Bioactive Compounds from the Mushroom-Forming Fungus Chlorophyllum molybdites

A novel compound (1) along with two known compounds (2 and 3) were isolated from the culture broth of Chlorophyllum molybdites, and three known compounds (4–6) were isolated from its fruiting bodies. The planar structure of 1 was determined by the interpretation of spectroscopic data. By comparing the specific rotation of the compound with that of the analog compound, the absolute configuration of 1 was determined to be R. This is the first time that compounds 2–4 were isolated from a mushroom-forming fungus. Compound 2 showed significant inhibition activity against Axl and immune checkpoints (PD-L1, PD-L2). In the bioassay to examine growth inhibitory activity against the phytopathogenic bacteria Peptobacterium carotovorum, Clavibacter michiganensis and Burkholderia glumae, compounds 2 and 3 inhibited the growth of P. carotovorum and C. michiganensis. In the bioassay to examine plant growth regulatory activity, compounds 1–4 showed a significant regulatory activity on lettuce growth.


Introduction
The fruiting body of certain kinds of eukaryotic, non-photosynthetic and aerobic fungi is generally known as a mushroom. The mushroom-forming fungi produce spores, and the spores germinate and create mycelia. The mycelia eventually produce primordia, which grow into new whole mushrooms, and the life cycle continues. Based on the taxonomic classification, the mushroom-forming fungi are divided into two groups, Basidiomycetes and Ascomycetes [1,2]. There is an expression that says, "plants act as producers, animals as consumers, and fungi as restorers or decomposers". In other words, plants create organic substances (carbohydrates) by photosynthesis and animals consume such plants. Then fungi, including mushroom-forming ones, play important roles in bringing the plants and animals back to the land. There are large differences in the structures of metabolites produced by mushroom-forming fungi compared to those produced by plants and animals, and biological activities indigenous to mushroom-forming fungi are often due to the differences [3,4]. Mushroom extracts and their secondary metabolites have been found human health due to their slow biodegradation [39]. Therefore, it is important to search for effective chemicals from natural sources to suppress phytopathogenic bacteria without environmental pollution. Recently, we found that anti-phytopathogenic-bacterial fatty acids were isolated from the mushrooms A. blazei [40].
On the other hand, C. molybdites is known to form fairy rings, and normally found in farmlands, lawns, etc. It is suitable for artificial cultivation [15]. Fairy rings are an interesting phenomenon in which the growth of grass is promoted and/or inhibited by the interaction between fungi and plants worldwide [41]. We discovered three plant growth regulators, 2-azahypoxanthine (AHX), 2-aza-8-oxohypoxanthine (AOH) and imidazole-4-carboxamide (ICA), as the fairy-ring-causing principles. Our study of fairy rings was covered in Nature and we named them "fairy chemicals" after the title of the article in the journal [42]. AHX and ICA were found from the culture broth of the fairy-ring-forming fungus Lepista sordida, and AOH was isolated from AHX-treated rice as a metabolite of AHX [43]. Recently, we also reported that AHX is a promising anti-angiogenic agent in retinal neovascularization by inhibiting the activation of hypoxia inducible factor [44]. AOH is effective as a cosmetic ingredient with a skin barrier function against water loss and skin lightening [45]. ICA inhibited the expression of Axl receptor tyrosine kinase and immune checkpoint molecules in melanoma cells in vitro and improved the therapeutic response to cisplatin in mouse melanoma xenografts in vivo [46]. Additionally, erinaceolactones A and B, erinachromanes A and B and erinaphenol A were isolated as plant growth inhibitors from the culture broth of Hericium erinaceus [2]. Plant growth regulators, armillariols A to C and sesquiterpene aryl esters, were isolated from the culture broth of Armillaria sp. They might play some roles in the Armillaria root disease [2]. We continue to search for the substances that interact between plants and fungi in C. molybdites.
