DPPH Radical Scavenging Activity of New Phenolics from the Fermentation Broth of Mushroom Morehella importuna

In recent years, wild morel mushroom species have begun to be widely cultivated in China due to their high edible and medicinal values. To parse the medicinal ingredients, we employed the technique of liquid-submerged fermentation to investigate the secondary metabolites of Morehella importuna. Two new natural isobenzofuranone derivatives (1–2) and one new orsellinaldehyde derivative (3), together with seven known compounds, including one o-orsellinaldehyde (4), phenylacetic acid (5), benzoic acid (6), 4-hydroxy-phenylacetic acid (7), 3,5-dihydroxybenzoic acid (8), N,N′-pentane-1,5-diyldiacetamide (9), and 1H-pyrrole-2-carboxylic acid (10), were obtained from the fermented broth of M. importuna. Their structures were determined according to the data of NMR, HR Q-TOF MS, IR, UV, optical activity, and single-crystal X-ray crystallography. TLC-bioautography displayed that these compounds possess significant antioxidant activity with the half DPPH free radical scavenging concentration of 1.79 (1), 4.10 (2), 4.28 (4), 2.45 (5), 4.40 (7), 1.73 (8), and 6.00 (10) mM. The experimental results would shed light on the medicinal value of M. importuna for its abundant antioxidants.


Introduction
The genera Morchella (Morel), which includes these species M. importuna, M. esculenta, M. sextalata, and M. eximia, are edible medicinal fungi of the phylum Ascomycota with high gastronomic quality and potential therapeutic use [1]. The fruiting body of these species contained abundant bioactivities substances [2]. It was reported that the aqueous extract from M. importuna possessed antileishmanial activity, a novel peptide from M. importuna could induce apoptosis in HeLa cells, phenolic compounds from M. esculenta with high antioxidant activity [3], and sterols and trilinolein showed significant inhibition of NF-κB activation [4]. However, the wild morel is rare and could not be artificially cultivated until recent years. To utilize the natural resources of this genus mushroom, mycelia prepared by microbial fermentation technology were an alternative product for the investigation of its chemical constitution with pharmaceutical potential. Some reports provided the convincing truth that mycelia and other products prepared from submerged culture are a valid alternative to the fruiting body, such as M. esculenta [5]. Microbial fermentation was generally used to explore the metabolites of some wild and rare mushrooms in our lab. In this paper, we mainly focus on the isolation, chemical structures, and antioxidant properties of the low-molecule metabolites of M. importuna by microbial fermentation. As a Molecules 2023, 28, 4760 2 of 10 result, two new isobenzofuranone derivatives and one new orsellinaldehyde derivative, together with five phenolics and two N-contained metabolites hexamethylene bisacetamide and 1H-pyrrole-2-carboxylic acid, were isolated from the fermentation broth. These natural metabolites show significant antioxidant activity.

