Lipid-Lowering Polyketides from the Fungus Penicillium Steckii HDN13-279

Seven new polyketides, named tanzawaic acids R–X (1–6, 11), along with seven known analogues (7–10 and 12–14), were isolated from Penicillium steckii HDN13-279. Their structures, including the absolute configurations, were elucidated by NMR, MS, X-ray diffraction, circular dichroism (CD) analyses and chemical derivatization. Five compounds (2, 3, 6, 10 and 12) significantly decreased the oleic acid (OA)-elicited lipid accumulation in HepG2 liver cells at the concentration of 10 μM, among which, four compounds (3, 6, 10 and 12) significantly decreased intracellular total cholesterol (TC) levels and three Compounds (3, 6, and 10) significantly decreased intracellular triglyceride (TG) levels. Moreover, the TG-lowering capacities of compounds 6 and 10 were comparable with those of simvastatin, with the TG levels being nearly equal to blank control. This is the first report on the lipid-lowering activity of tanzawaic acid derivatives.


Introduction
Metabolic syndrome (MetS), including obesity, insulin resistance (IR), dyslipidemia and hypertension, have long been a worldwide problem [1][2][3]. Dyslipidemia, as one of the most common causes of MetS, can further result in atherosclerosis, myocardial infarction and cerebrovascular diseases, seriously threatening human life [4][5][6]. Due to the current undesirable side-effects of lipid-regulating drugs, it is urgent to find new classes of bioactive compounds with lipid-lowering capacity and a safer profile [7][8][9][10]. Famous drugs for the treatment of dyslipidemia are lovastatin analogues, reported to be produced by various fungal species, including Aspergillus spp., Penicillium citrinum, Pleurotus spp., and Monascus ruber, thus, suggesting fungi as promising sources for the discovery of new drug leads against MetS [11,12].

