A new p-terphenyl derivative from the insect-derived fungus Aspergillus candidus Bdf-2 and the synergistic effects of terphenyllin

A new p-terphenyl derivative 4″-deoxy-2′-methoxyterphenyllin (1), along with six known p-terphenyl derivatives (2–7), a known flavonoid derivative dechlorochlorflavonin (8) and a known fellutanine A (9), were isolated from the insect-derived strain of the fungus Aspergillus candidus Bdf-2, associated with Blaptica dubia. The structure of 1 was established by the analysis of the 1D and 2D NMR and HR-ESI-MS spectra. Compounds 1–9 were evaluated for antibacterial activities against Staphylococcus aureus ATCC29213, Escherichia coli ATCC25922 and Ralstonia solanacearum, and for antioxidant activities. Synergistic effects of compound 2 with the other compounds were also investigated. As a result, compound 6 displayed the best antibacterial activities in all single compound with MIC value of 32 µg/mL against S. aureus ATCC29213 and R. solanacearum, respectively. However, no antibacterial effect against E. coli ATCC25922 was detected from any single compound. The combination of 2 + 6 exhibited obvious synergistic effect against S. aureus ATCC29213 and the MIC value was 4 µg/mL. Compound 6 also showed the best antioxidant activity as a single compound with an IC50 value of 17.62 µg/mL. Combinations of 5 + 6, 2 + 4 + 5 and 2 + 4 + 5 + 6 displayed synergistic effect and their antioxidant activities were better than that of any single compound.


INTRODUCTION
Cockroaches (Blattaria) are significant omnivores and have diverse hindgut microbiota encompassing hundreds of microbial species (Tinker & Ottesen, 2016). Furthermore, due to its unique niche, cockroach is likely to be the habitat for unique microorganism.
The fungus Aspergillus candidus Bdf-2 was isolated from the abdomen of Blaptica dubia. Although the genus Aspergillus have been proven to be a large reservoir of p-terphenyl derivatives and other biologically active compounds, not many insect-derived fungal strains of the Aspergillus candidus have been described. We wondered whether cockroaches which can survive in a specific environment, were related to the symbiotic microorganisms living in their bodies. Based on promising screening results in search of producers of biologically active compounds, the insect-derived fungus Aspergillus candidus Bdf-2 was selected for further studies. Here we deal with the description, isolation and characterization of a new p-terphenyl derivative (1) together with other eight previously known compounds 2-9 ( Fig. 1). All compounds (1)(2)(3)(4)(5)(6)(7)(8)(9) were evaluated for their antibacterial and antioxidant activities. The details of isolation, structure determination and biological activities of these compounds are presented herein.

General experimental procedures
The UV spectra were scanned on a UV-2600 instrument (Shimadzu, Kyoto, Japan). IR spectra were recorded on a Shimadzu Affinity-1 instrument (Shimadzu, Kyoto, Japan). HRESIMS spectra were obtained on a Q-TOF mass spectrometer from Bruker maXis, with an electrospray ionization (ESI) interface (Bruker, Fremont, CA, USA). Standard 1D and 2D NMR spectra were recorded on a Bruker Avance-600 NMR spectrometer ( 1 H at 600 MHz and 13 C at 151 MHz) (Bruker, Fremont, CA, USA). Silica gel Qingdao Marine Chemical Inc.,Qingdao,China), and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) were used for column chromatography. Semi-preparative HPLC separations were carried out on a semi-preparative HPLC instrument equipped with a UC-3281 pump and a UC-3292S UV detector, using an XB-C 18 column (250 mm × 10 mm, 10 µm, Welch, Shanghai, China). Precoated silica gel GF-254 plates (Qingdao Marine Chemical Inc., Qingdao, China) were used for analytical TLC and Spots were visualized under UV light (254 or 365 nm).

Fungal material
The fungus Aspergillus candidus Bdf-2 ( Fig. S1) was isolated from the abdomen of Blaptica dubia which were purchased from Lianwanjia Dubia exclusive store in Qingdao on November 30, 2016. The isolate was identified as Aspergillus candidus by analysis of its morphological characteristics and the rDNA gene internal transcribed spacer (ITS) sequence (GenBank accession number MH681592) (Shan et al., 2019), as well as betatubulin (tub2) gene sequence (GenBank accession number MN533958), RNA polymerase II second largest subunit (RPB2) gene sequence (GenBank accession number MN533959) and large subunit ribosomal RNA gene sequence (GenBank accession number MN533960) (Figs. S2-S5). The fungus was stored on potato dextrose agar (PDA) slants at 4 • C in the College of Forestry and Landscape Architecture, South China Agricultural University, Guangzhou, China. The culture was also stored at China Center for Type Culture Collection (CCTCC) and the strain preservation number was CCTCC AF 2019013.

