p-Terphenyls From Aspergillus sp. GZWMJZ-055: Identification, Derivation, Antioxidant and α-Glycosidase Inhibitory Activities

One new (1) and fifteen known (2–16) p-terphenyls were isolated from a solid culture of the endophytic fungus Aspergillus sp. GZWMJZ-055 by adding the leaves of its host Eucommia ulmoides. Furthermore, nine p-terphenyls (17–25) were synthesized from the main compounds (5–7), among which derivatives 18, 19, 21, 22, and 25 are new p-terphenyls. Compounds 15 and 16 were also, respectively, synthesized from compounds 6 and 7 by oxidative cyclization of air in the presence of silica gel. These p-terphenyls especially those with 4,2′,4″-trihydroxy (4–7, 20, 21) or 4, 4″-dihydroxy-1,2,1′,2′-furan (15, 16) substituted nucleus, exhibited significant antioxidant and α-glucosidase inhibitory activities and lower cytotoxicity to caco-2 cells. The results indicated their potential use as lead compounds or dietary supplements for treating or preventing the diabetes.


INTRODUCTION
Diabetes is a chronic metabolic disease characterized by high blood sugar (HBS). Long-term HBS causes the damage to blood vessels and endangers various organs such as the heart, brain, kidneys, peripheral nerves, and eyes, and thus seriously affects the life quality of patients. Studies show that oxidative stress may be one of the important causes for diabetes and its complications. Too much reactive oxygen species in the body will increase the maturation disorder and apoptosis of pancreatic β-cells, leading to decrease insulin synthesis and secretion. Hyperglycemia and hyperlipidemia in diabetic patients can promote the production of active oxides, causing oxidative stress, then oxidative stress and hyperglycemia promote each other, leading to a vicious circle (Karunakaran and Park, 2013). At present, the treatment of type 2 diabetes is based on oral drugs, mainly containing metformin, α-glucosidase inhibitors, dipeptidyl peptidase IV inhibitors, and sodium-glucose cotransporter 2 inhibitors. Among them, α-glucosidase inhibitors can inhibit the degradation of polysaccharides to glucose and delay the absorption of glucose in the small intestine to reduce blood sugar. Such drugs can effectively reduce postprandial hyperglycemia without causing symptoms of hypoglycemia and are highly beneficial to patients who use carbohydrates as their main source of calories.
The α-glucosidase inhibitors, such as acarbose, miglitol, and voglibose currently used clinically are all microbial metabolites or their derivatives. Therefore, discovery of the new α-glucosidase inhibitors from microbial natural products (NPs) has unique advantages. p-Terphenyls, as an important kind of fungal NPs, its chemical investigation could be dated back to 1877 (Liu, 2006). At present, over 230 p-terphenyls have been isolated from fungi and lichens (Li et al., 2018). In addition, some p-terphenyl derivatives were also total synthesized (Yonezawa et al., 1998;Takahashi et al., 2017;Zhang et al., 2018). As reported, p-terphenyls had a broad spectrum of biological properties, such as cytotoxic (Wang et al., , 2020, antimicrobial (Intaraudom et al., 2017), and phosphodiesterase inhibitory (El-Elimat et al., 2013) activities, but the most interesting bioactivities were antioxidative (Kuhnert et al., 2015) and αglucosidase inhibitory activities . Furthermore, p-terphenyls can also be isolated from edible mushroom (Liu et al., 2004;Ma et al., 2014;Wang et al., 2014), indicating that this kind of compounds have low toxicity in the human body and are very suitable for the research of anti-diabetic drugs.

General Experimental Procedures
UV spectra were measured on a Waters 2487 dual λ absorbance detector. IR spectra were recorded on a Nicolet Nexus 470 spectrophotometer as KBr disks. 1 H, 13 C NMR and 2D NMR spectra were recorded on Bruker-600 MHz using TMS as an internal standard. ESIMS and HR-ESIMS analysis were carried out on Waters Xevo TQS and Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MS, respectively. Column chromatography was performed on silica gel (200-300 mesh; Qingdao Puke Parting Materials Co., Ltd., China), Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden), silica gel H, and plates precoated with silica gel GF254 (Qingdao Puke Parting Materials Co., Ltd., China), respectively. HPLC separation was performed on HITACHI Primaide with an ODS column (YMC-pack ODS-A, 10 mm × 250 mm, 5 µm, 4 mL/min). Synthetic compounds were also purified using a SepaBean machine equipped with SepaFlash columns (Santai Technologies Inc., China).