Structure Identification
Ten compounds were purified from the organic extract prepared from the fermentation broth of M. importuna as reported in detail in the experimental part.
Inspection of the spectral data of compounds 1-2 (Table 1) indicated that they are derivatives of isobenzofuranone, which possess one phenyl group substituted by two meta phenolic hydroxyls according to two meta-coupled aromatic protons ( The key heteronuclear multiple-bond correlation (HMBC) spectrum of 1 exhibited correlations from H-7 to C-1, C-5, C-6, and C-3a, H-5 to C-4, C-6, C-7, and C-3a, and H-3 to C-1 and C-8 and allowed the establishment of isobenzofuranone substituted with two hydroxyls and one methoxyl groups in 1. The planar structure of 1 was characterized as one new derivative of isobenzofuranone, namely 4,6-dihydroxy-3methoxyisobenzofuran-1(3H)-one. The absolute configuration of C-3 was assigned by the optical rotation test. Rubralide A [6] isolated from Penicillium rubrum showed the dextral optical rotation with the value of [a] 20 D + 4.0 (c 0.5, EtOH), whose only one chiral carbon has an (R)-configuration at C-3. The sinistral rotation value of [a] 20 D − 23.0 (c 0.36, MeOH) of 1, indicated the (S)-configuration at C-3. Therefore, compound 1 was concluded as (S)-4,6-dihydroxy-3-methoxyisobenzofuran-1(3H)-one. Compound 2 was isolated as white amorphous substance with the UV (methanol) λ max , nm: 224. The molecular formula of 2 was determined to be C 9 H 8 O 5 by HR Q-TOF MS at 197.0443 for [M + H] + (calculated for C 9 H 9 O 5 , 197.0450). The IR spectra showed the absorptions for hydroxyl (3312 cm −1 ), ester carbonyl (1684 cm −1 ), and aromatic unsaturated double bond (1637 cm −1 ). The NMR spectrum of 2 was similar to those of 1, except for the presence of the proton at δH 10.33 (H-3) and carbon signal at δC 196.6 (C-3) ( Table 1). The downfield chemical shift of H-3 and C-3 indicated the presence of a formaldehyde group in compound 2, compared with the hemiacetal group at same position in compound 1. HMBC correlations from H-7 to C-5, C-6, and C-3a, H-5 to C-4, C-6, and C-3a, H-3 to C-1 and C-5, and H-8 to C-1 were observed in HMBC spectrum of compound 2, indicating the opening of the furanone ring of an isobenzofuranone skeleton. Compound 2 was deduced as methyl 2-formyl-3,5-dihydroxybenzoate according to the IUPAC system, a hydrolysis product of 1 ( Figure 1). Compound 2 was often used as the building block for the synthetic medicinal chemicals [7] and reported as a natural product for the first time in this paper. Isobenzofuranone is also known as phthalide and is widely distributed in microbes and plants [8]. In recent years, many natural molecules with this skeleton were discovered and shown diverse biological activities, such as antibacterial [9], antifungal [10], insecticidal [11], cytotoxic [12], anticyclooxygenase-2 [13], anti-acetylcholinesterase [14], α-glucosidase inhibitory effect [15], anticoagulation [16], hepatoprotective [17], and neurodegenerative prevention [18], etc. So, phthalide analogs exhibit a potential pharmacological value. Except for Hericium erinaceus [19,20] and Pleurotus djamor [21], few reports refer to phthalide-related metabolites isolated from mushrooms. New isobenzofuranone derivative 1 from mushroom M. importuna merits further investigation for its bioactivities. neurodegenerative prevention [18], etc. So, phthalide analogs exhibit a potential pharmacological value. Except for Hericium erinaceus [19,20] and Pleurotus djamor [21], few reports refer to phthalide-related metabolites isolated from mushrooms. New isobenzofuranone derivative 1 from mushroom M. importuna merits further investigation for its bioactivities.   ) and δH 2.53 (s, 3H) were observed in 1 H NMR spectra of 4. Based on the above 1 H and 13 C NMR data, 4 was identified as 2,4-dihydroxy-6-methylbenzaldehyde, namely o-orsellinaldehyde, which was confirmed by X-ray diffraction ( Figure 1). Crystallographic data Compound 3 was isolated as a white powder with UV (methanol) λ max , nm: 202. The molecular formula of compound 3 was determined to be C 9 H 10 O 4 based on the HR Q-TOF MS peak at m/z: 261.1823 (calculated for C 9 H 10 O 4 Na, 261.1830) and NMR data. The IR spectra showed the absorptions for hydroxyl (3433 cm −1 ) and an aromatic unsaturated double bond (1630 cm −1 ). Additionally, 1  . HMBC correlations from H 3 -8 to C-1/2/3, from H-7 to C-6, from H-5 to C-1/3/4/6, and from H 3 -9 to C-3 could be observed in HMBC spectra. Compound 3 was a new derivative of 4 and was identified as 4,6-dihydroxy-3-methoxy-2-methylbenzaldehyde ( Figure 1). o-Orsellinaldehyde was reported as a bioactive metabolite produced by the mushroom Grifola frondosa with a selective cytotoxic effect and anti-inflammatory and pro-apoptotic properties [22,23], and Phlebiopsis gigantea with antifungal activity [24].
Compound 10 was isolated as a white powder. Additionally, 1  The final ωR 2 was 0.1426 (all data) and R 1 was 0.0366 (I ≥ 2σ (I)). PCA was previously reported as an antimicrobial natural product obtained from bacteria, the sponge Agelas nakamurai, and the Chinese herb Pseudostellaria heterophylla [37,38]. It was discovered for the first time as a mushroom metabolite in this paper.