Therefore, we attempted to find Axl and immune checkpoint inhibitors, anti-phytopathogenic-bacterial and plant-growth-regulating compounds from both the culture broth and fruiting bodies of C. molybdites. As a result, a novel compound (1) along with two known compounds (2 and 3) were isolated from the culture broth, and three known compounds (4-6) were isolated from the fruiting bodies. Here, we describe the isolation, structure determination and Axl immune checkpoint inhibitory activities of the compounds. In addition, we report anti-phytopathogenic-bacterial activity and plant growth regulation activity of these compounds.

Results and Discussion
The culture broth of C. molybdites was partitioned between n-hexane and water, and then EtOAc and water, successively. The EtOAc-soluble parts were fractionated with repeated chromatography, and a novel compound (1) and two known compounds (2 and 3) were isolated (Figure 1). The fresh fruiting bodies of C. molybdites were extracted with EtOH and then with acetone. After the solutions were combined and concentrated, they were partitioned between n-hexane and water, EtOAc and water, and the water part concentrated under reduced pressure, and then extracted with EtOH, successively. The EtOAc-soluble part and the EtOH-soluble part were fractionated with repeated chromatography. As a result, three known compounds (4-6) were isolated ( Figure 1). Compound 1 was obtained as brown amorphous. The molecular formula was determined as C 10 H 11 NO 5 with HRESIMS (m/z 224.0584 [M-H] − ; calcd for C 10 H 10 NO 5 , 224.0565), indicating the presence of six degrees of unsaturation in the molecule. The structure of 1 was elucidated through the interpretation of NMR spectra including DEPT, HMQC, COSY and HMBC ( Figures S2-S5). The DEPT experiment indicated the presence of one methylene, five methines and four tetrasubstituted carbons including two carboxy groups (δ C 171.0, 173.6). The complete assignment of all the protons and carbons was accomplished as shown in Table 1. The 4-aminobenzoic acid group was constructed based on the characteristic 1 H and 13 Figure 2). Hence, its planar structure was determined to be 4-(2,3-dihydroxypropanamido)benzoic acid. To determine the absolute configuration of 1, the specific rotation {[α] D 23 +29 (c = 0.12, MeOH)} was compared with that of its analog, (R)-2,3-dihydroxy-N-(4-vinylphenyl)propenamide {[α] D 20 +59.2 (c = 1.30, acetone)}, whose absolute configuration has been determined [47]. All the data allowed us to conclude that compound 1 was a novel compound, (R)-4-(2,3-dihydroxypropanamido)benzoic acid ( Figure 1).  Compounds 2 and 3 were identified as fusaric acid and 9,10-dehydrofusaric acid by the comparison of their spectroscopic data with those reported [48]. Compounds 2 and 3 have been isolated from the culture filtrate of Fusarium nygamai, which showed a strong inhibition of root elongation of seedlings as well as wide chlorosis of tomato leaves rapidly evolving into necrosis [48]. Both the compounds were first isolated from mushroom-forming fungi. Compound 4 was identified as ethyl 2-acetylamino-2-deoxy-β-D-glucopyranoside, which was isolated from Aspergillus terreus and has growth-promoting activity for Lactobacillus bifidus var. pennsylvanicus [49]. This compound also was isolated from mushrooms for the first time. Compound 5, 4-ethoxy-4-oxobutanoic acid, has been isolated from the mushroom Trametes versicolor [50], which has insulinotropic action in rat islets [51]. Compound 6 was identified as methyl 4-hydroxyphenylacetate, which was isolated from the fungus Gloeophyllum odoratum and a marine fungus Penicillium oxalicum, and has potent inhibitory activity against tobacco mosaic virus [52,53].
The human A549 alveolar epithelial cell lines were treated with compounds 2 and 3. As shown in Figure 3, compound 3 showed no effects on all the gene expressions. However, compound 2 significantly inhibited the expression of Axl, PD-L1 and PD-L2. The difference in the activity between 2 and 3 indicated that a butyl group played an important role in the suppression of all three genes.
