Isolation and Characterization of Benzaldehyde Derivatives with Anti

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Isolation and Characterization of Benzaldehyde Derivatives with Anti-Inflammatory Activities from Eurotium cristatum, the Dominant Fungi Species in Fuzhuan Brick Tea Jie Shi,† Jianxin Liu,‡ Dingding Kang,† Yimin Huang,‡ Wenping Kong,† Yunxi Xiang,‡ Xiangcheng Zhu,†,§ Yanwen Duan,*,†,§,∥ and Yong Huang*,†,∥ †

Xiangya International Academy of Translational Medicine at Central South University, Changsha, Hunan 410013, China School of Pharmaceutical Sciences, Hunan University of Medicine, Huaihua, Hunan 418000, China § Hunan Engineering Research Center of Combinatorial Biosynthesis and Natural Product Drug Discovery, Changsha, Hunan 410013, China ∥ National Engineering Research Center of Combinatorial Biosynthesis for Drug Discovery, Changsha, Hunan 410011, China

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‡

S Supporting Information *

ABSTRACT: Eurotium cristatum is the dominant fungi species in Fuzhuan brick tea (FBT), which may be responsible for the color, taste, and associated health benefits. In this study, eight E. cristatum strains were first isolated from various brands of FBT in China. Six benzaldehyde derivatives 1−6 were then isolated and characterized from these FBT-derived E. cristatum, four of which were also present in the spores of E. cristatum. The anti-inflammatory activities of these benzaldehyde derivatives were evaluated in lipopolysaccharide-induced RAW264.7 cells. These compounds could inhibit the expression both of inducible nitroxide synthase and cyclooxygenase-2. Our results suggest that the benzaldehyde derivatives from E. cristatum may be at least partially responsible for the observed health benefits of FBT.

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INTRODUCTION

The unique postfermentation process of FBT using E. cristatum has enabled special phytochemical profiles of FBT extracts, including tea polyphenols,13 flavones,14 triterpenoids,15 norisoprenoid,16 and polysaccharides.17 These unique metabolites were suggested to play important roles to mediate these important health benefits in humans and experimental animals. To further understand the role of E. cristatum in the health benefits of FBT, we initiated a study to systematically characterize the E. cristatum strains isolated from eight different FBT manufacturers by 18S rRNA sequencing and analyzing the chemical composition of the spore and their fermentation. We have isolated and characterized six benzaldehyde derivatives from their fermentation extracts and discovered they possess anti-inflammatory activities. Using a liposaccharide-induced RAW264.7 cell model, several of these compounds were shown to selectively inhibit the expression of inducible nitric oxide synthase (iNOS).

1,2

Tea is one of the most popular beverages in the world. Human consumption of Fuzhuan brick tea (FBT) dates back to the 16th century.3 Due to the lack of fresh vegetables, people who live in high altitude in China, including QinghaiTibet Plateau, have heavily relied on high-fat food and have a long history to consume FBT.4 The traditional dietary consumption of FBT even led to dental caries and fluorosis in local children due to the presence of fluoride in FBT.4,5 However, proper consumption of FBT has also been observed to have antihyperglycemia, antihyperlipidemia, or antiobesity effects in humans and model animals.6−9 In contrast to green tea, FBT is a unique postfermentation tea, with the presence of “golden flora”,10 a dominant fungi species Eurotium cristatum (also named Aspergillus cristatus) in the fermentation and storage of the leaves, leaf buds, and stems of Camellia sinensis.9 The golden flora is an important indicator to evaluate the quality of FBT by local people with daily FBT consumption. At least 2 × 105 colony-forming units of E. cristatum should be present in one gram of FBT, based on the manufacturing protocol enforced by current regulations.11 We have recently discovered that the FBT-derived fungi may survive in the mouse gut and could normalize gut microbiota dysbiosis in obese mice.12 © 2019 American Chemical Society

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RESULTS AND DISCUSSION Isolation of Eight E. cristatum Strains from Different FBT Brands and Their 18S rRNA Analysis. Eight Received: March 3, 2019 Accepted: March 29, 2019 Published: April 10, 2019 6630

DOI: 10.1021/acsomega.9b00593 ACS Omega 2019, 4, 6630−6636

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Figure 1. Phylogenetic analysis of Eurotium spp. (A) The phylogenetic tree of isolated E. cristatum strains using the MEGA6 software. (B) The morphology of E. cristatum CB10001−CB10008.

