.0)	80.1	4.19 (1H, m) 3.87 (1H, d, 12.0)
3	42.5	2.15 (1H, m)	76.7	-	76.7	-
4	39.6	2.02 (1H, d, 12.0) 1.28 (1H, q, 12.0)	52.5	2.92 (1H, d, 12.0) 2.76 (1H, d, 12.0)	52.5	2.93 (1H, d, 12.0) 2.77 (1H, d, 12.0)
5	68.7	4.72 (1H, d, 11.0)	196.6	-	196.3	-
5a	138.7	-	127.6	-	127.9	-
6	124.7	7.48 (1H, s)	127.3	7.62 (1H, d, 2.3)	129.6	7.68 (1H, d, 2.3)
7	137.6	-	137.3	-	130.5	-
8	125.9	7.06 (1H, dd, 8.5, 2.0)	132.7	7.40 (1H, dd, 8.5, 2.3)	134.4	7.50 (1H, dd, 8.3, 2.3)
9	120.5	6.85 (1H, d, 8.1)	120.7	7.05 (1H, d, 8.5)	121.2	7.12 (1H, d, 8.3)
9a	156.5	-	162.3	-	163.2	-
10	62.8	3.31 (1H, m) 3.23 (1H, m)	67.1	3.37 (2H, m)	67.0	3.40 (2H, m)
11	63.4	4.43 (2H, s)	62.5	4.44 (2H, s)	66.0	5.11 (2H, d, 12.0)
2′					177.4	-
3′					29.3	2.10 (2H, m)
4′					25.0	2.33 (1H, m) 1.95 (1H, m)
5′					55.2	4.20 (1H, m)
6′					173.2	-

. The supplementary spectra of compound 1 are shown in Figures S1–S4.

(R)-3-Hydroxy-3,7-bis(hydroxymethyl)-3,4-dihydrobenzo[b]oxepin-5(2H)-one (2): Yellow oil (MeOH); [α${]}_{\mathrm{D}}^{25}$-13.0 (c 0.01, MeOH). HR-ESI-MS: m/z 261.0747 [M + Na]⁺ (calcd for C₁₂H₁₄O₅Na, 261.0750); IR (KBr): 2929, 2878, 1676 cm⁻¹; UV: 254 nm; the ¹H NMR and ¹³C NMR measured using the reagent DMSO-d₆ are shown in Table 1. The supplementary spectra of compound 2 are shown in Figures S5–S7.

(3-Hydroxy-3-(hydroxymethyl)-5-oxo-2,3,4,5-tetrahydrobenzo[b]oxepin-7-yl) methyl-2’-oxopyrrolidine-5’-carboxylate (3): Yellow oil (MeOH); [α${]}_{\mathrm{D}}^{25}$+ 6.0 (c 0.01, MeOH). HR-ESI-MS gave m/z 372.1043 [M + Na]⁺ (calcd for C₁₇H₁₉O₇NNa, 372.1040); IR (KBr): 2926, 1738, 1679 cm⁻¹; UV: 254 nm; the ¹H NMR and ¹³C NMR measured using the reagent DMSO-d₆ are shown in Table 1. The supplementary spectra of compound 3 are shown in Figures S8–S10.

(3S,8S)-3-(1-Hydroxy-but-3-ynyl)-3H-isobenzofuran-1-one (4): Yellow oil (MeOH); HR-ESI-MS m/z 225.0648 [M + Na]⁺ (calcd for C₁₂H₁₀O₃Na, 225.0630); IR (KBr): 3429, 1671, 1472, 1384, 1291, 1202, 1053, and 1017 cm⁻¹; UV peak at 347 nm and 205 nm; the ¹H NMR and ¹³C NMR measured using reagent DMSO-d₆ are shown in

Table 2. The ¹H-NMR (400 MHz) and ¹³C-NMR (100 MHz) spectrum data of compounds 4–5 in DMSO-d₆.