Fungal Material
The fungus Aspergillus sp. GZWMJZ-055 was isolated from the leaves of Eucommia ulmoides collected from Guiyang Medicinal Botanical Garden and was determined as Aspergillus sp. by the phylogenetic tree (Supplementary Figure 1) of the ITS sequence (GenBank No. KY038594). The strain was deposited in Guiyang laboratory in 20% glycerol at −80 • C.

Fermentation, Extraction and Isolation
The fungal strain GZWMJZ-055 was cultured on PDA at 28 • C for 3 days to prepare the seed culture. Spores were incubated at 28 • C for 2 days, a rotary shaker with shaking at 120 rpm in a 500 mL cylindrical flask containing 150 mL seed medium. The seed medium (5 mL) was added to the above rice fermentation medium in a 1000-mL Erlenmeyer flask. Totally, 100 Erlenmeyer flasks were incubated at room temperature (rt) under static conditions for 30 days. The cultures were then extracted by ethyl acetate (EtOAc) (500 mL for each) three times and the combined EtOAc extracts were dried in vacuo until constant weigh to yield 423.5 g of EtOAc extract.

Oxygen Radical Absorbance Capacity (ORAC) Assay
The anti-oxidative activity of compounds was evaluated by ORAC assay (Huang et al., 2002) that was carried out mainly by using 2,2 -azobis(2-amidinopropane) dihydrochloride (AAPH, 153.0 µM), fluorescein (FL, 81.6 nM), testing compounds, and trolox as a positive control, all of which were dissolved in phosphate buffer solution (PBS, 75 mM, pH 7.4). The concentrations were 6.25 µM for compounds 4-7 and trolox, 12.5 µM for compounds 1-3, 8-16 and trolox, and 25.0 µM for compounds 17-25 and trolox, respectively (Supplementary Figure 36). In short, each 25 µL of testing compounds, blank (PBS), negative (PBS) and trolox, and 150 µL of FL were added in each well and incubated at 37 • C for 10 min. Each 25 µL of AAPH was then added to the testing compounds, blank and trolox groups, and 25 µL of PBS was added to the negative group. Fluorescence intensity of each well was measured one time every 1 min for 90 cycles using a Fluoroskan Ascent FL plate-reader (Thermo Scientific Varioskan LUX) at excitation of λ 485 nm and emission of λ 530 nm. The relative fluorescence intensity f was equaled to the ratio of the absolute fluorescence reading to the initial fluorescence reading, and the net area under curve (AUC) was obtained by subtracting the AUC of the blank from that of the compound. The AUC was calculated as 0.5 + f 1 +... f i +... + f 89 + 0.5 × f 90 , in which f i means the ratio of fluorescence reading at time i to the initial fluorescence reading. The final ORAC values were calculated as micromole of trolox equivalents per micromole of the compound (µmole TE/µmole) by using a regression equation between the trolox concentration and the net area under the FL decay curve. That is, the relative ORAC value = (AUC compound -AUC blank )/(AUC trolox -AUC blank ).

DPPH Radical-Scavenging Assay
The anti-oxidative activity of compounds was also evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay (Wang et al., 2007). The experiment was divided into the following five groups, blank (methanol, MeOH), sample (mix compound and DPPH solution), background (pure compound solution), negative (pure DPPH solution), and positive [mix vitamin C (VC) and DPPH solution] controls. DPPH (0.15 mM), compounds (1-100 µM), and VC (1-100 µM) that was regarded as a compound sample in the following procedures all were dissolved in MeOH. Each 160 µL of MeOH was placed in negative control and blank groups, while each 160 µL of testing compounds or VC was placed in sample and background groups. Then, MeOH (each 40 µL) was, respectively, added to blank and background groups, while 40 µL of DPPH was, respectively, added to negative, positive, and sample controls. After 30-min incubation in the dark at rt, the decrease in DPPH radical concentration was monitored by measuring the absorbance at λ 517 nm with a microplate reader (Multiskan Spectrum, Thermo Scientific Varioskan LUX). The DPPH radical-scavenging rate was calculated as: The IC 50 (half maximal inhibitory concentration) values of compounds and VC were calculated by SPSS (Statistical Package for the Social Sciences) software from the radical-scavenging rates at the final concentrations of 100, 50, 10, 5, and 1 µ M.