Results and Discussion
The fungal strain P. steckii HDN13-279 was fermented (45 L) under shaking conditions at 28 °C for 9 days. The EtOAc extract (40 g) was fractionated by silica gel vacuum liquid chromatography (VLC), C-18 ODS column chromatography, Sephadex LH-20 column chromatography, ODS MPLC, and finally HPLC to yield compounds 1-14 ( Figure 1). Tanzawaic acid R (1) was obtained as a pale yellow oil with the molecular formula C18H26O4 analyzed by HRESIMS. The 1D NMR data (Table 1) indicated the presence of two methyls, three methylenes (with one oxygenized), 11 methines (including five sp 2 methines and one oxymethine), and two non-protonated carbons (including one carbonyl). The planar structure of compound 1 was proved to be the same as tanzawaic acid H [25], supported by the similar 1D NMR and 2D NMR data ( Figure 2   The relative configuration of 1 was assigned by NOESY spectroscopic data. The NOESY correlations from H-5 to H-7/H 3 -18, H-6 to H-8/H-12 indicated a trans fusion of the rings and placed H-7, Me-18 and the penta-2,4-dienoic acid moiety on the same side of the decalin ring, while H-6, H-8, H-12 on the other side. In addition, the NOESY correlations between H-9a (δ H 1.69) and H-8/H-11a (δ H 1.66), H-9b (δ H 0.74) and H-7, H-10 and H-8/H-12, H-11a and H-12/H-13, H-12 and H-13 indicate that the hydroxymethyl at C-10 and the hydroxyl at C-13 were located at the same side as the penta-2,4-dienoic acid moiety ( Figure 3). Thus, the NOESY data suggested that Compound 1 was a C-13 epimer of tanzawaic acid H. In a previous report [25], the absolute configuration of tanzawaic acid H was deduced based on the proposed biogenetic pathway. To make a solid evidence, we determined the absolute configuration of 1 by X-ray diffraction following chemical derivatization. Firstly, Compound 1 was esterified to generate the methyl ester (1a) and then the single crystal (CCDC 1537543) of 1a was successfully obtained (Figure 4) with Flack parameter = 0.02 (11). Consequently, the absolute configuration of 1 was elucidated to be 6R, 7R, 8R, 10S, 12S, 13S ( Figure 1).
Tanzawaic acid S (2) was isolated as a pale yellow oil. The molecular formula of 2 was established as C 19 (Table 1), and the only difference was the existence of signal for an additional methoxy (δ H 3.33). Further analysis of the 2D NMR spectra indicated the additional methoxy was attached at C-13 (δ H 76.3) in 2 ( Figure 2). The relative configuration of 2 cannot be completely assigned by NOESY data as the signals for H-7, H-8 and H-12 were overlapped. We further resorted to chemical derivatization. Both Compounds 1 and 2 were successfully transformed into Compound 1b when methylated by NaH and CH 3 I in DMF solution, which suggested they share the same relative configuration. Moreover, 1 and 2 showed almost the same CD curve ( Figure 5), further confirming their same absolute configuration.             Tanzawaic acids T (3) and U (4) were obtained as pale yellow oil, and had the same molecular formula (C 19 (Table 2) suggested that they possessed similar tanzawaic acid scaffold to Compound 2. Further analysis of the 2D NMR spectra indicated that Compounds 3 and 4 shared the same planar structure (Figure 2), and the structural difference between 3 (or 4) and 2 was that the hydroxymethyl in 2 was replaced by a methyl in 3 and 4 (δ H 0.89 in 3, δ H 0.88 in 4). The NOESY spectroscopic data of 3 and 4 indicated that they are C-13 epimers ( Figure 3). The NOESY correlations between H-7 and H-13 suggested the β orientation of H-13 in Compound 3. The NOESY correlations between H-13 and H-11a (δ H 1.57), H-11a and H-10, and between H-11b (δ H 1.16) and H 3 -17/13-OCH 3 in Compound 4 indicated an α oriented H-13 in 4 ( Figure 3). Although the relative configuration of C-13 in Compound 3 was different from 1, the CD curves of them are almost identical ( Figure 5), indicating that the cotton effects, especially those at 270 nm, were predominated by the (2E, 4E)-penta-2,4-dienoic acid moiety. Consequently, the absolute configurations of 3 and 4 were deduced as 6R, 7R, 8R, 10S, 12S, 13R and 6R, 7R, 8R, 10S, 12S, 13S, respectively.  (Table 3) indicated the presence of three methyls, two methylene, ten methines (including six sp 2 methines), and three non-protonated carbons (including one carbonyl and two oxygenated carbons). These data are similar to those of the isolated known compound, tanzawaic acid C (7) [19], except for the appearance of an additional oxygenated non-protonated carbon and the absence of a methine in 5, indicating that Compound 5 is a hydroxylated analogue of 7. Analysis of the 2D NMR spectra located the additional hydroxyl group on C-10 ( Figure 2), and the methyl group on C-10 in 5 oriented to the same side as in Compound 7 according to the NOESY correlation between H 3 -17 and H 3 -18 ( Figure 3). The absolute configuration of 5 was determined by comparing CD with that of 7 whose absolute configuration was determined using X-ray diffraction with Flack parameter = 0.06 (9) in this work ( Figure 4). Their identical CD curve indicated the same absolute configuration ( Figure 5).
Tanzawaic acid W (6) was isolated as a pale yellow oil, whose molecular formula was established as identical to that of compound 5 based on the HRESIMS ions detected at m/z 305.1752 [M − H] − . The 1D and 2D NMR spectra indicated that the difference to 5 was the lack of the hydroxyl group at C-10 and its presence at C-15, and the methyl on C-15 faced to the same orientation to H-7 according to the NOESY correlations between H-7 and H 3 -16. Compound 6 also had similar CD curve with 7, which indicated the same absolute configuration ( Figure 5).
Tanzawaic acid X (11) was isolated as pale yellow oil with the molecular formula of C 18 (Table 3) indicated the presence of two methyls, three methylenes (with one oxygenated), eight methines (including six sp 2 ones), and five non-protonated carbons (including one carbonyl). Further analysis of the 2D NMR spectra indicated that the planar structure 11 was similar to tanzawaic acid A (13) [19] and the only difference was the methyl at C-10 in 13 was replaced by a hydroxymethyl. The relative configuration of 11 was determined by NOESY correlations between H-8 (δ H 3.26) and H-10 (δ H 1.85), which suggested the cofacial of H-8 and H-10. Moreover, the coincident CD curves of 11 and 13 indicated the 8R, 9S absolute configuration of 11 ( Figure 5). No. The seven known Compounds 7-10 and 12-14 were identified (Figure 1) as tanzawaic acid C (7), tanzawaic acid B (8), tanzawaic acid M (9), tanzawaic acid E (10), arohynapene B (12), tanzawaic acid A (13) and tanzawaic acid D (14) by comparing NMR data and optical rotation with those reported in the literature [15,[19][20][21][22]26]. As determining the absolute configuration of tanzawaic acid derivatives always be a challenging work, the stereochemistry of Compounds 7-9, 12 and 14 were not confirmed by solid evidence in literature reports. Herein, we present the determination of the absolute configurations assisted by X-ray differentiation, CD data and chemical transformation. The absolute configurations of Compounds 7 and 8 were determined under X-ray (CCDC numbers 1537544 and 1537542) with the Flack parameters = 0.06 (9) and 0.07 (10), respectively (Figure 4). The absolute configurations of Compounds 9 and 14 were determined by comparing CD spectra with Compounds 8 and 13, respectively ( Figure 5). The absolute configuration of Compound 12 was assigned by chemical transformation. We found the Pd/C reduction products (established as 12a) of Compounds 12 and 13 showed the same 1D NMR data and similar optical rotations ([α] 20 D −7.78 (c 0.042, MeOH) and [α] 20 D −6.09 (c 0.042, MeOH), respectively), which suggested Compounds 12 and 13 had the same relative and absolute configurations, which is also in accord with the same CD curves of 12 and 13 ( Figure 5).
All the compounds were evaluated for their cytotoxicity (on HL-60, HCT-116, K562, Hela and A549 cell lines), but none of them presented a cytotoxic effect at 30 µM. The antiviral (influenza A H1N1 virus) and NF-κB inhibitory activities were also evaluated, with no activity detected under the concentration of 30 µM. In light of the structural similarity with lovastatin, with the exception of Compound 9 (limited quantity), all the compounds were evaluated for their lowering effects against oleic acid (OA)-elicited lipid accumulation in HepG2 liver cells. Five compounds (2, 3, 6, 10 and 12) significantly decreased the lipid accumulation elicited by OA, determined by oil-red O staining, at the concentration of 10 µM. Compounds 6 and 12 showed comparable efficiency with simvastatin ( Figure 6). Additionally, four compounds (3, 6, 10 and 12) could significantly decreased intracellular total cholesterol (TC) levels and three compounds (3, 6, and 10) significantly decreased intracellular triglyceride (TG) levels ( Figure 7). It's worth mentioning that the TG-lowering efficiency of Compounds 6 and 10 were comparable with simvastatin and the TG levels were nearly equal to blank control (p > 0.05) ( Figure 7B). , respectively), which suggested compounds 12 and 13 had the same relative and absolute configurations, which is also in accord with the same CD curves of 12 and 13 ( Figure 5). All the compounds were evaluated for their cytotoxicity (on HL-60, HCT-116, K562, Hela and A549 cell lines), but none of them presented a cytotoxic effect at 30 μM. The antiviral (influenza A H1N1 virus) and NF-κB inhibitory activities were also evaluated, with no activity detected under the concentration of 30 μM. In light of the structural similarity with lovastatin, with the exception of compound 9 (limited quantity), all the compounds were evaluated for their lowering effects against oleic acid (OA)-elicited lipid accumulation in HepG2 liver cells. Five compounds (2, 3, 6, 10 and 12) significantly decreased the lipid accumulation elicited by OA, determined by oil-red O staining, at the concentration of 10 μM. Compounds 6 and 12 showed comparable efficiency with simvastatin ( Figure 6). Additionally, four compounds (3, 6, 10 and 12) could significantly decreased intracellular total cholesterol (TC) levels and three compounds (3, 6, and 10) significantly decreased intracellular triglyceride (TG) levels ( Figure 7). It's worth mentioning that the TG-lowering efficiency of compounds 6 and 10 were comparable with simvastatin and the TG levels were nearly equal to blank control (p > 0.05) ( Figure 7B).