	4		5
NO	δC	δH (m.; J in Hz)	δC	δH (m.; J in Hz)
1	170.4	-	170.1	-
2	-	-	-	-
3	82.4	5.67 (1H, d, 2.0)	82.4	5.77 (1H, d, 5.6)
4	123.3	7.72 (1H, d, 6.9)	124.3	7.73 (1H, d, 6.9)
5	134.4	7.77(1H, td, 7.1, 1.0)	134.4	7.77 (1H, td, 7.1, 1.0)
6	129.5	7.58 (1H, t, 7.6)	129.7	7.61 (1H, t, 7.6)
7	125.0	7.81 (1H, d, 7.6)	125.2	7.83 (1H, d, 7.6)
3a	148.3	-	147.6	-
7a	126.6	-	126.4	-
8	69.3	4.19 (1H, qd, 7.1, 2.0)	70.1	3.91 (1H, m, 11.6, 5.6)
9	24.2	2.48 (2H, m)	23.8	2.48 (2H, m)
10	81.6	-	81.3	-
11	73.6	2.95 (1H, t, 2.8)	73.6	2.89 (1H, t, 2.8)
8-OH	-	5.28 (1H, d, 6.4)	-	5.53 (1H, d, 5.6)

. The supplementary spectra of compound 4 are shown in Figures S11–S13.

(3S,8R)-3-(1-Hydroxy-but-3-ynyl)-3H-isobenzofuran-1-one (5): Yellow oil (MeOH); HR-ESI-MS m/z 225.0648 [M + Na]⁺ (calcd for C₁₂H₁₀O₃Na, 225.0630); IR (KBr): 3429, 1671, 1472, 1384, 1291, 1202, 1053, 1017 cm⁻¹; UV: 347 nm, 205 nm; the ¹H NMR and ¹³C NMR measured using the reagent DMSO-d₆ are shown in Table 2. The supplementary spectra of compound 5 are shown in Figures S14–S17.

2.6. Antioxidant Activity Assays

A 150 μMol/L DPPH solution was diluted with MeOH, and the final concentration reached an absorbance of 1.0 at a wavelength of 517 nm. Then, 3 mL of DPPH solution diluted with 1 mL of sample was mixed on 96-well plates. The absorbance was measured using a Varoskan flash multimode reader. DPPH absorbance inhibition rate (%) = (Acontrol − Atest) × 100/Acontrol [23].

2.7. Measurement of Nitrite Production and TNF-α

After 48 h of culture, the RAW264.7 cells produced NO in the medium, and 100 mL of supernatant was collected and reacted with 100 mL of Griess reagent. Indomethacin served as a positive control. The amount of NO produced at 570 nm was measured. The TNF-α levels were measured using an ELISA kit. The TNF-α detection method used was based on the literature [24,25].

2.8. MTT Assay

The activity of RAW264.7 cells was tested by MTT (Sigma Aldrich, Missouri, USA) colorimetry after 48 h of incubation, after which different concentrations of compounds were added. Then, 10 mL of 5 mg/mL MTT was added to the 96-well plates. The cells were incubated at 37 °C for 4 h, after which the medium was removed. The formazan produced by the reduction dye from live cells was lysed with 0.04 mol/L HCl isopropanol solutions. Finally, the optical density was measured. When the optical density of the experimental compound group was less than 80% of that of the control group, the compounds in the experimental group were considered to be cytotoxic.

3. Results

3.1. Structure Elucidation of Components

The non-polysaccharide components of the fruiting bodies and mycelia of Lentinus edodes were studied. Ten compounds were isolated, respectively (Figure 1). Among them, there were 5 new compounds and 15 known compounds (The supplementary spectra of known compounds are shown in Figures S18–S32) [26,27,28,29,30,31]. Next, the structure of the new compound was elucidated.

