Secondary Metabolites and Antiradical Activity of Liquid Fermentation of Morchella sp. Isolated from Southwest China

Morels famous for their taste and nutrition are in short supply all over the world although they were considered as one of the most highly prized edible and medicinal mushrooms. Because of the limitation of resource and cultivation technology, fermentation of edible mushroom was gradually applied to nutrient, bioactivity and breeder seed preparation. At present, there are more reports on sugar and amino acid but less on other components. Morchella sp. YDJ-ZY-1 was isolated from the wild fruiting body by the spores releasing method in Zunyi Guizhou province in Southwest China and identified based on phenotype and genotype characteristics. Chemical compositions of YDJ-ZY-1 were investigated from liquid fermentation that will lay the foundation for further development and utilization. Four pyranoids (1–4) and 2-(1-oxo-2-hydroxyethyl) furan (5), linoleic acid (6), Morelin (2-hydroxy-cinnamic acid methyl ester, (7) and 1-O-β-d-ribofuranose-Morelin (8) were obtained from EtOAc extraction and elucidated by spectral data. Product 4 and 8 were new compounds and 7 was isolated from nature for the first time. Antiradical activity was evaluated by free radical scavenging effect on DPPH (1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl). Compound 5 exhibited strong antiradical activity while compounds 1 and 2 exhibited moderate activity. Thus, incubation of Morchella sp YDJ-ZY-1 separated from the wild fruit body afforded eight compounds. Secondary metabolites with new structures were mined from fermentation of Morchella sp. and antiradical activity was evaluated.


Introduction
Morels are some of the most highly prized edible and medicinal mushrooms, and outdoor cultivation has been achieved in recent years. These edible mushrooms appreciated worldwide have been used in traditional medicine for centuries, due to their health-related benefits. In recent research, the anti-oxidative, anti-inflammatory bioactivities, immunostimulatory, anti-tumor and antimicrobial properties were reported [1][2][3]. It was considered that morel's health benefits were attributed mainly to sugar (polysaccharides) and to various constituents such as compounds, amino acids, important vitamin, fatty, organic acid and mineral profile [2,4]. The crude polysaccharide isolated from the fruiting bodies of Morchella importuna showed a neuroprotective effect against H 2 O 2 -induced PC12 cell cytotoxicity by reducing oxidative stress [5]. Due to unique flavor, taste and texture, morels are used in different recipes all over the world. Additionally, morels are used as a laxative, purgative, emollient, laxative, purgative, emollient, body tonic, and for stomach problems, healing wound and for general weakness [6]. Benefited from its high price, morels play a very important role in the economy of the country. Due to the high demand for morels and their increasing economic importance, research on its cultivation, bioactivity, functional compositions is being investigated more and more [6]. Anti-tumor bioactivity-guided isolation of the MeOH extraction of Morchella esculenta afforded eight compounds including three fatty acids and five sterols. Three compounds exhibited the most potent cytotoxicity to human lung cancer cell lines [7]. Morchella protein hydrolyzate (MPH) produced by M. esculenta exhibits antioxidant activity [8]. The carboxymethylated polysaccharide (CPMEP) derived from the purified polysaccharide from Morchella angusticepes Peck (PMEP) showed stronger cholesterol-lowering activity than PMEP in rats [9]. Because of the limitation of wild Morchella resources, the cultivation of Morchella is increasingly popular for consumption as a functional food and for food-flavoring. Due to the limitation of strict techniques and difficulty in large-scale cultivation, fermentation has attracted more and more attention. It is of significance and potential to develop approaches to replace the traditional supply chain. However, there is no systematic research on the chemistry of liquid fermentation of Morchella.
Herein, a strain, YDJ-ZY-1, was isolated from wild Morchella in Zunyi Guizhou province and fermented in PDA media. Eight compounds were obtained from EtOAc extraction. Products 4 and 8 were new compounds and 7 was a new natural product. The antiradical activity was evaluated.

Identification of Strain
The strain YDJ-ZY-1 was obtained from wild Morchella by the spore releasing method. Hyphae were thick, strong, white mycelium at the beginning while yellowish in the middle and later stage, then the color changed to deeper and brownish in the later stage ( Figure 1). Brownish sclerotia with different sizes were easily produced. The capsule is cylindrical. Eight ascospores arranged in a single row are elliptical and colorless (400×) (Figure 2). Genomic DNA of the strain was extracted by CTAB (cetyltriethylammnonium bromide) method and identified as Morchella sp. with 99% similarity based on internal transcribed spacer (ITS) of ribosomal DNA (rDNA). Phylogenetic tree by the Neibor-joining method was constructed by Mega 5.1 (Figure 3).