Figure 2. Structure of benzaldehyde compounds. Six benzaldehyde compounds 1−6 were isolated from E. cristatum CB10006. Compounds 7−9 were from other Eurotium spp.

amount of E. cristatum spores in FBT and the observed health benefits of the consumption of the extracts of Ganoderma lucidum spores,20 we first evaluated the presence of secondary metabolites in the methanol extracts of E. cristatum CB10001− CB10008 using high-performance liquid chromatography (HPLC) analysis (Figure 3A). The spore methanol extracts of the tested strains showed similar metabolite profile with the same retention time and UV spectra, indicating the presence of the same series of metabolites. We next fermented E. cristatum CB10001−CB10008 in three different solid media and their methanol extracts were similarly analyzed. All the fermented eight E. cristatum strains had similar fermentation profiles, which were also similar to the metabolite profiles observed from their spore extracts (Figure 3B). To isolate these metabolites, E. cristatum CB10006 was fermented in 1.2 kg solid corn medium for 45 days. The resulting E. cristatum CB10006 were then extracted with methanol to obtain 23 g crude extract. The extract was

representative brands of commercial FBTs were purchased, and a large amount of golden flora could be observed inside each FBT (Figure S1). The golden flora was then picked up using tooth sticks and spread on to M40Y agar plates to obtain individual fungi colonies CB10001−CB10008 (Figure 1).18 Their genomic DNAs were extracted and subjected to 18S rRNA sequencing. Saccharomyces cerevisiae was used as an outgroup to construct the phylogenetic tree. All the isolated strains CB10001−CB10008 fell into the same clade with A. cristatus and A. flavus, indicating their closer evolutionary relationships. The stain CB10006 was close to A. cristatus AB002073.1, which was also isolated from FBT, with its whole genome sequenced recently.19 Based on the above analysis, all the isolated strains CB10001−CB10008 belonged to E. cristatum, consistent with the presence of the dominant E. cristatum in FBT. Isolation and Identification of Benzaldehyde Derivatives from E. cristatum. Due to the presence of a large 6631

DOI: 10.1021/acsomega.9b00593 ACS Omega 2019, 4, 6630−6636

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Figure 3. Analysis of benzaldehyde derivatives in E. cristatum. (A) HPLC analysis of the benzaldehydes from E. cristatum spores. E. cristatum CB10001 (i), E. cristatum CB10002 (ii), E. cristatum CB10003 (iii), E. cristatum CB10004 (iv), E. cristatum CB10005 (v), E. cristatum CB10006 (vi), E. cristatum CB10007 (vii), and E. cristatum CB10008 (viii). 1, ■; 2, ⧫; 4, ▼; 5, ●. (B) HPLC analysis of the benzaldehydes from E. cristatum CB10006. Spores (i), corn medium (ii), MAY medium (iii), and rice medium (iv). (C) The quantity of benzaldehydes from E. cristatum spores.

(LPS)-stimulated RAW264.7 cell model, which have been used to evaluate many natural products derived from the traditional Chinese medicine plants (Figure 4).31−33 Compounds 1 and 6 decreased the iNOS protein expression, but they did not significantly inhibit cyclooxygenase-2 (COX-2) protein expression. In contrast, compounds 2−5 were shown to moderately inhibit LPS-induced iNOS and COX-2 protein expression. It appeared that the aliphatic chains in these benzaldehydes played certain roles in the determination of the binding affinity toward either iNOS or COX-2. For example, a larger conjugation system in 1 or 6 led to a significantly decreased inhibition against COX-2, whereas compounds 1 and 2 with two conjugated double bonds showed stronger inhibitory effects against iNOS than compounds 4 or 5. Interestingly, previous radioligand binding assay suggested that compounds 4 or 5, instead of 1, could bind to human opioid and cannabinoid receptors.23 Therefore, these compounds, especially 1, could be used to explore the interactions between COX-2 and iNOS, since they are closely associated.34−36 This was consistent with the recent report by Liu and co-workers that compounds 1−5 were able to inhibit the production of NO in the Griess assay activated by LPS.22 Antibacterial Activities. The antibacterial activities of compounds 1−6 were evaluated against several Gram-positive pathogens using paper disk assay, with linezolid as the positive control (5 μg per paper disk) (Table 1). All tested compounds were used at 50 μg per paper disk. Compounds 1, 2, 4, and 5 showed weak antibacterial activities against Staphylococcus aureus ATCC 29213, methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), and Bacillus subtilis,