We also examined effect of compounds 2 and 3 on the growth of Pectobacterium carotovorum, Clavibacter michiganensis and Burkholderia glumae. Pectobacterium species are economically important plant pathogens and cause soft rot and blackleg disease on a range of plant species around the world [54,55]. Among the Pectobacterium species, P. carotovorum is a Gram-negative plant-specific pathogen and has the widest host range that causes soft rot disease in diverse plants [56]. Potato is the most important crop affected in temperate regions [54,57]. A Gram-positive plant pathogenic bacterium, C. michiganensis, is one of the most disruptive bacterial diseases of tomato [58]. This bacterium gives a serious threat to the processing and fresh market tomato industries and causes catastrophic epidemics in most tomato-growing areas of the world. In general, this vascular pathogen causes wilt and canker symptoms by invading and diffusing in the xylem through natural openings or wounds [59,60]. B. glumae, as a Gram-negative bacterium, was first described in Japan leading to grain rotting and seedling blight on rice [61,62], and is an emerging rice disease that greatly limits the productivity of rice [63]. We used ampicillin as a positive control that has anti-phytopathogenic-bacterial activity. As a result, compounds 2 and 3 inhibited the growth of P. carotovorum and C. michiganensis at 0.1 µmol/paper disc, while showing no activity against the growth of B. glumae ( Figure 4). Compounds 2 and 3 were identified as fusaric acid and 9,10-dehydrofusaric acid by the comparison of their spectroscopic data with those reported [48]. Compounds 2 and 3 have been isolated from the culture filtrate of Fusarium nygamai, which showed a strong inhibition of root elongation of seedlings as well as wide chlorosis of tomato leaves rapidly evolving into necrosis [48]. Both the compounds were first isolated from mushroom-forming fungi. Compound 4 was identified as ethyl 2-acetylamino-2-deoxy-β-D-glucopyranoside, which was isolated from Aspergillus terreus and has growth-promoting activity for Lactobacillus bifidus var. pennsylvanicus [49]. This compound also was isolated from mushrooms for the first time. Compound 5, 4-ethoxy-4-oxobutanoic acid, has been isolated from the mushroom Trametes versicolor [50], which has insulinotropic action in rat islets [51]. Compound 6 was identified as methyl 4-hydroxyphenylacetate, which was isolated from the fungus Gloeophyllum odoratum and a marine fungus Penicillium oxalicum, and has potent inhibitory activity against tobacco mosaic virus [52,53].
The human A549 alveolar epithelial cell lines were treated with compounds 2 and 3. As shown in Figure 3, compound 3 showed no effects on all the gene expressions. However, compound 2 significantly inhibited the expression of Axl, PD-L1 and PD-L2. The difference in the activity between 2 and 3 indicated that a butyl group played an important role in the suppression of all three genes.