DPPH Free Radical Scavenging Activity
In the TLC-bioautography experiments, the various degrees of white spot observed in the different lanes indicated that these compounds 1, 2, 4, 5, 7, 8, and 10 possessed significant antioxidant activity (Figure 2). Their antioxidant activities were further assessed by the Brand-Williams' method. DPPH radical scavenging rate of these compounds with the different doses were shown in Figure 3. The half DPPH scavenging concentration (SC 50 ) of these compounds were 1.79 (1), 4.10 (2), 4.28 (4), 2.45 (5), 4.40 (7), 1.73 (8), and 6.00 (10) mM, compared with the value of 0.216 mM of vitamin C, which were estimated by using Probit analysis in SPSS 18.0 with p value less than 0.01 except for 0.064 for 2. Most of these compounds displayed significant anti-DPPH radical potency. These data suggest that M. importuna could be used as a source of abundant natural antioxidants. that M. importuna could be used as a source of abundant natural antioxidants.
The DPPH radical (DPPH•) bearing a stable unpaired electron is regarded as one convenient method for determining the antioxidant activity of a wide variety of organic molecules [39]. The intracellular free radicals were believed to be involved in a diverse range of diseases and accelerating the aging process [40]. Antioxidants with the ability to scavenge free radicals and reduce oxidative stress are reported to be consecrated with all types of pharmacological applications [41]. In recent years, the antioxidant substance obtained from mushrooms were discovered to confer a beneficial effect on human health, such as antidiabetic and antihyperglycemic [42], neuroprotective [43], hepatoprotective [44], anti-inflammatory [45], immunomodulatory [46], anti-aging [47], anticancer [48], antimicrobial [49], anti-melanogenic [50] and hypopigmentation [51], anticoagulant [52], nephroprotective [53], and hyperuricemia treatment [54], etc. The abundant natural antioxidants of M. importuna will lay the foundation for its medicinal benefits.   that M. importuna could be used as a source of abundant natural antioxidants.
The DPPH radical (DPPH•) bearing a stable unpaired electron is regarded as one convenient method for determining the antioxidant activity of a wide variety of organic molecules [39]. The intracellular free radicals were believed to be involved in a diverse range of diseases and accelerating the aging process [40]. Antioxidants with the ability to scavenge free radicals and reduce oxidative stress are reported to be consecrated with all types of pharmacological applications [41]. In recent years, the antioxidant substance obtained from mushrooms were discovered to confer a beneficial effect on human health, such as antidiabetic and antihyperglycemic [42], neuroprotective [43], hepatoprotective [44], anti-inflammatory [45], immunomodulatory [46], anti-aging [47], anticancer [48], antimicrobial [49], anti-melanogenic [50] and hypopigmentation [51], anticoagulant [52], nephroprotective [53], and hyperuricemia treatment [54], etc. The abundant natural antioxidants of M. importuna will lay the foundation for its medicinal benefits.   The DPPH radical (DPPH•) bearing a stable unpaired electron is regarded as one convenient method for determining the antioxidant activity of a wide variety of organic molecules [39]. The intracellular free radicals were believed to be involved in a diverse range of diseases and accelerating the aging process [40]. Antioxidants with the ability to scavenge free radicals and reduce oxidative stress are reported to be consecrated with all types of pharmacological applications [41]. In recent years, the antioxidant substance obtained from mushrooms were discovered to confer a beneficial effect on human health, such as antidiabetic and antihyperglycemic [42], neuroprotective [43], hepatoprotective [44], anti-inflammatory [45], immunomodulatory [46], anti-aging [47], anticancer [48], antimicrobial [49], anti-melanogenic [50] and hypopigmentation [51], anticoagulant [52], nephroprotective [53], and hyperuricemia treatment [54], etc. The abundant natural antioxidants of M. importuna will lay the foundation for its medicinal benefits.

General Experimental Procedures
NMR spectra were acquired on a Bruker AVANCE III 500 spectrometer operating at 500/125 MHz and a Bruker AVANCE NEO 600 spectrometer operating at 600/150 MHz.