α-Glucosidase Inhibitions in Saccharomyces cerevisiae
The inhibitions of the compounds against α-glucosidase from Saccharomyces cerevisiae were assayed by reported method (Xu et al., 2018). The testing compounds were dissolved in dimethyl sulfoxide (DMSO) to obtain stock solution (10 mM) and then diluted into the concentrations by PBS (pH 6.8), while α-glucosidase (2.0 U/mL, Sigma), 4-nitrophenyl-α-Dglucopyranoside (PNPG, 2.5 mM, Macklin), Na 2 CO 3 (0.2 M), and acarbose (2.5 mg/mL, Sigma) were directly dissolved in PBS. 20 µL of the compound solution and acarbose were, respectively, mixed in a 96-well microplate with 20 µL of α-glucosidase and 60 µL of PBS as the drug and positive groups, while the pure PBS solution was used as the blank group. After incubation for 15 min at 37 • C, 20 µL of PNPG solution was added to each well of testing groups and further incubated at 37 • C for 30 min. Finally, 80 µL of Na 2 CO 3 solution was added to each well to stop the reaction and the absorbance was measured by a microplate reader (Multiskan Spectrum, Thermo Scientific Varioskan LUX) at λ 405 nm. The inhibitory rate (%) was calculated as [1 − (A drug /A blank )] × 100%. The IC 50 values were calculated by SPSS software from the drug inhibitory rates at the final concentrations of 500, 250, 50, 25, 5, and 1 µM ( Table 3).

α-Glucosidase Inhibitions in Caco-2 Cell Line
The α-glucosidase inhibition assay was also carried out in caco-2 cell line (Hansawasdi and Kawabata, 2006). Caco-2 cells at logarithmic growth stage were inoculated in a 6-well plate with an inoculation density of 4000/cm 2 and cultured in an incubator with 5% CO 2 at 37 • C in a Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1% non-essential amino acid, 1% penicillin/streptomycin, 1% L-glutamine, and 0.25 mg plasmocin. The DMEM medium was changed one time every 2 days for 24 days. Then, the culture medium was removed, and the cell surface was washed three times by a PBS solution (pH 7.4) at 37 • C. 1.0 mL of sucrose/maltose (both 28 mM) PBS solution was added to the control well, 1 mL of PBS was added to the blank well, 0.2 mL of compounds or acarbose (positive control) with different concentration and 0.8 mL of above sucrose/maltose solution were added to the drug well. The final concentration gradients of compounds and acarbose were 1.0, 0.3, 0.1, 0.03, 0.01 µM and 10000, 3000, 1000, 300, 100 µg/mL, respectively. The obtained solutions for the enzymatic hydrolysis reactions of sucrose and maltose were incubated at 37 • C for 60 min. After terminating the reactions in an ice bath for 10 min, 10 µL of the reaction mixture was added into 1 mL of the glucose kit (Nanjing Jiancheng Bioengineering Institute Co., Ltd.) and maintain 10 min at 37 • C. The α-glucosidase inhibitory activity of the compounds was then determined by measuring the glucose content in the reaction solution (pipette 100 µL reaction solution into 96-well plate) via the absorbance at λ 505 nm with a microplate reader (BioTek Synergy H1, BioTek, VT, United States). The α-glucosidase inhibitory rate (%) was calculated as [1 − (A drug -A blank )/ (A control -A blank )] × 100%. The IC 50 values were calculated as showed in Table 3 by SPSS software.
The cytotoxic effects on the coca-2 cells were evaluated by the CTG assay (Elisia and Kitts, 2008;Wang et al., 2019). Briefly, coca-2 cells were seeded in 96-well plates at a density of 2 × 10 3 cells/well and treated with the final concentration of 1.0 µM of the compounds. After 72 h incubation, 100 µL of CTG solution (Promega) was added into each well. The luminescence value was tested by using a microplate reader (BioTek Synergy H1) after staying at rt for 10 min.