Fermentation, extraction, and isolation
The fungus Aspergillus candidus Bdf-2 was cultured on potato dextrose agar (PDA) (potato 200 g/L, dextrose 20 g/L, and agar 20 g/L) medium in Petri dishes at 28 • C for 5 days. Then, three agar plugs (0.5 × 0.5 cm) were inoculated in a 500 mL Erlenmeyer flask containing 200 mL of potato dextrose broth (PDB) (potato 200 g/L and dextrose 20 g/L) medium and incubated on a rotary shaker at 150 rpm and 28 • C for 5 days. The obtained liquid seeds were added into the sterilized rice culture medium (6.0 kg) in the incubators at 28 • C for 60 days before harvest. The leavening was extracted with ethyl acetate (EtOAc). Then, the EtOAc extracts were dissolved in 1 L methanol (MeOH) and extracted with petroleum ether at room temperature. The petroleum ether and MeOH extracts were dried using vacuum concentration to give crude extracts of 20.7 g and 113.5 g, respectively.

Antibacterial assay
One Gram-positive (S. aureus ATCC29213) and two Gram-negative (E. coli ATCC25922 and R. solanacearum) bacteria were selected for antibacterial activity assay. Amikacin was used as a positive control. The minimum inhibitory concentrations (MIC) of the compounds and positive control were determined in sterile 96-well plates using the microculture tetrazolium (MTT) assay as described previously (Ouyang et al., 2018;Zhao et al., 2010). The synergistic antibacterial effects of terphenyllin (2) with different compounds were determined by the same method. The compounds in different combinations were mixed together at the same concentration and volume.

Antioxidant assay
Radical scavenging assay was determined by a microplate spectrophotometric method based on the reduction of a methanol solution of DPPH according to our previous reports (Ouyang et al., 2018). Inhibition (%) of free radical (DPPH) in percent was determined as where A control is the absorbance of the control reaction containing all reagents except the test sample, and A sample is the absorbance of the test compounds. Tests were carried out in triplicate. BHT was used as a positive control. The IC 50 value was calculated using linear relation between the compound concentration and probability of the percentage of DPPH inhibition. The synergistic antioxidant effects of terphenyllin (2) with different compounds were determined by the same method. The compounds in different combinations were mixed together at the same concentration and volume as well as the synergistic antibacterial effects assay.

Purification and characterization
The solid fermentation product of Aspergillus candidus Bdf-2 was extracted with ethyl acetate (EtOAc) and the resulting extracts were partitioned into petroleum ether-and methanol (MeOH)-soluble fractions. The petroleum ether and MeOH fractions were further purified by conventional chromatographic techniques. One new p-terphenyl derivative (1) and eight known compounds (2-9) were structurally characterized (Fig. 1). Among them, compounds 1-7 were p-terphenyl derivatives. Interestingly, terphenyllin (2) (43.5 g/113.5 g) was the dominant compound in the EtOAc crude extracts of Aspergillus sp. Bdf-2.
The IR spectrum exhibited the absorption bands due to the presence of hydroxyl (3354 cm −1 ), and aromatic (1591 and 1520 cm −1 ) moieties. The 13 C-NMR (  H-4 ). The existence of a para-substituted benzene ring was also suggested by the 1 H NMR data of δ H 7.22 (2H, d, 8.4, H-2, 6) and 6.89 (2H, d, 8.4, H-3, 5). Therefore, an aromatic proton of pentasubstituted benzene at δ H 6.77 (1H, s, H-5 ) was identified based on the left 4 degrees of unsaturation and the 13 C NMR. The NMR spectral data of 1 indicated the presence of the p-terphenyl skeleton with one hydroxyl and three methoxy groups. The HMBC correlations (Fig. 2) of 2 -OCH 3 with C-2 , 3 -OCH 3 with C-3 , 6 -OCH 3 with C-6 , and H-5 with C-1 , C-3 , C-4 , and C-6 and the NOE between 3 -OCH 3 and 4 -OCH 3 , H-5 to 6 -OCH 3 suggested that the structure of the pentasubstituted benzene ring was 2 , 3 and 6 three methoxyl substituted. So the hydroxyl group was set at the para-substituted benzene ring unit. The HMBC signals of H-5 with C-1 , H-2 with C-4 , H-2 with C-1 established the structure of compound 1, and which was named 4 -dehydroxy-2 -methoxyterphenyllin.
The known compounds were identified according to their spectroscopic data ( 1 H NMR, 13 C NMR and HR-ESI-MS) as well as comparison with literature data. The clear HR-ESI-MS and NMR spectra of known compounds were also provided in Supplementary Materials to further confirm the structure (Figs. S15-S38). antioxidant activity with an IC 50 value of 17.62 µg/mL. Compounds 5 and 2 also displayed stronger antioxidant activity than BHT with the IC 50 value of 25.93 µg/mL and 39.03 µg/mL, respectively. Compound 4 displayed relatively weak antioxidant activity with an IC 50 value of 130.68 µg/mL. Other compounds indicated inactivity and the IC 50 values were larger than 200 µg/mL. Combinations of 5 + 6, 2 + 4 + 5 and 2 + 4 + 5 + 6 displayed synergistic effect and the antioxidant activities were better than the single compound. While combinations of 2 + 1, 2 + 3 and 2 + 4 showed minus effect and the IC 50 values were all larger than 200 µg/mL.