separated successively by repeated column chromatography (CC) over silica gel, preparative thin-layer chromatography, and semipreparative HPLC, affording compounds 1−6, whose structures were similarly established as previously reported (Figure 2).21,22 Compounds 1−6 were all benzaldehyde derivatives, including dihydroauroglaucin (DAG) (1), isodihydroauroglaucin (IDAG) (2), 2-(2′,3-epoxy-1′,3′-heptadienyl)6-hydroxy-5-(3-methyl-2-butenyl) benzaldehyde (3), flavoglaucin (FG) (4), tetrahydroauroglaucin (TAG) (5), and 2(2′,3-epoxy-1′,3′,5′-heptatrienyl)-6-hydroxy-5-(3-methyl-2butenyl)benzaldehyde (6). Despite some of these benzaldehyde derivatives have been isolated from other Eurotium strains, it was the first time that all of the six benzaldehyde derivatives were discovered in all E. cristatum from Fuzhuan brick tea. In addition, the discovery of the other benzaldehyde derivatives 7−9, from other Eurotuim spp.,23−25 suggested that Eurotuim spp. were rich sources of benzaldehydes natural products. The amount of the benzaldehyde derivatives was next determined (Figure 3C). It appeared that compound 1 showed the highest concentrations among of all tested strains, whereas E. cristatum CB10008 showed the highest production of compound 1. Anti-Inflammatory Activities. Some of these benzaldehyde compounds exhibited anti-inflammatory activities,23,26 αglucosidase inhibitory activities,24 radical-scavenging activity,27,28 and binding affinity for human opioid or cannabinoid receptors.29 Dark brick tea, such as Pu-erh tea, were also reported to have certain anti-inflammatory function.30 Therefore, we evaluated the anti-inflammatory activity of isolated benzaldehyde compounds 1−6, using lipopolysaccharide 6632

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Figure 4. Effect of compounds 1−6 on iNOS and COX-2 protein expression in LPS-stimulated RAW264.7 cells. (A) Effect of compounds 1−6 on iNOS and COX-2 protein expression. (B) The inhibitory effects of compounds 1−6 on iNOS protein expression. (C) The inhibitory effects of compounds 1−6 on COX-2 protein expression. Cells were seeded in 12-well plate for 24 h and then pretreated with tested compounds or dexamethason (DEX) for 1 h, followed stimulation with LPS (100 ng/mL) for another 18 h. Total proteins of cells were prepared and analyzed for iNOS, COX-2, and β-actin (loading control) by Western blotting. The protein level was quantitated by ChemiDoc XRS+ with Image Lab software (Wuhan Baileizhen Biotechnology Co., Ltd, China) and to count the density ratio of iNOS/COX-2 to β-actin. The density ratio of LPS group (blank control) was set to 1. Results are the mean ± standard deviation (SD), n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, vs LPS alone.

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METHODS General Experimental Procedure. NMR experiments were conducted on a Bruker spectrometer (500 MHz), chemical shifts were reported in parts per million relative to CDCl3 (δH = 7.26 ppm) for 1H NMR and CDCl3 (δC = 77.23 ppm) for 13C NMR spectroscopy. Column chromatography (CC) was carried out on silica gel (100−200 and 300−400 mesh, Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai, China) and polyamide (100−200 mesh, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). Analytical and preparative thin-layer chromatography (TLC) was carried out on silica gel (Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai, China). Semipreparative reversed phase-high-performance liquid chromatography was performed using a Waters 1525 Binary HPLC Pump equipped with a Waters 2489 UV/ visible detector and using a Welch Ultimate AQ-C18 column (250 × 10 mm2, 5 μm). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and other tissue culture reagents were purchased from Gibco BRL Co. (Grand Island, NY). Lipopolysaccharide (LPS, Escherichia coli 055: B5) and dexamethason (DEX) were obtained from Sigma Chemical Co. (St. Louis, MO). Inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) antibodies were purchased from Cell Signaling Technology (Boston, MA), and β-actin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Goat antimouse IgG and goat antirabbit IgG secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fungi Strains. FBTs were purchased from commercial sources (Table S1). The spores from different brands of