Compound 1 was a yellow oil (MeOH), HR-ESI-MS data (m/z 247.0941 [M + Na]⁺), with a molecular formula of C₁₂H₁₆O₄, with five degrees of unsaturation. The ¹H NMR spectrum (Table 1, Figure S1) displayed three hydroxy hydrogen signals at δ_H 5.40 (1H, s), 5.10 (1H, s), and 4.62 (1H, s); six oxymethylene hydrogen signals at δ_H 4.43 (2H, s, H-11), 4.31 (1H, dd, J = 11.0, 3.8 Hz, H-2), 3.31 (1H, m, H-10), 3.23 (1H, m, H-10), and 3.17 (1H, d, J = 11.0 Hz, H-2); two methylene hydrogen signals at δ_H 2.02 (1H, d, J = 12.0 Hz, H-4) and 1.28 (1H, q, J = 12.0 Hz, H-4); one oxymethine at δ_H 4.72 (1H, d, J = 11.0 Hz, H-5); and one methine at δ_H 2.15 (1H, m, H-3). In addition, the hydrogen signals at δ_H 7.48 (1H, s, H-6), 7.06 (1H, dd, J = 8.5, 2.0 Hz, H-8), and 6.85 (1H, d, J = 8.1 Hz, H-9) suggested that compound 1 contains a set of ABX diphenyl ring proton signals. ¹³C-NMR (150 MHz, DMSO-d₆) combined with the HSQC spectrum revealed aromatic carbons at δ_C 156.5 (C-9a), 138.7 (C-5a), 137.6 (C-7), 125.9 (C-8), 124.7 (C-6), and 120.5 (C-9); four oxygen-aliphatic signals at δ_C 75.0 (C-2), 68.7 (C-5), 63.4 (C-11), and 62.8 (C-10); and two aliphatic signals at δ_C 42.5 (C-3) and 39.6 (C-4).

The HMBC (Figure 2 and Figure S3) correlations from H-6 to C-5/C-5a/C-11, from H-8 to C-6/C-11, and from H-9 to C-7/C-9a indicated a benzene ring. C-7 was linked to C-11, and C-5 was linked to C-5a. The correlations from H-10 to C-2/C-3/C-4, from H-4 to C-3/C-5, and from H-2 to C-10/C-9a indicated a seven-membered ring structure, and C-3 was connected to C-10. Consequently, compound 1 was elucidated.

The relative structure of compound 1 could be determined by analyzing its NOESY correlation spectrum (Figure S4). H-5 was correlated with H-3 in the NOESY spectrum; therefore, H-3 and H-5 were on the same side. After complexation with a metal rhodium salt, the absolute configuration of C-5 was elucidated by the CD spectrum (Figure 3). According to the bulkiness rule, there was a positive Cotton effect at 350 nm [32,33]; hence, C-5 was elucidated as S. Combined with the relative configuration; C-3 was S-type.

Thus, compound 1 was elucidated, and the systematic name was (3S,5S)-5-hydroxy-2,3,4,5-tetrahydrobenzo[b]oxepine-3,7-diyl.

Compound 2 was a yellow oil (MeOH), HR-ESI-MS (m/z 261.0747 [M + Na]⁺), with a molecular formula of C₁₂H₁₄O_5, with six degrees of unsaturation. ¹H and ¹³C NMR (Table 1; Figure S5) suggested that compounds 1 and 2 have very similar structures, with the only significant difference being the substituents on the seven-membered ring. δ_C 196.6 (C-5) was a ketocarbonyl group, and δ_C 76.7 (C-3) was a quaternary carbon with oxygen. The HMBC (Figure 2) correlations from H-6 to C-5, from H-4 to C-2/C-3/C-5/C-5a/C-10, from H-10 to C-2/C-3, and from H-2 to C-3/C-9a confirmed the planar structure of compound 2. The absolute configuration of C-3 was determined by the CD spectrum (Figure 3 and Figure S7) after complexation with a metal rhodium salt [32,33]. A negative Cotton effect at 350 nm elucidated the absolute configuration of C-3 as R.