Isolation and Identification of Secondary Metabolites
Eight compounds were isolated and purified by repeated column chromatography with different eluting agents from fermentation ( Figure 4). The antiradical activity was evaluated by the DPPH method. Compound 1 was obtained as brown oil. The 13 C-NMR spectrum (183.1, 171.6, 169.2, 114.3, 111.5) revealed the parent skeleton of pyrone type of which indicated two substituted carbons signals (171.6, 169.2). There were also oxygen-bearing methylene carbon signals at δ 61.1, as well as a methyl carbon at δ 19.6. Two aromatic ring protons signals 6.18 (s), 6.36 (s) provided evidence that 1

Isolation and Identification of Secondary Metabolites
Eight compounds were isolated and purified by repeated column chromatography with different eluting agents from fermentation ( Figure 4). The antiradical activity was evaluated by the DPPH method. Compound 1 was obtained as brown oil. The 13 C-NMR spectrum (183.1, 171.6, 169.2, 114.3, 111.5) revealed the parent skeleton of pyrone type of which indicated two substituted carbons signals (171.6, 169.2). There were also oxygen-bearing methylene carbon signals at δ 61.1, as well as a methyl carbon at δ 19.6. Two aromatic ring protons signals 6.18 (s), 6.36 (s) provided evidence that 1

Isolation and Identification of Secondary Metabolites
Eight compounds were isolated and purified by repeated column chromatography with different eluting agents from fermentation ( Figure 4). The antiradical activity was evaluated by the DPPH method.

Isolation and Identification of Secondary Metabolites
Eight compounds were isolated and purified by repeated column chromatography with different eluting agents from fermentation ( Figure 4). The antiradical activity was evaluated by the DPPH method. Compound 1 was obtained as brown oil. The 13 C-NMR spectrum (183.1, 171.6, 169.2, 114.3, 111.5) revealed the parent skeleton of pyrone type of which indicated two substituted carbons signals (171.6, 169.2). There were also oxygen-bearing methylene carbon signals at δ 61.1, as well as a methyl carbon at δ 19.6. Two aromatic ring protons signals 6.18 (s), 6.36 (s) provided evidence that 1 Compound 1 was obtained as brown oil. The 13 C-NMR spectrum (183.1, 171.6, 169.2, 114.3, 111.5) revealed the parent skeleton of pyrone type of which indicated two substituted carbons signals (171.6, 169.2). There were also oxygen-bearing methylene carbon signals at δ 61.1, as well as a methyl carbon at δ 19.6. Two aromatic ring protons signals 6.18 (s), 6.36 (s) provided evidence that 1 was structure with two substituted pyrone consistent with 13 C-NMR. Two methoxy protons signals at δ 4.41 (s, 2H) and three methyl protons signals at δ 2.32 (s, 3H) were observed in 1 H-NMR. From HMBC (Heteronuclear Multiple-Bond Correlation) data, methoxy protons signals at δ 4.41 were correlated with carbons signals δ C 111.5 and δ C 171.6. Methyl protons signals at δ 2.32 were related with δ C 114.3 and δ C 169.2. Aromatic ring protons at δ H 6.18 was related with δ C 19.6, δ C 111.5 and δ C 169.2 while δ H 6.36 was related with δ C 114.3, δ C 171.6 and δ C 61.1 in HMBC ( Figure 5). Compared 1 H-NMR and 13 C-NMR spectral data with the literature [10,11], its structure was confirmed as 1.
Compound 2 was isolated as brown oil. In the 1 H-NMR spectrum, one olefinic protons signals at δ 5.22 (s, 1H), two oxygen-bearing protons signals at δ 4.44 (m, 1H) and 3.75 (t, 1H), three methyl protons signals at δ 1.98 (s, 3H) were observed. The 13 C-NMR spectrum of 2 showed one carbonyl signal at δc 192.1. Corresponding to the 1 H-NMR spectrum, there were two oxygen-bearing carbon signals δc 80.9 and δc 65.6 in 13 C-NMR. Compared NMR data with those reported in the literatures [12], the structure of 2 was determined to be Erinapyrone B.
Compound 2 was isolated as brown oil. In the 1 H-NMR spectrum, one olefinic protons signals at δ 5.22 (s, 1H), two oxygen-bearing protons signals at δ 4.44 (m, 1H) and 3.75 (t, 1H), three methyl protons signals at δ 1.98 (s, 3H) were observed. The 13 C-NMR spectrum of 2 showed one carbonyl signal at δc 192.1. Corresponding to the 1 H-NMR spectrum, there were two oxygen-bearing carbon signals δc 80.9 and δc 65.6 in 13 C-NMR. Compared NMR data with those reported in the literatures [12], the structure of 2 was determined to be Erinapyrone B.
Compound 6 was isolated as white powder. 13 C-NMR spectra of 6 were typical of a fatty acid. The 13 C-NMR spectrum revealed four olefinic carbons signals at δ 130.3, 130.1, 128.2 and 128.0, as well as a carboxyl carbon signal at δ 180.6. Additionally, there were serials of methylene carbons signals at δ 24.8-34.3. The 1 H-NMR spectrum exhibited the presence of olefinic protons signals at δ 5.37 (m) and methyl group 0.88 (m). By a comparison with data available in the literature, compound 6 was determined to be linoleic acid [15].  Table 1. Oxygen-bearing methylene were observed at δ H 3.76 (2H), δ C 62.9 in compound 8, suggesting the ribofuranose. The chemical shifts were consistent with β-d-ribofuranose. H1 was related to 155.6 (C1) revealed the sugar was connected to C1. The HR-ESI-MS indicated the molecular formula C 15