Metabolic Regulation of the Fungus
After adding the leaves of Eucommia ulmoides in the rice medium, both the production and the α-glucosidase inhibitory activity of the EtOAc extracts of the solid-state fermentation increased significantly from 5.3 to 9.5 g/kg and from the IC 50 value of 15.0 to 2.0 µg/mL, respectively. The original p-terphenyl products 5-7, 10, and 12 in the rice medium also largely increased by adding the leaves of E. ulmoides. In addition, the number of the p-terphenyl-type chromophores also increased significantly. For example, p-terphenyls 1-4, 8, 9, 11, and 13-16 were newly produced after adding the leaves of E. ulmoides (Figure 1). The results indicated that the content and diversity of the microbial natural products could increase highly by adding the host materials to the culture media of the microorganisms via the chemical microbehost interaction.

Synthesis of p-Terphenyls 17-25
As shown in Scheme 1, compounds 17-19 were synthesized from compounds 5-7 by demethylation reaction using BBr 3 , which were further transformed to compounds 20-22 by oxidation of air in the system of silica gel and MeOH. The acetylation of compounds 5-7 provided compounds 23-25 by Ac 2 O/DMAP. It is interesting that compounds 15 and 16 could be synthesized from compounds 6 and 7 by an oxidative dehydrocyclization of air in the system of silica gel and MeOH. But compound 5 cannot undergo the same reaction to form the corresponding 2,2 -oxygen bridged p-terphenyl derivative, indicating that the oxidative cyclization might be carried out by a radical process. That is, compounds 6 and 7 formed a radical intermediate a which underwent an intramolecular cyclization to generate the keto intermediate b in the presence of SiO 2 and O 2 . Compounds 15 and 16 were then yielded by a keto-enol tautomerization of the intermediate b in the SiO 2 and MeOH (Scheme 2). To confirm the effect of silica gel and O 2 , the reaction of compound 6 was carried out in the four conditions, i.e., O 2 , silica gel/argon, silica gel/air, and silica gel/O 2 . The results showed that compound 6 could not be converted to compound 15 without silica gel. Both the reaction and conversion rates increased in the order of O 2 , silica gel/argon, silica gel/air, and silica gel/O 2 (Supplementary Figure 37). And the silica gel acted as a catalyst to accelerate the tautomerization between keto and enol. The fact that a little compound 15 was also formed in the silica gel/argon system could be explained from the air adsorbed in the silica gel.
Frontiers in Microbiology | www.frontiersin.org that 4-and 2 -hydroxys are very important active sites of the p-terphenyls. As the disappearance of the two hydroxys, changing to hydrogens, methoxyls or acetoxyls, for example, the DPPH radical-scavenging activity of p-terphenyls was greatly reduced. However, the activity is still maintained when the 2 -hydroxy formed a furan ring with C-6.

CONCLUSION
There is a mutually beneficial relationship between endophytes and their host plants. Adding the host plants to the culture medium of endophytes could enhanced the metabolic potential of the endophytic strains and thus enriched the chemodiversity of the microbial natural products. p-Terphenyls, especially those 4,2 ,4 -trihydroxy or 4,4 -dihydroxy-1,2,1 ,2 -furan substituted ones, have a stronger antioxidant activity, α-glucosidase inhibitory activity and lower cytotoxicity, implying their potential use in the fight against diabetes as the drug leads or dietary supplements.

DATA AVAILABILITY STATEMENT
Publicly available datasets were analyzed in this study. This data can be found here: GenBank No. KY038594.

AUTHOR CONTRIBUTIONS
YX isolated the fungus and compounds, performed the structure elucidation, and assayed part of the bioactivity. YW synthesized the compounds. DW assayed part of the bioactivity. WH did fermentation and extraction. LW directed the implementation of the study and prepared the manuscript. WZ designed the study and revised the manuscript. All authors contributed to the article and approved the submitted version.