Thus, the structure of compound 2 was determined, and the systematic name was (R)-3-hydroxy-3,7-bis(hydroxymethyl)-3,4-dihydrobenzo[b]oxepin-5(2H)-one.

Compound 3 was a white powder, HR-ESI-MS (m/z 372.1043 [M + Na]⁺), with a molecular formula of C₁₇H₁₉O₇N and eight degrees of unsaturation. The ¹H NMR data suggested (Table 1; Figure S8) that compounds 2 and 3 have very similar structures, with the only significant difference being the substituent on the benzene ring. The HMBC (Figure 2 and Figure S10) correlations from N-H to C-3′/C-4′/C-5′, from H-3′ to C-2′/C-5′, and from H-11 to C-6′/C-7/C-8 and the ¹H-¹H COSY correlations of H-3′ with C-4a’/C-4b’ and H-4a’ with C-5′/C-4b’ established a five-membered nitrogen heterocyclic ring linked to C-11. The planar structure of compound 3 was elucidated, and the systematic name was (3-hydroxy-3-(hydroxymethyl)-5-oxo-2,3,4,5-tetrahydrobenzo[b]oxepin-7-yl) methyl-2’-oxopyrrolidine-5’-carboxylate.

Compound 4 was a yellow oil (MeOH) with a molecular formula of C₁₂H₁₀O₃ according to its HR-ESI-MS (m/z 225.0648 [M + Na]⁺), implying eight degrees of unsaturation. ¹H NMR (400 MHz) (Table 2; Figure S11) combined with HSQC revealed four aromatic protons at δ_H 7.72 (1H, d, J = 6.9 Hz, H-4), 7.77 (1H, td, J = 7.1, 1.0 Hz, H-5), 7.58 (1H, t, J = 7.6 Hz, H-6), and 7.81 (1H, d, J = 7.6 Hz, H-7); one hydroxy hydrogen at δ_H 5.28 (1H, d, J = 6.4 Hz, 8-OH); two oxymethine hydrogen at δ_H 5.67 (1H, d, J = 2.0 Hz, H-3) and 4.19 (1H, qd, J = 7.1, 2.0 Hz, H-8); one methine at δ_H 2.95 (1H, t, J = 2.8 Hz, H-11); and two methylene at δ_H 2.48 (2H, m, H-9). The ¹³C NMR (100 MHz) of DMSO-d₆ revealed one ester carbonyl carbon signal at δ_C 170.4 (C-1); six sp² hybrid carbons at δ_C 148.3 (C-3a), 134.4 (C-5), 129.5 (C-6), 126.7 (C-7a), 125.0 (C-7) and 123.3 (C-4); two oxygen-aliphatic signals at δ_C 82.4 (C-3) and 69.3 (C-8); two sp hybrid carbon signals at δ_C 81.6 and 73.6 (C-10 and C-11); and one saturated carbon at δ_C 24.2 (C-9).

The HMBC (Figure 2) correlations from H-6 to C-4/C-5/C-7a, from H-4 to C-3/C-6/C-7a, from H-5 to C-7/C-3a, from H-7 to C-1/C-5/C-3a, and from H-3 to C-1/C-3a/C-4/C-7a/C-8/C-9 established the 4a fragment. Correlations from 8-OH to C-3/C-8/C-9, from H-8 to C-3/C-3a/C-9/C-10, and from H-9 to C-3/C-8/C-11 implied the presence of a 4b fragment. Moreover, correlations between H-8 and C-3/C-3a and between H-3 and C-8/C-9 revealed the connecting position of segments 4a and 4b. The plane structure of compound 4 was elucidated.

The position of H-3 and H-8 of compound 5 was confirmed by the coupling constant of the compound. H-3 was δ_H 5.67 (1H, d, J = 2.0 Hz), and H-8 was δ_H 4.19 (1H, qd, J = 7.1, 2.0 Hz). Due to its coupling constant J = 2.0 Hz, the dominant conformation was obtained based on the carbon chain according to the Capon rule. The simulation confirmed that H-3 and H-8 of the compound were on the same side, which is a red configuration.