Fungus Material
The strain M. importuna was collected by Xie Bao-gui (Fungal Research Centre, Fujian Agriculture and Forestry University, Fuzhou, China) and was further identified by the DNA sequence of internal transcribed spacers (ITS) region ( Figure S21). The strain has been deposited in College of Life Sciences, Fujian Normal University and has been deposited in the China Centre for Type Culture Collection (CCTCC M 2014324).

Fermentation and Preparation of Extracts
M. importuna was cultured by submerged liquid fermentation [55], which was carried out in Erlenmeyer flasks (250 mL) containing 100 mL of potato dextrose medium with a total volume of 40 L. These flasks were incubated at 28 • C with a shaking speed of 180 rpm. The culture broth was centrifuged at 5000 rpm for 30 min to remove the mycelia and then extracted in batches with equal volumes of ethyl acetate. The organic phase was concentrated under reduced pressure by a rotary evaporator to afford the crude extract E1 (3.4 g). Following the same step, another potato dextrose medium which added 1 g bran and 20 g bean sprout juice per liter was used to culture M. importuna with a total volume of 36 L and obtained the crude extract E2 (8.8 g).  (20 mg). E1.11 was subjected to silica gel (1.0 g) chromatography using a CHCl 3 -CH 3 OH solvent gradient to yield compound 7 (5 mg). E1.2 was separated by column chromatography over Sephadex LH-20 (120 g) with methanol eluant and afforded the subfractions E1.21 (22 mg). E1.21 was next separated by column chromatography over Sephadex LH-20 (120 g) with acetone eluant and afforded E1.211 (11.4 mg) and E1.212 (7.9 mg). Compounds 5 (5.5 mg) and 6 (5.4 mg) were, respectively, purified from E1.211 and E1.212 by column chromatography over silica gel (1 g) with the eluant of CHCl 3 -CH 3 OH solvent gradient. E1.3 was separated by column chromatography over Sephadex LH-20 (120 g) with methanol eluant and afforded E1.31 (5.4 mg) and E1.32 (7.9 mg). Then, E1.31 and E1.32 were, respectively, subjected to column chromatography over Sephadex LH-20 (120 g) with acetone eluant and yielded pure compounds 4 (2 mg) and 3 (0.7 mg).

DPPH Free Radical Scavenging Activity
TLC-bioautography [56] was used to determine the free radical scavenging activity of compounds 1-10. These compounds were dispensed to the concentration of 2 mg/mL. Then, 2 µL solution of each compound was spotted onto a precoated silica gel GF254 plate. The TLC plate was left to let the solvent evaporate completely. Then, the plate was sprayed with 0.04% DPPH in ethanol and incubated at 40 • C for 30 min. Any antioxidant compounds could be seen as white spots against the blue background. Ascorbic acid was set as the positive control sample.
The DPPH scavenging rate of these compounds was further assayed by the method reported by Blois [57] and modified by Brand-Williams [58]. Each compound was dissolved in methanol and diluted to 6 different concentrations with the same volume of 94 µL in a 96-well microtiter plate. Then, 20 µL of DPPH dissolved in methanol with a concentration of 0.714 µg/mL was added to these wells. The final concentration of these compounds in each well was 9.8, 19. where A 1 is the absorbance of the samples and the standards, and A 0 is the absorbance of the vehicle.

X-ray Single Crystal Diffraction for Compound 4 and 10
The single crystal of 4 and 10 was obtained from aqueous acetone. A suitable crystal was selected and subjected to an Oxford Gemini S Ultra diffractometer using Cu-Kα (λ = 1.54184 Å) radiation at 99 K for 4 and using Mo-Kα (λ = 0.7073 Å) radiation at 273 K for 10. Their structures were determined using the direct method and refined with fullmatrix least-squares calculations on F 2 using olex2-2.1 [59]. Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre (CCDC).

Conclusions
In summary, M. importuna can produce abundant phenolics, including two new isobenzofuranone derivatives and one new orsellinaldehyde derivative. These natural metabolites show the significant antioxidant activity of TLC-bioautography and DPPH free radical scav-