Table 1. Antimicrobial Activities of Compounds 1−6 against Gram-Positive Bacteria Pathogens at 50 μga diameter of the inhibition zone (mm) compounds

S. aureus

MRSA

MSSA

B. subtilis

1 2 3 4 5 6 linezolid DMSO

7.3 ± 0.4 7.7 ± 0.3 7.4 ± 0.5 6.9 ± 0.1 19.8 ± 0.6 -

6.6 ± 0.4 7.1 ± 0.1 6.9 ± 0.3 7.2 ± 0.2 18.0 ± 1.3 -

7.2 ± 0.1 7.4 ± 0.5 7.7 ± 0.3 6.5 ± 0.1 17.8 ± 0.5 -

6.7 ± 0.4 7.3 ± 0.5 7.2 ± 0.1 6.3 ± 0.1 19.8 ± 0.6 -

a

All experiments were repeated thrice. -: No cytotoxicity observed.

which were consistent with previous reported antibacterial activities.22 In conclusion, eight E. cristatum strains were isolated from various brands of FBT. Six known benzaldehyde compounds 1−6 were isolated from E. cristatum CB10006. Compounds 1, 2, 4, and 5 were also present in the spores of E. cristatum. Based on these results, these compounds could inhibit the expression of iNOS and COX-2 in LPS-stimulated RAW264.7 cells. Compounds 1, 2, 4, and 5 showed weak antibacterial activities against S. aureus, MSSA, MRSA, and B. subtilis. Our results suggested that the benzaldehydes from the golden flora in FBT may be at least partially responsible for the observed health benefits of FBT. These benzaldehydes could be developed as selective iNOS inhibitors. 6633

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Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated FBS (Gibco BRL Co, Grand Island, NY), penicillin G (10 000 units/mL), streptomycin (10 μg/mL), and L-glutamine (29.2 mg/mL) (Gibco BRL Co, Grand Island, NY). Cells were grown at 37 °C in a humidified atmosphere containing 5% CO2. All of tested compounds were dissolved in dimethyl sulfoxide (DMSO) in 100 mM and the working concentration was 100 μm (DMSO concentration was less than 0.1% in assay). RAW264.7 cells were plated at a density of 2 × 105 cells/wells in 12-well plates for 24 h. The cells were pretreated with the tested compounds for 1 h and then stimulated with LPS (100 ng/mL) for another 18 h. At the end of incubation time, the total proteins in the cells were prepared and the expression of iNOS, COX-2, and β-actin was analyzed by Western blotting. DEX was chosen as the positive control with the concentration of 0.5 μm for the inhibition of iNOS and COX-2 expression. Cells stimulated by LPS without any intervention were observed as blank control. Cells incubated by DMEM medium were used as normal control. Western Blot Analysis. After stimulation of RAW264.7 cells by LPS for 18 h, the total cellular proteins were extracted using radioimmunoprecipitation assay lysis buffer (Cell Signaling technology, Boston, MA) with 1× protease inhibitor mix (Roche Applied Science, Germany). Protein concentration was determined by the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Thirty micrograms of proteins per lane was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and followed by transferring to a nitrocellulose membrane (GE Healthcare Life Sciences, Buckinghamshire, U.K.). The membrane was blocked in 5% skimmed milk and then incubated with the primary antibody (iNOS and COX-2) and mouse antibodies specific for β-actin at 4 °C overnight. The membrane was subsequently incubated with goat antimouse IgG and goat antirabbit IgG secondary antibodies at room temperature for 1 h. The protein expression level was quantitated using ChemiDoc TM XRS+ with Image Lab TM software (Wuhan Baileizhen Biotechnology Co., Ltd., China). Statistical Evaluation. All data are given as the mean ± SD for the three independent experiments. Statistical significance was compared between treated groups and the blank control and determined by using paired Student’s t test. P values less than 0.05 were accepted as statistically significant. Antimicrobial Assays. Compounds 1−6 were evaluated for their antibacterial activities against S. aureus ATCC 29213, methicillin-sensitive S. aureus, methicillin-resistant S. aureus, and B. subtilis. Linezolid and compounds 1−6 were individually dissolved and diluted with DMSO to obtain concentrations of 1 and 10 mg/mL. All tested compounds were used at 5 μL. Linezolid was used as the positive control at 5 μL. The tested plates were incubated at 37 °C overnight. The inhibition zones were then measured.