Antiradical Activity of Compounds
Ability of scavenging effect on DPPH free radical was accomplished to evaluate the antioxidant activity. Five concentrations were tested in the experiment, Vitamin C (VC) was the positive control ( Figure 6). The data obtained in the present study suggests that compound 5 showed strong scavenging effect on DPPH free radical activity while compounds 1 and 2 exhibited moderate activity against free radicals. Compounds 3, 4, 7 and 8 almost exhibited no effect. For compounds 1 and 2, the radical scavenging capacity increased as the increasing of compound concentration. Compared to 1 and 2, compounds 3 and 4 exhibited less activity while the difference was the one more substitute side chain at C3 in compounds 3 and 4. The antiradical activity may be affected by the side chain. Moreover, the length of the side chain has little effect on the activity. Statistical analysis indicated that clearance of different compounds at the same concentration is different. The clearance of the same compound is different at different concentrations. Compound 5 showed the best clearance at 0.25 mg/mL.
Molecules 2019, 24, x FOR PEER REVIEW 6 of 9 analysis indicated that clearance of different compounds at the same concentration is different. The clearance of the same compound is different at different concentrations. Compound 5 showed the best clearance at 0.25 mg/mL.

Discussion
Morchella are famous rare edible fungi with high medicinal and economic value that show great potential in food, health products, pharmaceutical and cosmetics. The effects of Morchella on immune regulation, preventing decrepitude, anti-tumor, anti-fatigue, protection of the cardiovascular system and liver are proven in pharmacological tests. With the development of modern techniques, new effective metabolites and active monomers are eager to be discovered. Polysaccharide of Morchella, the main active ingredient, was mostly investigated. Additionally, there are many other kinds of chemical constituents (amino acids, sterols, organic acids et al.) found from the fruiting body of the Morchella species. However, there are no reports on the chemistry of liquid fermentation. Strain Morchella sp.YDJ-ZY-1 was isolated from wild fruiting body in Zunyi Guizhou province, Southwest China. Chemical compositions of EtOAc extraction from liquid fermentation were investigated and eight secondary metabolites were obtained. Compound 1-4 were structures of pyrone, compound 5 was furan derivative, compound 6 was unsaturated fatty acid, and compounds 7 and 8 were methyl cinnamate derivatives. Product 4 and 8 were new compounds while product 7 was a new natural product isolated from nature for the first time. Metabolite 5 with strong radical scavenging capacity was found the highest yield (3.4%). Product 6 linoleic acid, one of essential fatty acids that must be obtained through diet was proved important in metabolism and health. It could effectively reverse the inflammatory responses induced by palmitic acid treatment in microglial cells [16]. Research points to linoleic acid's anti-inflammatory, acne reductive, skin-lightening and moisture retentive properties when applied topically on the skin [17]. Product 7 and 8 with the structure of methyl cinnamate is mainly used in the daily chemical and food industry. Methyl cinnamate a flavor agent, is commonly used as fixative or edible flavor, but is also an important organic synthetic raw material. Methyl cinnamate is used as a chemical identification and distinguishing Tricholoma matsutake and Agaricus blazei and it could also provide a reference for evaluating the quality of edible fungi Tricholoma matsutake and Agaricus blazei [18]. Because of it is rich in bioactive ingredients and has many pharmacological effects, Morchella has broad prospects for development and utilization.