We also examined effect of compounds 2 and 3 on the growth of Pectobacterium carotovorum, Clavibacter michiganensis and Burkholderia glumae. Pectobacterium species are economically important plant pathogens and cause soft rot and blackleg disease on a range of plant species around the world [54,55]. Among the Pectobacterium species, P. carotovorum is a Gram-negative plant-specific pathogen and has the widest host range that causes soft rot disease in diverse plants [56]. Potato is the most important crop affected in temperate regions [54,57]. A Gram-positive plant pathogenic bacterium, C. michiganensis, is one of the most disruptive bacterial diseases of tomato [58]. This bacterium gives a serious threat to the processing and fresh market tomato industries and causes catastrophic epidemics in most tomato-growing areas of the world. In general, this vascular pathogen causes wilt and canker symptoms by invading and diffusing in the xylem through natural openings or wounds [59,60]. B. glumae, as a Gram-negative bacterium, was first described in Japan leading to grain rotting and seedling blight on rice [61,62], and is an emerging rice disease that greatly limits the productivity of rice [63]. We used ampicillin as a positive control that has anti-phytopathogenic-bacterial activity. As a result, compounds 2 and 3 inhibited the growth of P. carotovorum and C. michiganensis at 0.1 µmol/paper disc, while showing no activity against the growth of B. glumae (Figure 4). In addition, the effect of compounds 1-6 and the analog compounds of 4 and 6 [Nacelyl-D-glucosamine and methyl 2-(3-hydroxyphenyl)acetate] on lettuce growth was evaluated ( Figure 5). We used 2,4-dichlorophenoxyacetic acid (2,4-D) as a positive control, which inhibited the root and hypocotyl growth of lettuce dose-dependently. Compound 1 weakly promoted the root growth at 1 µmol/paper, while it inhibited the hypocotyl growth of lettuce at 1, 10 and 100 nmol/paper ( Figure 5A). As for the root and hypocotyl growth of lettuce, compounds 2-4 and methyl 2-(3-hydroxyphenyl)acetate showed inhibition activity at 100 and 1000 nmol/paper, respectively( Figures 5B and 5C). Compounds 5, 6 and N-acelyl-D-glucosamine exhibited no activity ( Figure 5C). Interestingly, a comparison of the structures between 4 and N-acelyl-D-glucosamine indicated that the ethoxy group at C-2 strengthened the lettuce-growth-inhibitory activity. The comparison between 6 and methyl 2-(3-hydroxyphenyl)acetate suggested that the hydroxy group at the meta position played an important role in the stronger inhibitory activity than at the para position.   In addition, the effect of compounds 1-6 and the analog compounds of 4 and 6 [N-acelyl-D-glucosamine and methyl 2-(3-hydroxyphenyl)acetate] on lettuce growth was evaluated ( Figure 5). We used 2,4-dichlorophenoxyacetic acid (2,4-D) as a positive control, which inhibited the root and hypocotyl growth of lettuce dose-dependently. Compound 1 weakly promoted the root growth at 1 µmol/paper, while it inhibited the hypocotyl growth of lettuce at 1, 10 and 100 nmol/paper ( Figure 5A). As for the root and hypocotyl growth of lettuce, compounds 2-4 and methyl 2-(3-hydroxyphenyl)acetate showed inhibition activity at 100 and 1000 nmol/paper, respectively( Figure 5B,C). Compounds 5, 6 and Nacelyl-D-glucosamine exhibited no activity ( Figure 5C). Interestingly, a comparison of the structures between 4 and N-acelyl-D-glucosamine indicated that the ethoxy group at C-2 strengthened the lettuce-growth-inhibitory activity. The comparison between 6 and methyl 2-(3-hydroxyphenyl)acetate suggested that the hydroxy group at the meta position played an important role in the stronger inhibitory activity than at the para position.  In addition, the effect of compounds 1-6 and the analog compounds of 4 and 6 [Nacelyl-D-glucosamine and methyl 2-(3-hydroxyphenyl)acetate] on lettuce growth was evaluated ( Figure 5). We used 2,4-dichlorophenoxyacetic acid (2,4-D) as a positive control, which inhibited the root and hypocotyl growth of lettuce dose-dependently. Compound 1 weakly promoted the root growth at 1 µmol/paper, while it inhibited the hypocotyl growth of lettuce at 1, 10 and 100 nmol/paper ( Figure 5A). As for the root and hypocotyl growth of lettuce, compounds 2-4 and methyl 2-(3-hydroxyphenyl)acetate showed inhibition activity at 100 and 1000 nmol/paper, respectively( Figures 5B and 5C). Compounds 5, 6 and N-acelyl-D-glucosamine exhibited no activity ( Figure 5C). Interestingly, a comparison of the structures between 4 and N-acelyl-D-glucosamine indicated that the ethoxy group at C-2 strengthened the lettuce-growth-inhibitory activity. The comparison between 6 and methyl 2-(3-hydroxyphenyl)acetate suggested that the hydroxy group at the meta position played an important role in the stronger inhibitory activity than at the para position.

