Two new compounds from edible mushroom Sarcomyxa edulis

Abstract Chemical investigation of the edible mushroom Sarcomyxa edulis led to the isolation of one new highly degraded sterol (1), and one new β-carboline alkaloid (2), along with nine known compounds (3-11) for the first time from this mushroom. The structures of new compounds were elucidated using HR-ESI-MS data and NMR spectroscopy. In addition, anti-inflammatory activity of new compounds was evaluated against lipopolysaccharide-induced NO production in RAW 264.7 macrophages. Compound 2 exhibited a good anti-inflammatory activity with IC50 value of 9.88 ± 0.48 μM, and compound 1 exhibited a weak inhibitory effect with IC50 value of 71.36 ± 5.11 μM. Graphical Abstract


Introduction
Mushrooms have been consumed by humans for thousands of years, which is due to their abundant nutritional value and potential medicinal value Venditti et al. 2017). They are a rich source of bioactive compounds and possess numerous bioactivities, including anti-hyperglycemic, anti-oxidant, anti-inflammatory, anti-tumor, and anti-diabetic activities (Choi et al. 2019;Pandya et al. 2019;Chen et al. 2020;Raju et al. 2021;Lv et al. 2021b). Mushroom act as a prebiotic to modulate the gut microbiome that confer nutritional and health benefits to the host. (Jayachandran et al. 2017). Sarcomyxa edulis (Y.C. Dai, Niemel€ a & G.F. Qin) T. Saito, T. Tonouchi & T. Harada, also known as "Huangmo", "Dongmo" or "Yuanmo" in China, is an important edible and medicine mushroom, mainly distributed in northeastern China, Korea, Japan, the far eastern areas of Russian, and other cold regions. And it has been used as a folk medicine for the treatment of epilepsy and rheumatism for a long time (Cao et al. 1996). Previous literature reports indicated that S. edulis has multiple pharmacological activities, including anticancer (Sergeev et al. 1985;Ma et al. 1991;Rha et al. 1999), antioxidant (Ji et al. 2011), hypoglycemic (Inoue et al. 2013), hepatoprotective (Inafuku et al. 2012), and radioprotective activities 2015). However, there have been few studies have focused on the isolating and identifying compounds from the S. edulis; to date, only three reports were available, and several steriods and fatty acids have been isolated (Yaoita et al. 2002a;2002b).
Thus, we investigated the chemical constituents of S. edulis. As a result, one new highly degraded sterol (1), and one new b-carboline alkaloid (2), along with nine known compounds (3-11) were isolated for the first time. Compound 2 exhibited a good anti-inflammatory activity with IC 50 value of 9.88 ± 0.48 lM. The present study describes the isolation, structural elucidation, and anti-inflammatory activities of new compounds.  (Table S1, supplementary material) showed a total of 21 carbon signals, including a lactone carbonyl carbon at d C 172.5 (s, C-1), six olefinic carbon signals due to three double bonds, and 14 sp 3 carbons in the up-field region. Three double bonds and a carbonyl group contributed four degrees of unsaturation, so there must be three rings in the structure. The above NMR features were similar to those of demethylincisterol A 3 , a nor-ergosteroid isolated from the mushroom Agrocybe chaxingu (Kawagishi et al. 2006;Yajima et al. 2012). The main difference was that a newly arisen double bond group replaced the hemiketal carbon and a methylene carbon in demethylincisterol A 3 , which suggested that 1 should be a dehydrated derivative. The 1 H, 1 H-COSY correlations ( Figure S1, supplementary material) of 1 established the sidechain moiety of the nor-ergosteroid skeleton. The HMBC correlations ( Figure S1, supplementary material) from H-5 [d H 5.73 (1H, ddd, J ¼ 6.9, 2.4, 1.9 Hz)] to C-3 [d C 161.0 (s)], C-4 [d C 152.1 (s)] and C-7 [d C 47.9 (s)], confirmed the presence of a double bond located at the C-4-C-5 position. The stereochemistry of the new nor-ergosteroid was verified by the NOESY correlations ( Figure S2, supplementary material) of H-8$H-6a/ H-11 and H 3 -12$H-6b/H-13. Therefore, the structure of 1 was established as shown in Figure 1 and named as eduline A.