Isolation and Identification of Morchella sp. YDJ-ZY-1
Species of wild Morchella were isolated by spore releasing method by a self-made isolation device [19]. The sprayed spores were collected at different times by agar water medium, placed directly below and around the isolation device. When the spores were germinated, hyphae were transformed and purified into PDA (Potato Dextrose Agar) slant and then stored in 4 °C. The

Discussion
Morchella are famous rare edible fungi with high medicinal and economic value that show great potential in food, health products, pharmaceutical and cosmetics. The effects of Morchella on immune regulation, preventing decrepitude, anti-tumor, anti-fatigue, protection of the cardiovascular system and liver are proven in pharmacological tests. With the development of modern techniques, new effective metabolites and active monomers are eager to be discovered. Polysaccharide of Morchella, the main active ingredient, was mostly investigated. Additionally, there are many other kinds of chemical constituents (amino acids, sterols, organic acids et al.) found from the fruiting body of the Morchella species. However, there are no reports on the chemistry of liquid fermentation. Strain Morchella sp.YDJ-ZY-1 was isolated from wild fruiting body in Zunyi Guizhou province, Southwest China. Chemical compositions of EtOAc extraction from liquid fermentation were investigated and eight secondary metabolites were obtained. Compound 1-4 were structures of pyrone, compound 5 was furan derivative, compound 6 was unsaturated fatty acid, and compounds 7 and 8 were methyl cinnamate derivatives. Product 4 and 8 were new compounds while product 7 was a new natural product isolated from nature for the first time. Metabolite 5 with strong radical scavenging capacity was found the highest yield (3.4%). Product 6 linoleic acid, one of essential fatty acids that must be obtained through diet was proved important in metabolism and health. It could effectively reverse the inflammatory responses induced by palmitic acid treatment in microglial cells [16]. Research points to linoleic acid's anti-inflammatory, acne reductive, skin-lightening and moisture retentive properties when applied topically on the skin [17]. Product 7 and 8 with the structure of methyl cinnamate is mainly used in the daily chemical and food industry. Methyl cinnamate a flavor agent, is commonly used as fixative or edible flavor, but is also an important organic synthetic raw material. Methyl cinnamate is used as a chemical identification and distinguishing Tricholoma matsutake and Agaricus blazei and it could also provide a reference for evaluating the quality of edible fungi Tricholoma matsutake and Agaricus blazei [18]. Because of it is rich in bioactive ingredients and has many pharmacological effects, Morchella has broad prospects for development and utilization.

Isolation and Identification of Morchella sp. YDJ-ZY-1
Species of wild Morchella were isolated by spore releasing method by a self-made isolation device [19]. The sprayed spores were collected at different times by agar water medium, placed directly below and around the isolation device. When the spores were germinated, hyphae were transformed and purified into PDA (Potato Dextrose Agar) slant and then stored in 4 • C. The genomic DNA was extracted and ITS (internal transcribed spacer) sequence was applied to identify strain combined microstructure. The primers ITS1F (5 -CTTG GTCA TTTA GACG AAGTAA ) and ITS 4(5 -TCCT CCGC TTAT TGAT ATGC-3 ) were used to amplify the ITS rDNA. The PCR (Polymerase Chain Reaction) reaction was performed with the following cycles: (1) 94 • C for 3 min; (2) 30 cycles of 94 • C for 1 min, 55 • C for 1 min and 72 • C for 2 min, and (3) 72 • C for 5 min. Sequences were accomplished by Beijing Boyoushun Bio-ulab Technology Co., LTD. Homologous analysis was carried out by BLAST in NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic tree was constructed based on the Neighbor-joining method by Mega 5.1.

Fermentation of Strain
The purified cultures were incubated in 500 mL Erlenmeyer flasks that contain 200 mL PDA liquid media at 18-20 • C and 135 rpm on a shaker. And then cultures were transformed into twenty 2000 mL flasks containing 1000 mL PDA media for another 10 days.