General Experimental Procedures
1 H NMR spectra (one-and two-dimensional) were recorded on a Jeol lambda-5 spectrometer or a JNM-ECZ500R spectrometer at 500 MHz, and 13 C NMR spectra w recorded on the same instrument at 125 MHz (JEOL, Tokyo, Japan). HRESIMS spec were measured on a JMS-T100LP mass spectrometer (JEOL, Tokyo, Japan). The spec rotation values were measured with a Jasco DIP-1000 polarimeter (Jasco, Tokyo, Japa HPLC separations were performed with a Jasco Chromatography Data Station Chro NAV system using reverse-phase HPLC columns (CAPCELL PAK C18 AQ, Osaka so Osaka, Japan; COSMOSIL PBr, nacalai tesque, Kyoto, Japan; InerSustain Amide, InertS tain Phenyl, GL Science, Tokyo, Japan). Silica gel plate (Merck F254), ODS gel plate (Mer F254) and silica gel 60 N (Kanto Chemical, Tokyo, Japan) were used for analytical T and for flash column chromatography. All solvents used throughout the experime were obtained from Kanto Chemical Co. (Tokyo, Japan).

Fungal Material
Fresh fruiting bodies of C. molybdites were collected at Tsu, Mie Prefecture, Japan 2015. The culture mycelium was isolated from the fruiting bodies successfully and th identified as C. molybdites by determining the internal transcribed spacer (ITS) regions nuclear ribosomal DNA (rDNA) sequences deposited at NCBI BLA (http://blast.ncbi.nlm.nih.gov/). The mycelia of C. molybdites were pre-cultured on pot dextrose agar (PDA), and the inoculated mycelia were incubated at 25°C for two wee After growth, 10 pieces (6 mm diameter) cut from the two-week-cultured mycelia w

General Experimental Procedures
1 H NMR spectra (one-and two-dimensional) were recorded on a Jeol lambda-500 spectrometer or a JNM-ECZ500R spectrometer at 500 MHz, and 13 C NMR spectra were recorded on the same instrument at 125 MHz (JEOL, Tokyo, Japan). HRESIMS spectra were measured on a JMS-T100LP mass spectrometer (JEOL, Tokyo, Japan). The specific rotation values were measured with a Jasco DIP-1000 polarimeter (Jasco, Tokyo, Japan). HPLC separations were performed with a Jasco Chromatography Data Station ChromNAV system using reverse-phase HPLC columns (CAPCELL PAK C18 AQ, Osaka soda, Osaka, Japan; COSMOSIL PBr, nacalai tesque, Kyoto, Japan; InerSustain Amide, InertSustain Phenyl, GL Science, Tokyo, Japan). Silica gel plate (Merck F254), ODS gel plate (Merck F254) and silica gel 60 N (Kanto Chemical, Tokyo, Japan) were used for analytical TLC and for flash column chromatography. All solvents used throughout the experiments were obtained from Kanto Chemical Co. (Tokyo, Japan).

Fungal Material
Fresh fruiting bodies of C. molybdites were collected at Tsu, Mie Prefecture, Japan, in 2015. The culture mycelium was isolated from the fruiting bodies successfully and then identified as C. molybdites by determining the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA) sequences deposited at NCBI BLAST (http://blast.ncbi. nlm.nih.gov/). The mycelia of C. molybdites were pre-cultured on potato dextrose agar (PDA), and the inoculated mycelia were incubated at 25 • C for two weeks. After growth, 10 pieces (6 mm diameter) cut from the two-week-cultured mycelia were inoculated into 500 mL Erlenmeyer flasks containing 300 mL of PDB medium (n = 5), and the cultures were incubated for 4 weeks (25 • C, 120 rpm). Lettuce seeds (Lactuca sativa L. cv. Cisko; Takii Co., Ltd., Tokyo, Japan) were used in this study.