Fuzhuan brick tea were individually picked by toothsticks and then cultured in M40Y agar (2% malt extract, 5% yeast extract, 40% sucrose, 1.5% agar) plate to obtain spores. Genomic DNA from fungal mycelia was extracted.18 The 18S rRNA of E. cristatum was amplified and sequenced by polymerase chain reaction. The phylogenetic relationships between filamentous fungi were established based on 18S rRNA sequencing and then submitted to the software MEGA6. The 18S rRNA sequences of E. cristatum CB10001−CB10008 were deposited to GenBank (MK371789, MK377302, MK372215, MK377017, MK377016, MK377030, MK377018, and MK377020). Fermentation, Isolation, and Purification. E. cristatum CB10001−CB10008 were grown in M40Y medium at 28 °C on rotary shakers (230 rpm) for 60 h.10 Their fresh mycelia were then inoculated into 250 mL Erlenmeyer flasks for 30 days in MAY medium, rice medium, or corn medium. The MAY medium contained (w/v) 2% malt extract, 0.5% yeast extract, 3% sucrose, and 2% agar. The rice medium consisted of 30 g rice and 40 mL of distilled water, whereas the corn medium consisted of 30 g corn and 50 mL distilled water. For a large-scale fermentation, the fresh mycelia from M40Y medium were inoculated into 10 × 1 L flasks containing corn (120 g) and distilled H2O (200 mL). After incubation for 45 days at 28 °C, the fermented solid mash was extracted with EtOH (10 L × 3) to obtain the crude extract. The crude extract was redissolved with H2O (1 L) and extracted with petroleum ether (PE)/EtOAc (1:3) to remove certain lipids. The organic phase layer was evaporated to dryness under reduced pressure to yield the crude extract (23 g). Then, it was subjected to silica gel chromatography (CC) using PE/EtOAc (10−100%) to give 10 fractions (Fr.1−10). Fr.7 (435 mg) was separated by polyamide CC eluted with H2O/EtOH (2:8) to yield 11 subfractions (Fr.7-1−Fr.7-11). Fr.9-2−Fr.11-1 (152.7 mg) were further purified by silica gel CC eluting with PE/EtOAc (9:1) to yield 1 (26.4 mg). Fr.8 (251.7 mg) was separated by silica gel CC eluting with PE/ EtOAc (8:2) to yield six subfractions (Fr.8-1−Fr.8-6). Fr.8-4 (119 mg) was further purified by silica gel CC to yield 2 (PE/ EtOAc (15:1), 19.9 mg) and 6 (PE/EtOAc (6:1), 1.7 mg). Fr.9 was separated by silica gel CC eluting with PE/EtOAc (49:0.5) to yield Fr.9-3 (21.3 mg). Fr.9-3 was further purified by semipreparative RP-C18 HPLC with a flow rate of 3 mL/ min and a gradient elution of CH3CN/H2O (containing 0.2% formic acid) in 20 min (50−95% for 15 min, followed by 95% for 2 min, and 95−5% for 1 min, followed by 5% for 2 min) to afford compounds 3 (tR = 20.0 min, 7.2 mg) and 6 (tR = 19.4 min, 2.1 mg). Fr.6-4 (785.1 mg) was purified by preparative TLC on a silica gel plate (20 cm × 20 cm) with a mobile phase of n-hexane and MeOH (8:2.75) to yield 4 (10.1 mg) and 5 (21.8 mg). Quantitative Analysis of Benzaldehyde Compounds in the Spores of E. cristatum. The spores of individual E. cristatum strain (0.5 mL in 20% glycerol) were extracted with MeOH (5 mL × 3) to obtain the crude extract. The extracts were analyzed by HPLC. To quantify the benzaldehyde compounds in E. cristatum spores, a standard curve was prepared based on compound 1. All experiments were repeated three times, and the titers of the individual metabolites were presented as mean ± SD. Anti-Inflammatory Assay. The murine macrophages RAW264.7 cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00593. E. cristatum strains derived from different brands of Fuzhuan brick tea; E. cristatum in Fuzhuan brick tea; standard calibration curve; HPLC analysis of the crude fermentation products; diameter of the inhibition zone of compounds 1−6 (PDF) 6634