DISCUSSION
In our previous study, the EtOAc extract of Aspergillus candidus Bdf-2 from Blaptica dubia displayed the best antibacterial activity and excellent antioxidant activity among the isolated strains (Shan et al., 2019). Thus Aspergillus candidus Bdf-2 was selected for the further isolation and identification of the secondary metabolites. Based on the results presented here, we describe a new p-terphenyl derivative 4 -deoxy-2 -methoxyterphenyllin (1), along with eight known compounds were isolated from the insect-derived strain of the fungus Aspergillus candidus Bdf-2. Compounds 1-6 were all p-terphenyl derivatives and compound 7 was candidusin derivatives. Candidusin can be deduced to be a cyclization product of terphenyllin between C-6 and C-2 via an oxygen atom (Wang et al., 2017). Based on the antibacterial and antioxidant activities results (Tables 3 and 4), some structure-activity relationships among the p-terphenyl derivatives were found. Compound 6 displayed the strongest antibacterial and antioxidant activities and there were five hydroxyls in its structure. Compound 5 also showed good antibacterial and antioxidant activities and had four hydroxyls. Compounds 1 and 3 only had one hydroxyl in their structures and didn't show any bioactivity at the test concentrations. Above all, the hydroxyl was presumed as the active functional group in p-terphenyl derivatives and the strength of activity depending on the number of hydroxyl groups. In addition, compound 9 showed no antibacterial or antioxidant activity, which was consistent with the results reported in the literature (Zin et al., 2016;Kumla et al., 2017). Compound 2 which was the dominant compound didn't show strong individual bioactivity in this study. To date, there have been a lot of reports about the biological activities of single p-terphenyl derivatives, while no reports on the synergistic antibacterial and antioxidant activities. Hence, the synergistic effects of compound 2 with other compounds were also investigated. According to the results in Tables 3 and 4, parts of p-terphenyl derivatives displayed obvious synergistic effects. In this study, the compounds in different combinations were just mixed together at the same concentration and volume. Different proportions of the compounds may have a great impact on bioactivity. Therefore, the relative content of different compounds in metabolites of Aspergillus candidus Bdf-2 would deserve further study.

CONCLUSIONS
Aspergillus candidus Bdf-2 associated with Blaptica dubia was a strain rich in p-terphenyl derivatives and terphenyllin (2) was the dominant compound in the EtOAc crude extracts. Antibacterial and antioxidant activities of single compound were not as good as synergistic effect. Maybe this was a good explanation for why the crude extract had good bioactivities. We can still find some patterns in the antibacterial and antioxidant activities of monomer compounds. The hydroxyl was presumed as the active functional group in p-terphenyl derivatives and the strength of activity depending on the number of hydroxyl groups. Compound 6 displayed the best antibacterial and antioxidant activities, which may be a promising candidate for further studies in the development of a potent antibacterial and antioxidant.