Results and discussion
Compound 2 (Table S2, supplementary material) showed a total of 14 carbon signals, including a conjugated ketone carbon at d C 201.2 (s, C-14), a conjugated carboxylic carbonyl at d C 166.4 (s, C-16), 11 aromatic or olefinic carbon signals due to a b-carboline basic skeleton, and a methyl carbon at d C 25.8 (q, C-15). A b-carboline skeleton, a ketone and a carboxylic group just matched the degrees of unsaturation. The 1 H and 13 C NMR spectroscopic data of 2 were similar to those of a b-carboline alkaloid dichotomide VI , and the main differences were from different substituent groups and positions. The HMBC correlations ( Figure  S11, supplementary material) from H-4 to C-10, C-12, and C-16 [166.4 (s)], and from H-5 to C-7 [159.5 (s)], C-11 and C-13, confirmed a carboxylic group and a hydroxy group substituted at C-3 and C-7, respectively. While, the HMBC correlations from H 3 -15 to C-1 and C-14 revealed that an acetyl group was located at C-1. Therefore, the structure of 2 was established as 1-acetyl-7-hydroxy-b-carboline-3-carboxylic acid, shown in Figure 1.
First, the effects of the new compounds at different concentrations (20, 40, and 80 mM) on the viability of RAW264.7 macrophages were evaluated by CCK8. And the results showed that compounds 1 and 2 did not show significant cytotoxic effects (Table S3). Next, the new compounds 1 and 2 were tested for their anti-inflammatory activity against lipopolysaccharide-induced NO production in RAW 264.7 macrophages, and aminoguanidine was used as a positive control. The results are illustrated in Table  S3 (supplementary material). Compound 2 exhibited a good anti-inflammatory activity with IC 50 value of 9.88 ± 0.48 lM, which was comparable with the aminoguanidine (IC 50 ¼ 8.92 ± 0.66 lM). And compound 1 exhibited a weak inhibitory effect with IC 50 value of 71.36 ± 5.11 lM. Alkaloids have been reported to possess anti-oxidant and anti-inflammatory effects, such as berberine, discretine, gentianadine, gentianamine, jatrorrhizine (Perez 2001). These compounds have poor bioavailabilities, and their metabolisms and pharmacokinetics are related to the activity of the microbiota (Feng et al. 2010). In addition, accumulating evidence has demonstrated that the anti-inflammatory potential of bioactive ingredients is related to its antioxidant and microbiota modulation. They can modulate the gut microbiota structure and composition, and increase production of short-chain fatty acids to promote health (Chen et al. 2011;Habtemariam 2020). In this study, we determined the anti-inflammatory activity of compounds through in vitro experimentation. The expression of bioactivity depends on the assimilation of different classes of natural substances in vivo (Dabulici et al. 2020). It should be further evaluated by an in vivo animal study.

General experimental procedures
See supplementary material.

Mushroom material
The fresh fruiting bodies of S. edulis were collected from Suihua City, Heilongjiang Province, China, in September 2020 and identified based on morphological characteristic and molecular analysis (ITS). ITS sequence of S. edulis has been uploaded on GeneBank (accession number OK485911). Voucher specimen (No. 20200920) was deposited at the College of Chinese Medicinal Material, Jilin Agricultural University.

Extraction and isolation
The detailed procedure of extraction and isolation are reported in supplementary material.

Cell viability assay
See supplementary material.

Determination of NO production
See supplementary material.

Conclusion
One new highly degraded sterol (1), and one new b-carboline alkaloid (2), along with nine known compounds (3-11) were isolated from the edible mushroom S. edulis for the first time. Compound 2 exhibited a good anti-inflammatory activity. It could be an effective natural anti-inflammation compound with considerable pharmaceutical value.

Geolocation information
Changchun (E: 125.24 , N: 43.48 ) is the capital of Jilin Province which located in the north of China. This region has a typical temperate continental monsoon climate with few heat resources.

Disclosure statement
No potential conflict of interest was reported by the authors.