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(12) Kang, D.; Su, M.; Duan, Y. W.; Huang, Y. Eurotium cristatum, a new fungi probiotic from Fuzhuan brick tea, alleviated obesity in mice by modulating gut microbiota. bioRxiv 2019, 1−29. (13) Zhu, Y. F.; Chen, J. J.; Ji, X. M.; Hu, X.; Ling, T. J.; Zhang, Z. Z.; Bao, G. H.; Wan, X. C. Changes of major tea polyphenols and production of four new B-ring fission metabolites of catechins from post-fermented Jing-Wei Fu brick tea. Food Chem. 2015, 170, 110− 117. (14) Luo, Z. M.; Du, H. X.; Li, L. X.; An, M. Q.; Zhang, Z. Z.; Wan, X. C.; Bao, G. H.; Zhang, L.; Ling, T. J. Fuzhuanins A and B: the Bring fission lactones of flavan-3-ols from Fuzhuan brick-tea. J. Agric. Food Chem. 2013, 61, 6982−6990. (15) Ling, T. J.; Wan, X. C.; Ling, W. W.; Zhang, Z. Z.; Xia, T.; Li, D. X.; Hou, R. Y. New triterpenoids and other constituents from a special microbial-fermented tea-Fuzhuan brick tea. J. Agric. Food Chem. 2010, 58, 4945−4950. (16) Luo, Z. M.; Ling, T. J.; Li, L. X.; Zhang, Z. Z.; Zhu, H. T.; Zhang, Y. J.; Wan, X. C. A new norisoprenoid and other compounds from Fuzhuan brick tea. Molecules 2012, 17, 3539−3546. (17) Xie, G.; Ye, M.; Wang, Y.; Ni, Y.; Su, M.; Huang, H.; Qiu, M.; Zhao, A.; Zheng, X.; Chen, T.; Jia, W. Characterization of pu-erh tea using chemical and metabolic profiling approaches. J. Agric. Food Chem. 2009, 57, 3046−3054. (18) Rogers, S. O.; Bendich, A. J. Extraction of Total Cellular DNA from Plants, Algae and Fungi. In Plant Molecular Biology Manual; Gelvin, S. B., Schilperoort, R. A. Ed.; Springer: Dordrecht, 1994; pp 183−190. (19) Ge, Y.; Wang, Y.; Liu, Y.; Tan, Y.; Ren, X.; Zhang, X.; Hyde, K. D.; Liu, Y.; Liu, Z. Comparative genomic and transcriptomic analyses of the Fuzhuan brick tea-fermentation fungus Aspergillus cristatus. BMC Genomics 2016, 17, No. 428. (20) Chang, C. J.; Lin, C. S.; Lu, C. C.; Martel, J.; Ko, Y. F.; Ojcius, D. M.; Tseng, S. F.; Wu, T. R.; Chen, Y. Y.; Young, J. D.; Lai, H. C. Ganoderma lucidum reduces obesity in mice by modulating the composition of the gut microbiota. Nat. Commun. 2015, 6, No. 7489. (21) Peng, X. Y.; Liang, F. L.; Li, D. L.; Chen, Y. C.; Tao, M. H.; Zhang, W. M.; Zhao, Y. L. Secondary metabolites of Eurotium cristatum from Fu brick tea and their biological activities. Chin. Tradit. Herb. Drugs 2013, 44, 1881−1886 (In Chinese) . (22) Zhang, P.; Jia, C.; Deng, Y.; Chen, S.; Chen, B.; Yan, S.; Li, J.; Liu, L. Anti-inflammatory prenylbenzaldehyde derivatives isolated from Eurotium cristatum. Phytochemistry 2019, 158, 120−125. (23) Gao, J.; Leon, F.; Radwan, M. M.; Dale, O. R.; Husni, A. S.; Manly, S. P.; Lupien, S.; Wang, X.; Hill, R. A.; Dugan, F. M.; Cutler, H. G.; Cutler, S. J. Benzyl derivatives with in vitro binding affinity for human opioid and cannabinoid receptors from the fungus Eurotium repens. J. Nat. Prod. 2011, 74, 1636−1639. (24) Kim, K. S.; Cui, X.; Lee, D. S.; Ko, W.; Sohn, J. H.; Yim, J. H.; An, R. B.; Kim, Y. C.; Oh, H. Inhibitory effects of benzaldehyde derivatives from the marine fungus Eurotium sp. SF-5989 on inflammatory mediators via the induction of heme oxygenase-1 in lipopolysaccharide-stimulated RAW264.7 macrophages. Int. J. Mol. Sci. 2014, 15, 23749−23765. (25) Li, D. L.; Li, X. M.; Li, T. G.; Dang, H. Y.; Proksch, P.; Wang, B. G. Benzaldehyde derivatives from Eurotium rubrum, an endophytic fungus derived from the mangrove plant Hibiscus tiliaceus. Chem. Pharm. Bull. 2008, 56, 1282−1285. (26) Wu, M. D.; Cheng, M. J.; Hsieh, S. Y.; Yuan, G. F. Chemical constituents of the fungus of Eurotium chevalieri BCRC 07F0022. Chem. Nat. Compd. 2014, 49, 1175−1176. (27) Wang, S.; Li, X. M.; Teuscher, F.; et al. Chaetopyranin, a benzaldehyde derivative, and other related metabolites from Chaetomium globosum, an endophytic fungus derived from the marine red alga Polysiphonia urceolata. J. Nat. Prod. 2006, 69, 1622−1625. (28) Kwon, J.; Lee, H.; Ko, W.; Kim, D. C.; Kim, K. W.; Kwon, H. C.; Guo, Y.; Sohn, J. H.; Yim, J. H.; Kim, Y. C.; et al. Chemical constituents isolated from antarctic marine-derived Aspergillus sp. SF5976 and their anti-inflammatory effects in LPS-stimulated RAW 264.7 and BV2 cells. Tetrahedron 2017, 73, 3905−3912.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.D.). *E-mail: [email protected] (Y.H.). ORCID