Axl and Immune Checkpoint Molecule Assay [35]
The human A549 alveolar epithelial cell line was purchased from the American Type Culture Collection (Rockville, MD, USA) and cultured in DMEM, supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine and 100 U mL penicillin plus 100 µg/mL streptomycin. All cells were cultured at 37 • C in 75 cm 2 flasks in an atmosphere composed of 5% CO 2 and 95% air. Confluent cells were passaged after 5-7 days.
A549 cells in 0.1% BSA-DMEM were seeded in 24-well plates. Test compounds (20 µg/mL) were added to the wells, and the plates were incubated for 24 h. The total RNA was extracted using Sepasol®-RNA I Super G (Nacalai) following the instructions of the manufacturer. One µg of total RNA was denatured at 65 • C for 10 min and then reverse-transcribed using ReverTra Ace Reverse Transcriptase (TOYOBO) and oligo (dT) primer in a volume of 20 µL according to the manufacturer's protocol.
Each gene contains forward and reverse sequences (5 > 3 ), which are, respectively, GGAGCGAGATCCCTCCAAAAT and GGCTGTTGTCATACTTCTCATGG for the GADPH gene, TGCCATTGAGAGTCTAGCTGAC and TTAGCTCCCAGCACCGCGAC for the Axl gene, GGACAAGCAGTGACCATCAAG and CCCAGAATTACCAAGTGAGTCCT for the PD-L1 gene, and ACCGTGAAAGAGCCACTTTG and GCGACCCCATAGATGATTATGC for the PD-L2 gene. The cDNA was amplified using PCR and the conditions were as follows: 94 • C, 1 min; 60 • C, 1 min; and 72 • C, 1 min for 28-35 cycles. The PCR products were electrophoresed on a 1.5% agarose gel and then stained with an ethidium bromide solution. The semi-quantitative RT-PCR results were quantified using ImageJ software.
The data are expressed as the mean ± standard error of the mean (SEM). The statistical difference was calculated using analysis of variance with post hoc analysis using Fisher's predicted least significant difference test. All statistics were performed using the StatView 5.0 package (Abacus Concepts, Berkeley, CA, USA).

Antibacterial Assay [40]
Each bacterium (C. michiganensis, B. glumae and P. carotovorum) was taken from the slant using an inoculation loop and suspended in 1 mL of sterile water in a 1.5 mL Eppendorf tube, and a suspension of 10 8 colony forming units (CFU)/mL was made with reference to the OD 600 . YP medium (yeast extract 5 g/L, peptone 10 g/L, agar 15 g/L) in a test tube was autoclaved for 20 min at 121 • C. The medium was left to stand until the temperature reached about 30 • C, and 100 µL of each bacterium suspension was added to the medium, and the mixture was poured into a Petri dish.
A total of 40 µL of a solution of each compound (0.1, 0.05 and 0.01 µmol in MeOH) and MeOH only (as control) were put on a paper disc (8 mm in diameter). After the discs were dried in the air, they were put on the medium. They were incubated for 3 days to evaluate their antibacterial activity.

Plant-Growth-Regulating Assay
Lettuce seeds were put on filter paper (Advantec No. 2, φ 55 mm; Toyo Roshi Kaisha, Japan), soaked in distilled water in a Petri dish (φ 60 × 20 mm) and incubated in a growth chamber in the dark at 25 • C for 1 day. Each sample was dissolved in 1 mL of MeOH (1, 10, 100 and 1000 nmol/mL) and then poured on filter paper (φ 55 mm) in a Petri dish (φ 60 × 20 mm). After the sample-loaded paper had been air-dried, 1 mL of distilled water was poured on the sample-loaded paper or intact filter paper (control). The preincubated lettuce seedlings (n = 9 in each Petri dish) were transferred onto the sample-loaded filter paper or control filter paper and incubated in a growth chamber in the dark at 25 • C for 3 days. The lengths of the hypocotyl and the root were measured using a ruler.