Identification of metabolites from edible mushroom Morchella sextelata and their biological evaluation

Abstract To identify bioactive metabolites from the fruiting body of Morchella sextelata, fourteen metabolites (1–14) including one undescribed morchesexten A (1) were isolated. Their structures including absolute configurations were assigned on the basis of spectroscopic data and quantum chemical computational methods. Furthermore, the anti-inflammatory and antioxidant activities of the isolated compounds were evaluated. Compounds 10–12 showed inhibitory effects on nitric oxide (NO) production with IC50 values of 15.2 ± 2.7, 10.2 ± 1.9 and 35.3 ± 10.5 μM, respectively. Compounds 7 and 9 exhibited strong antioxidant effect with IC50 values of 6.7 ± 0.4 and 7.3 ± 0.8 μM compared with Vit C (IC50 15.4 ± 0.2 μM). Graphical Abstract


Introduction
Morchella (Morchella spp., Pezizales, Ascomycota) are highly treasured as a dietary supplement for their savoury favour and health benefits throughout the globe, especially in Asia and Europe (Tietel and Masaphy 2018). In traditional Chinese medicine, it was prescribed for excessive phlegm, indigestion, shortness of breath as well as brain and kidney tonifying. Over 2000 papers with the topic 'morchella' are presented in SciFinder database, however, only 30 research papers discuss the bioactivities of morchella, which demonstrate its activities of anti-inflammatory, antioxidant, immunostimulant, antitumor and anticancer properties (Wu et al. 2021;Sambyal and Singh 2021). In addition, small amounts of secondary metabolites from Morchella have been elucidated, including terpenoids, sterols, fatty acids, pyrones (Yang et al. 2019a(Yang et al. , 2019bYang et al. 2020). Hence, Morchella sextelata was chosen to explore its pharmacological compositions by performing in vitro anti-inflammatory and antioxidant assays. As a result, the isolation and structural elucidation of compounds 1-14 (Figure 1), and their anti-inflammatory effects against the NO production of RAW264.7 cells and ABTSþ (2,2 0 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation) radical-scavenging activity assays were presented in this article. The results are expected to provide important scientific data for health product development of M. sextelata.

Results and discussion
Phytochemical investigation on the ethyl acetate fraction of the ethanol extract of the fruiting bodies of M. sextelata led to the obtainment of fourteen metabolites (1-14), including one undescribed morchesexten A (1).

Discussion
In recent years, secondary metabolites from wild edible mushrooms consumed mainly by local communities were identified. And their bioactivity results disclosed that these edible mushrooms as a part of natural diet with excellent antioxidant effects (Vamanu 2018). However, the effective substances of M. sextelata are still unclear. In this article, a partial chemical composition profile of M. sextelata was established. And compounds 2 and 3 had been reported with strong anticancer effect, respectively (Ping et al. 2020;Zhang et al. 2020). Furthermore, the inhibitory activities of all compounds on lipopolysaccharide (LPS)-induced NO production in RAW 264.7 cells were evaluated. As shown in Figure S3, compounds 10-12 inhibited NO production with IC 50 values ranging from 10.2 to 35.3 lM, while indomethacin showed an IC 50 value of 14.2 lM. In addition, at tested concentrations of 5-100 lM, all compounds except 7 and 9 showed no effects on ABTSþ radical scavenging ability ( Figure S4). Based on literature investigation (Sarikurkcu et al. 2022), 9 exhibited the antioxidant activity with IC 50 values of 7.3 lM. And our biological experiment result showed that 7 exhibited the ABTSþ radical scavenging activity with IC 50 values of 6.7 lM.

Mushroom material
Dried Morchella sextelata were purchased from QingPing Traditional Medicine Market in Guangzhou City, Guangdong province, China, in June 2020, and authenticated by Prof. Qi Luo. In addition, a voucher specimen (NFYLQ-002) was deposited at Syndrome Laboratory of Integrated Chinese and Western Medicine, Southern Medical University, China.

Computational Studies
Briefly, the conformational search generated low-energy conformers within a 10 kcal/ mol energy and was finished with the software Conflex 8. The predominant conformers were optimised by DFT calculation at the B3LYP/6-311G(d,p) level. ECD calculations were conducted at the B3LYP/6-311G(d,p) level with the PCM in MeOH solution. Detailed methods could be referred to previously published paper (Luo et al. 2017).
3.5. Anti-inflammatory assay RAW 264.7 cells were screened for anti-inflammatory activity by inhibiting the production of NO after LPS exposure (Tang et al. 2020). Briefly, RAW 264.7 cells were gifted from prof. X.-S. Zhao and cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum with 1% penicillin-streptomycin (100 U/ml penicillin G and 100 lg/mL streptomycin) and incubated in 37 C with humidified air atmosphere with 5% CO 2 . The cells were harvested with trypsin-EDTA and concentrated by centrifugation at 1000 Â g for 5 min and suspended in fresh medium. The suspended cells were diluted and seeded onto 96-well plates (2 Â 10 4 /well) in triplicate and allowed to adhere for 24 h. All tested samples were dissolved in DMSO and diluted with DMEM medium (DMSO final concentration < 0.1%). To determine the inhibitory activity of each compound, the cells were pretreated with fresh medium (200 ll/well) with the tested compounds at various final concentrations (0 À 100 lM) for 2 h, then the LPS (1 lg/mL, Biosharp, Hefei, Anhui, China) was added and cultured for another 24 h. Indomethacin was used as a positive control, and NO productions were measured using a NO detection kit (S0021, Beyotime, Shanghai, China) according to the manufacturer's manual. The optical density was measured at 540 nm throughout a microplate reader (Bio-Tek Synergy H1, Inc., Vermont, U.S.). All tests were performed in triplicate.

Assay for radical scavenging activity of ABTS
Using the ABTS free radical scavenging capacity assay, Trolox equivalent antioxidant activity was determined (Ma et al. 2021). Briefly, 200 ll of both 7 mM ABTS aqueous solution and 4.9 mM of potassium persulfate aqueous solution were mixed at dark for 12 h. The resulting ABTS radical cation was then diluted until the absorbance at 734 nm was within 0.7 ± 0.02. Different samples were then mixed at 0-100 lM and reacted at room temperature for 6 min. Followed by detecting the absorbance at 734 nm within 10 min. Ethanol was used as the blank control and Vit C was used as the positive control.

Conclusion
In summary, this report describes the isolation and structural elucidation of fourteen compounds 1-14 from the fruiting bodies of M. sextelata. To best of our knowledge, all known compounds except compounds 9-12 were reported for the first time in this species. Furthermore, compounds 10-12 showed inhibitory effects on NO production with IC 50 values of 15.2, 10.2, and 35.3 lM, respectively. In addition, compounds 7 and 9 exhibited strong antioxidant effect with IC 50 values of 6.7 and 7.3 lM compared with Vit C (IC 50 15.4 lM).