New Drimane Sesquiterpenes and Polyketides from Marine-Derived Fungus Penicillium sp. TW58-16 and Their Anti-Inflammatory and α-Glucosidase Inhibitory Effects

Marine fungi-derived natural products represent an excellent reservoir for the discovery of novel lead compounds with biological activities. Here, we report the identification of two new drimane sesquiterpenes (1 and 2) and six new polyketides (3–8), together with 10 known compounds (9–18), from a marine-derived fungus Penicillium sp. TW58-16. The planar structures of these compounds were elucidated by extensive 1D and 2D NMR, which was supported by HR-ESI-MS data. The absolute configurations of these compounds were determined by experimental and calculated electronic circular dichroism (ECD), and their optical rotations compared with those reported. Evaluation of the anti-inflammatory activity of compounds 1–18 revealed that compound 5 significantly inhibited the release of nitric oxide (NO) induced by lipopolysaccharide (LPS) in RAW264.7 cells, correlating with the inhibition of expression of inducible nitric oxide synthase (iNOS). In addition, we revealed that compounds 1, 3–6, 14, 16, and 18 showed strong α-glucosidase inhibitory effects with inhibition rates of 35.4%, 73.2%, 55.6%, 74.4%, 32.0%, 36.9%, 88.0%, and 91.1%, respectively, which were comparable with or even better than that of the positive control, acarbose. Together, our results illustrate the potential of discovering new marine-based therapeutic agents against inflammation and diabetes mellitus.


Introduction
Marine-derived natural products (MNPs) represent a new and promising source of therapeutic agents [1]. As the discovery of new natural products from terrestrial sources is shrinking, large quantities of MNPs have been reported, especially those from marine microorganisms. The extreme marine environment, including high salinity, intensely high pressure, absence of sun light, and deficiency of nutrients, endows marine microorganisms with unique biodiversity and metabolic pathways, leading to the production of structurally unique and biologically diverse MNPs [2]. Recently, the upward trend in the discovery of new MNPs from marine microorganisms continues unabated. For instance, they represented around 60% of all newly reported MNPs in 2017, indicating that marine microorganisms hold great potential in innovative compounds discovery [3]. Polyketides and terpenes, two structurally diverse groups of MNPs, are major secondary metabolites and terpenes, two structurally diverse groups of MNPs, are major secondary metabolites found in marine microorganisms. They have attracted significant attention due to their diverse biological functions [4,5].
Compound 3 was isolated as a brown solid powder. It has the molecular formula of C 13  in the 1 H-1 H COSY spectrum and HMBC correlation from H-9 to C-7, a 4,6-heptadien-2-ol fragment was ascertained. Furthermore, according to the HMBC correlations of H-7 with C-4 and C-5 and of H-6/H-4 with C-7, the 4, 6-heptadien-2-ol fragment was attached at C-5 of the 1,3,5-trisubstituted aromatic ring ( Figure 2). Thus, the planar structure of compound 3 was deduced.
Compound 5 was obtained as a brown solid powder. The HR-ESI-MS showed a quasimolecular ion at m/z 249.1133 [M + H] + (calcd. 249.1127), indicating a molecular formula of C 14 H 16 O 4 , one oxygen atom less than 4, and accounting for seven degrees of unsaturation. The 1 H and 13 C NMR spectra of 5 showed great similarities with those of 4 and the main difference laid in the 1 H and 13 C resonances of C-12. The upfield shift of 1 H and 13 C resonances of C-12 from δ H 3.77 (1H, m, H-12)/δ C 68.6 (C-12) in 4 to δ H 1.41 (2H, m, H 2 -12)/δ C 23.6 (C-12) in 5 indicated that the oxymethine at C-12 in 4 was replaced by a methylene in 5. The planar structure of 5 was confirmed by the 1 H-1 H COSY and HMBC correlations ( Figure 2). In addition, the double bonds of ∆ 7,8 and ∆ 9,10 were determined as Z and E configurations, respectively, by the coupling constants of 7,8 J at 10.4 Hz and 9,10 J at 14.8 Hz. Therefore, the structure of 5 was elucidated as 4-carboxy-5-((1Z,3E)-1,3-heptadien-1-yl)-1,3-benzenediol.