Yong Huang: 0000-0002-3163-1716 Funding

This research was supported in parts by NSFC grants 81473124 (to Y.H.); the Chinese Ministry of Education 111 Project B0803420 (Y.D.); and the Fundamental Research Funds for the Central Universities of Central South University 2017zzts869 (to J.S.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Center for Advanced Research in Central South University for the NMR experiment. We are also grateful to all the panelists in Hunan Engineering Research Center of Combinatorial Biosynthesis and Natural Product Drug Discovery.

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ABBREVIATIONS FBT, Fuzhuan brick tea; DAG, dihydroauroglaucin; IDAG, isodihydroauroglaucin; FG, flavoglaucin; TAG, tetrahydroauroglaucin

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REFERENCES

(1) Zheng, W. J.; Wan, X. C.; Bao, G. H. Brick dark tea: a review of the manufacture, chemical constituents and bioconversion of the major chemical components during fermentation. Phytochem. Rev. 2015, 14, 499−523. (2) Wang, Y.; Ho, C. T. Polyphenolic chemistry of tea and coffee: a century of progress. J. Agric. Food Chem. 2009, 57, 8109−8114. (3) Mukhtar, H.; Ahmad, N. Tea polyphenols: prevention of cancer and optimizing health. Am. J. Clin. Nutr. 2000, 71, 1698S−1702S. (4) Zhang, R.; Cheng, L.; Zhang, T.; Xu, T.; Li, M.; Yin, W.; Jiang, Q.; Yang, Y.; Hu, T. Brick tea consumption is a risk factor for dental caries and dental fluorosis among 12-year-old Tibetan children in Ganzi. Environ. Geochem. Health 2018, 1−13. (5) Cao, J.; Zhao, Y.; Liu, J. Brick tea consumption as the cause of dental fluorosis among children from Mongol, Kazak and Yugu populations in China. Food Chem. Toxicol. 1997, 35, 827−833. (6) Yamashita, Y.; Wang, L.; Tinshun, Z.; Nakamura, T.; Ashida, H. Fermented tea improves glucose intolerance in mice by enhancing translocation of glucose transporter 4 in skeletal muscle. J. Agric. Food Chem. 2012, 60, 11366−11371. (7) Li, Q.; Liu, Z.; Huang, J.; Luo, G.; Liang, Q.; Wang, D.; Ye, X.; Wu, C.; Wang, L.; Hu, J. Anti-obesity and hypolipidemic effects of Fuzhuan brick tea water extract in high-fat diet-induced obese rats. J. Sci. Food Agric. 