To clarify the absolute configuration of the C-3 position in compound 4, CD spectroscopy was performed [34]. Its CD spectrum (Figure 4 and Figure S13) at 220 nm showed a positive Cotton curve, so the C-3 position was S-type. Moreover, H-3 and H-8 of compound 4 were red, so the C-8 position was the S-type. The absolute configuration of C-8 was further verified by the rhodium salt collation method, and there was a positive Cotton effect at 350 nm. Based on the above evidence, the C-8 position was S-type.

Compound 4 was elucidated and systemically named (3S,8S)-3-(1-hydroxy-but-3-ynyl)-3H-isobenzofuran-1-one.

Compound 5 was a yellow oil (MeOH) with a molecular formula of C₁₂H₁₀O_3, as the HR-ESI-MS (m/z 225.0648 [M + Na]⁺), with eight degrees of unsaturation. The ¹H NMR and ¹³C NMR data (Table 2; Figure S14) indicated that compounds 4 and 5 have very similar structures. The only difference is the absolute configuration difference between them.

The position of H-3 and H-8 of compound 5 was confirmed by the coupling constant of the compound [34]. H-3 (δ_H 5.77) and H-8 (δ_H 3.91) showed the same coupling constant (J = 5.6 Hz). Due to its coupling constant J = 5.6 Hz, the dominant conformation was determined based on the carbon chain according to the Capon rule. The simulation confirmed that H-3 and H-8 of the compound were on the opposite side.

To clarify the absolute configuration of the C-3 position in compound 5, CD spectroscopy was performed [34]. Its CD spectrum (Figure 4 and Figure S16) at 220 nm showed a positive Cotton effect, so we inferred that the C-3 position was S-type. Moreover, H-3 and H-8 of compound 5 were in the Threo form, so the C-8 position was inferred to be R. The absolute configuration of C-8 was further verified by the rhodium salt collation method, and there was a negative Cotton effect at 350 nm. Based on the above evidence, the C-8 position was ultimately determined to be the R-type.

Compound 5 was elucidated and systemically named (3S,8R)-3-(1-hydroxy-but-3-ynyl)-3H-isobenzofuran-1-one.

3.2. Antioxidant and TNF-α and NO Inhibitory Activities of the Components

In the DPPH assays, compounds 11 and 16 exhibited moderate activity, as shown in

Table 3. Determination of DPPH antioxidant activity (IC₅₀).

Compounds	DPPH (IC50, μg/mL)
4	54.3 ± 1.8
7	78.9 ± 1.7
8	69.5 ± 0.9
9	54.3 ± 1.8
10	48.6 ± 1.2
11	25.2 ± 0.5
12	54.3 ± 1.8
14	52.8 ± 1.4
15	48.4 ± 2.1
16	34.7 ± 2.5
17	46.2 ± 0.9
18	>100
19	>100
20	98.7 ± 3.6
Trolox	10.8 ± 0.5

.

The LPS stimulation of NO production was also performed. Compounds 4, 9, 11 and 18 exhibited stronger inhibitory effects (IC₅₀ < 35 μM), and indomethacin was the positive control (IC₅₀ = 26.8 ± 1.3 μM). TNF-α is the main proinflammatory cytokine produced by macrophages and it has different proinflammatory effects on various cell types. In our research, the results showed that compounds 10 and 11 had better anti-inflammatory effects than the positive control group did. Compound 16 inhibits TNF-α production. The inhibitory activity of compound 16 was equivalent to that of the positive control, as shown in

Table 4. Inhibition on NO production of isolated compounds from Shiitake mushrooms (Lentinus edodes).