Bioactivities
The inhibitory effects of compounds 1-18 on NO production induced by LPS in murine macrophage RAW264.7 cells were evaluated. Dexamethasone (DXM) and curcumin (Cur) were used as positive controls. Our results show that compounds 5-7 and 16, especially 5, significantly inhibited NO production induced by LPS ( Figure 5A). Meanwhile, these compounds did not show obvious cytotoxicity toward RAW264.7 cells at 50 µM ( Figure 5B). Mechanistic studies showed that compound 5 significantly inhibited the expression of iNOS ( Figure 6B), the gene that is responsible for the production of NO. IN addition, compounds 1, 9 and 11 displayed a moderate inhibitory effects on the expression of iNOS ( Figure 6A). In contrast, all of the tested compounds did not obviously inhibit the expression of COX-2 at the indicated concentration, indicating specific effects on iNOS expression.  We then explored whether these compounds could also inhibit diabetes. To this end, we carried out an in vitro hypoglycemic assay to determine the effects of compounds 1-18 on the α-glucosidase activity. Acarbose was used as a positive control. The results show that compounds 1, 3-6, 14, 16, and 18 exhibited strong α-glucosidase inhibitory activities with inhibition rates of 35.4%, 73.2%, 55.6%, 74.4%, 32.0%, 36.9%, 88.0%, and 91.1%, respectively, which were comparable with or better than that of acarbose (Table 1). Preliminary structure-activity relationship (SAR) analysis revealed that substituents at C-13 of drimane sesquiterpenes may be crucial for their α-glucosidase inhibitory effects since compound 1 exhibited stronger activity than 2. In addition, the lactone in C-15 and C-11 seems to unfavour the α-glucosidase inhibitory activity since compound 9 exhibited poor activity compared with 1 even though they shared the same substituent at C-13. Further, the strong α-glucosidase inhibitory activities of polyketides 3-5, 16 and 18 indicate the necessity to further study the anti-diabetic activities of these compounds. Values are expressed as Mean ± SEM, n = 3; Acarbose used as positive control.

Discussion
Polyketides are a large family of natural products that are derived from acetate building blocks [22]. Due to their diverse activities, especially antibiotic, anti-tumor, immunosuppressive, etc., polyketides such as doxorubicin, erythromycin A, and rapamycin, have attracted much attention and been applied in the clinic [22][23][24][25]. In this study, we report the isolation and characterization of 11 polyketides, including six new compounds (3)(4)(5)(6)(7)(8), from the marine-derived fungus Penicillium sp. TW58-16. We first presented data to show that these compounds inhibited inflammation as they suppressed LPS-stimulated NO production in macrophages. Consistently, compounds 5-7 and 16, and 5 in particular, greatly inhibited the expression of iNOS, the enzyme that produces NO. We expect to perform structure modification and further explore the anti-inflammatory effects of these compounds.
In addition, scattered reports showed α-glucosidase inhibitory activities of polyketides [26][27][28][29], indicating the potential of this compound class in diabetes treatment. Consistent with previous reports, our in vitro pharmacological assay showed that compounds 3-6, 14, 16, and 18 exhibited potent α-glucosidase inhibitory activities at levels that were comparable with or better than acarbose, a known α-glucosidase inhibitor. Among them, the new polyketides 3-6 share structural similarities, but display distinct α-glucosidase inhibitory activities. Structure-activity relationship (SAR) analysis revealed that the length of the side chain or the introduction of carboxylic acid in the side chain may have a crucial effect on their activities, as compounds 3-5 exhibited significantly stronger α-glucosidase inhibitory effects than compound 6. To our surprise, compound 16 baring 3,4-dihydroxyl groups in the aromatic ring and an acetic acid side chain also exhibited significant α-glucosidase inhibitory activity, suggesting the favour of ortho-dihydroxyl groups for the α-glucosidase inhibitory activity. These were the first report to show a potent α-glucosidase inhibitory activity of the polyketide ε-caprolactone derivative 18.
Drimane sesquiterpenes are widely distributed in metabolites of higher plants and terrestrial and marine fungi. They also demonstrate a broad range of bioactivities, including antifungi and antibacteria, cytotoxicity, piscicidal and molluscicidal activity, etc. [30]. Here we found the new drimane sesquiterpene 1 and the known analogue 3 demonstrated strong anti-inflammation and α-glucosidase inhibitory activities. SAR analysis indicated that carboxy substituent in C-13 and lactone in C-15 and C-11 unfavour for the α-glucosidase inhibitory activity of these compounds.
To sum up, these discoveries of new α-glucosidase inhibitors may promote the study and development of new derivatives of this compound class for the treatment of inflammation and diabetes mellitus.