2013, 93, 1310−1316. (8) Xiao, J. B.; Capanoglu, E.; Jassbi, A. R.; Miron, A. Advance on the flavonoid C-glycosides and health benefits. Crit. Rev. Food Sci. Nutr. 2016, 56, S29−S45. (9) Tian, Y. Z.; Liu, X.; Liu, W.; Wang, W. Y.; Long, Y. H.; Zhang, L.; Xu, Y.; Bao, G. H.; Wan, X. C.; Ling, T. J. A new anti-proliferative acylated flavonol glycoside from Fuzhuan brick-tea. Nat. Prod. Res. 2016, 30, 2637−2641. (10) Xu, A.; Wang, Y.; Wen, J.; Liu, P.; Liu, Z.; Li, Z. Fungal community associated with fermentation and storage of Fuzhuan brick-tea. Int. J. Food Microbiol. 2011, 146, 14−22. (11) Man, Y.; Hao, B. X.; Tian, H. X.; Li, S.; Wang, C. L. The optimization of method of plate culture count for Eurotium cristatum. Food Ferment. Technol. 2017, 53, 22−25 (In Chinese) . 6635

DOI: 10.1021/acsomega.9b00593 ACS Omega 2019, 4, 6630−6636

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(29) Gao, J.; Francisco, L.; Radwan, M. M.; Dale, O. R.; Husni, A. S.; Manly, S. P.; Shari, L.; Xiaoning, W.; Hill, R. A.; Dugan, F. M.; et al. Benzyl derivatives with in vitro binding affinity for human opioid and cannabinoid receptors from the fungus Eurotium repens. J. Nat. Prod. 2011, 74, 1636−1639. (30) Zhang, L.; Shao, W. F.; Yuan, L. F.; Tu, P. F.; Ma, Z. Z. Decreasing pro-inflammatory cytokine and reversing the immunosenescence with extracts of Pu-erh tea in senescence accelerated mouse (SAM). Food Chem. 2012, 135, 2222−2228. (31) Hou, X. L.; Tong, Q.; Wang, W. Q.; Shi, C. Y.; Xiong, W.; Chen, J.; Liu, X.; Fang, J. G. Suppression of inflammatory responses by dihydromyricetin, a flavonoid from ampelopsis grossedentata, via inhibiting the activation of NF-κB and MAPK signaling pathways. J. Nat. Prod. 2015, 78, 1689−1696. (32) Liu, Z. G.; Li, Z. L.; Bai, J.; Meng, D. L.; Li, N.; Pei, Y. H.; Zhao, F.; Hua, H. M. Anti-inflammatory diterpenoids from the roots of Euphorbia ebracteolata. J. Nat. Prod. 2014, 77, 792−799. (33) Li, M. M.; Su, X. Q.; Sun, J.; Gu, Y. F.; Huang, Z.; Zeng, K. W.; Zhang, Q.; Zhao, Y. F.; Ferreira, D.; Zjawiony, J. K.; et al. Antiinflammatory ursane- and oleanane-type triterpenoids from Vitex negundo var. cannabifolia. J. Nat. Prod. 2014, 77, 2248−2254. (34) Tinker, A. C.; Wallace, A. V. Selective inhibitors of inducible nitric oxide synthase: potential agents for the treatment of inflammatory diseases? Curr. Top. Med. Chem. 2006, 6, 77−92. (35) FitzGerald, G. A.; Patrono, C. The coxibs, selective inhibitors of cyclooxygenase-2. N. Engl. J. Med. 2001, 345, 433−442. (36) Kim, S. F.; Huri, D. A.; Snyder, S. H. Inducible nitric oxide synthase binds, S-nitrosylates, and activates cyclooxygenase-2. Science 2005, 310, 1966−1970.

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