Compounds	NO Inhibitory Assay (IC50) μM	TNF-α Inhibitory Assay (IC50) μM
4	34.7 ± 2.5	>100
7	48.6 ± 3.4 *	>100
8	52.1 ± 3.6 *	>100
9	30.2 ± 2.5	94.5 ± 1.6
10	35.8 ± 1.7	75.3 ± 2.7 *
11	28.1 ± 2.2 *	68.1 ± 1.8 *
12	58.4 ± 1.9 *	>100
14	83.1 ± 3.1 **	98.4 ± 1.3
15	79.8 ± 2.8 **	-
16	45.7 ± 1.2 *	89.3 ± 1.8
17	>100	>100
18	29.9 ± 1.2	>100
19	89.6 ± 1.7 **	-
20	91.8 ± 2.3 **	-
Indomethacin	26.8 ± 1.3	88.5 ± 2.1

Significantly different compared to the indomethacin * p < 0.05, ** p < 0.01.

.

3.3. Chemical Composition Comparison

The compounds isolated from the rice fermentation of Lentinus edodes mycelium were compared with those obtained from the fruit body of Shiitake mushrooms. The secondary metabolites involved in the fermentation of Lentinus edodes mycelium are mainly isocoumarin compounds and their derivatives, which widely exist in nature but were isolated from Lentinus edodes. The chemical composition of Shiitake mushrooms was mainly composed of simple compounds with simple structural frameworks, and there were certain differences. In terms of activity, isocoumarin compounds have both anti-inflammatory and antioxidant activities. Moreover, mycelium produced more functional compounds with better activity.

4. Discussion

Shiitake mushrooms (Lentinus edodes) are rich in bioactive substances, which not only have high nutritional value, but also have medicinal properties. Therefore, this topic is of concern for researchers. At present, research on the active components of Shiitake mushrooms mainly focuses on the primary metabolites (polysaccharides, proteins, and polyunsaturated fatty acids) of Lentinus edodes mycelia and the nutritional components of the fruiting body itself [14]. We studied the fermentation metabolites of mushroom mycelia and the bioactive components of mushroom fruiting bodies and compared the compounds isolated from the two. In terms of the compound structure types, the metabolites produced during the fermentation of mushroom mycelia are mainly isocoumarin compounds and their derivatives. The chemical components isolated from the fruiting bodies of Lentinus edodes were mainly complexes with simple structural frameworks, followed by macrocyclic compounds. This indicates that there are differences in the main components of the two forms of shiitake mushrooms (fruiting bodies and mycelia).

Shiitake mushrooms have antioxidant, anti-aging, anti-inflammatory, and human immune functions. Shiitake mushrooms are widely used in food, health products, and medicine, and have broad research and development prospects [35,36,37,38,39,40]. In this study, the activity of the main compounds in the two forms of Lentinus edodes was tested, and the biological activity of the main compounds was analyzed and compared. In terms of compound activity, most of the isocoumarin compounds in mycelium metabolites have anti-inflammatory and antioxidant activities. In addition, the mycelia produced more functional compounds and exhibited better activity. This suggests that the metabolites of edible fungal mycelia are a new source of bioactive compounds.

In this study, new bioactive compounds of shiitake mushrooms with antioxidant and anti-inflammatory potential were identified, which were expected to provide a valuable theoretical reference for the rational development and utilization of edible and medicinal fungi represented by Lentinus edodes. In addition, we hope to provide new ideas for researchers to explore natural products with novel structures and excellent activities. However, it is undeniable that although the bioactive compounds in shiitake mushrooms have medicinal potential, whether there are side effects and whether they have medicinal properties still needs to be further explored by researchers.

In a word, shiitake mushrooms are a rich source of bioactive compounds with a high potential for development.

5. Conclusions

Our study systematically investigated the chemical composition of the Shiitake mushroom fruiting body and the solid-state fermentation of Lentinus edodes mycelium, and 5 new compounds and 15 known compounds were discovered. By analyzing the obtained compounds, we found that the non-polysaccharide components in the mycelium and the fruiting body of Shiitake mushrooms were different, and their activities were also different. Our results will provide a basis for the rational development of the functional constituents of the non-polysaccharides in Shiitake mushrooms and even the development of medicinal edible fungi.