Fungal Material
The fungus strain TW58-16 was isolated from hydrothermal vent sediment, collected from Kueishantao, Taiwan, and identified as Penicillium sp. according to the morphological characteristics and the internal transcribed spacer (ITS) sequence (MZ558028), which is 100.00% similar to Penicillium citrioviride isolate D5 (GU388431.1). The strain was deposited at Ocean College, Zhejiang University, Zhejiang, China.

Cell Culture
The murine macrophage RAW 264.7 cells were from the American Type Culture Collection (ATCC, USA) and cultured in DMEM supplemented with 10% FBS at 37 • C in a 98% humidified incubator with 5% CO 2 . The cells in the logarithmic phase were used for the following experiments.

Measurement of Cell Viability
Cell viability was assessed by the MTT assay. In brief, cells with a density of 1.5 × 10 5 cells/mL were seeded in each well of a 96-well culture plate (100 µL) and cultured overnight. Cell-free wells were set as blank controls. After attachment, the cells were co-treated with tested compounds (50 µM) and LPS (100 ng/mL) for 24 h. Then, the culture medium was replaced with DMEM full media containing 0.5 mg/mL MTT (100 µL) and incubated for another 2 h. After aspiration of the culture medium, DMSO (150 µL) was added to dissolve the formazan. Finally, the optical densities (OD) were measured at 490 nm.

NO Inhibition Assay
The NO concentrations were measured by the Griess method. After being cultured in 96-well plates overnight, cells (1.5 × 10 5 cells/well) were co-treated with tested compounds and LPS (100 ng/mL) for 24 h. Finally, the nitrite concentration in the culture supernatants was taken by the NO assay kit.

Western Blotting Assay
After treatment with the tested compounds, the cells were collected and centrifuged. The cell pellets were lysed by lysis buffer containing 1 mM phenylmethylsulfonyl fluoride on ice for 30 min, and the cells were sonicated on an ice bath. Total proteins were obtained by centrifuging the cell suspension. Protein concentrations were measured by BCA protein assay kit. Next, equal amount of proteins from each group were separated by 6-10% SDS-PAGE, transferred onto polyvinylidenedifluoride (PVDF) membranes, and blocked with 5% skim milk in TBST solution for 1 h at room temperature. Finally, the membranes were sequentially incubated with primary and secondary antibodies, followed by chemiluminescence detection.

α-Glucosidase Inhibitory Assay
The α-glucosidase inhibitory assay was conducted as per a previous report [34]. Acarbose was used as the positive control. Firstly, 25 µL of 1.6 mM samples and 50 µL of 0.2 U/mL α-glucosidase were mixed in 96-well plates. After preincubation at 37 • C for 10 min, 25 µL of 5 mM p-NPG was added to each well. Then, the reaction mixture was incubated at 37 • C for 5 min. Finally, the reaction was stopped by adding 100 µL of 0.1 M Na 2 CO 3 . The optical density was measured at 405 nm using a Synergy HT microplate reader. The α-glucosidase inhibition percentage (I%) was calculated using the following equation: I% = [(∆Abs control − ∆Abs sample )/∆Abs control ] × 100.

Statistical Analysis
GraphPad Prism software version 5 (GraphPad Software, Inc., San Diego, CA, USA) were used to perform the statistical analyses. Each experiment was conducted in triplicate, and the final data were expressed as mean ± standard error of mean (SEM). Multiple comparisons were carried out by one-way ANOVA, followed by Tukey's test. p < 0.05 was considered statistically significant.