Fungal Material
The fungal strain P. steckii HDN13-279 was isolated from the leaf of Sonneratia caseolaris collected from mangrove conservation area of Hainan, China. It was identified by ITS sequence and the sequence data have been submitted to GenBank (accession number: KY399997). The voucher specimen was deposited in our laboratory at −20 °C.

Fermentation and Extraction
The fungus P. steckii HDN13-279 was cultured at 28 °C on a rotary platform shakers at 180 rpm for 9 days in 500 mL Erlenmeyer flasks containing 150 mL of liquid culture medium, composed of maltose (20.

Fungal Material
The fungal strain P. steckii HDN13-279 was isolated from the leaf of Sonneratia caseolaris collected from mangrove conservation area of Hainan, China. It was identified by ITS sequence and the sequence data have been submitted to GenBank (accession number: KY399997). The voucher specimen was deposited in our laboratory at −20 • C.

Fermentation and Extraction
The fungus P. steckii HDN13-279 was cultured at 28 • C on a rotary platform shakers at 180 rpm for 9 days in 500 mL Erlenmeyer flasks containing 150 mL of liquid culture medium, composed of maltose (20. with EtOAc. The water-containing mycelia was extracted three times with acetone, and the mixed solution was concentrated under reduced pressure to afford an aqueous solution without acetone, then extracted three times with EtOAc. Both EtOAc solutions were combined and concentrated under reduced pressure to give the organic extract (40 g) [28].

Isolation
The organic extract was subjected to vacuum liquid chromatography over a silica gel column using a gradient elution with petroleum ether-CHCl 3 -MeOH to give six fractions (fractions 1-6).     Crystals of 1a, 7 and 8 were obtained in the mixed solvent of CHCl 3 -MeOH, and crystallographic data for 1a, 7 and 8 (Cu Kα radiation) have been deposited in the Cambridge Crystallographic Data Center with the deposition numbers CCDC 1537543, 1537544 and 1537542, respectively. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via the link of reference [29].

Esterification of 1
To a solution of 1 (3.8 mg) in MeOH (0.5 mL) was added excess TMS-CHN 2 in n-hexane (300 µL), and the mixture was stirred at r. t. for 30 min. Then the reaction mixture was concentrated in vacuo and the residue was purified by preparative HPLC (MeOH: H 2 O = 50-100%) to yield methyl ester 1a (2.0 mg). 1a: 1

Methylation of 1 and 2
To a solution of 1 (1.6 mg) or 2 (1.6 mg) and excess NaH in DMF (0.2 mL) were added excess CH 3 I, and the mixtures were stirred at 80 • C for 6 h. Then the reaction mixtures were filtered and purified by preparative HPLC (MeOH:H 2 O = 50-100%) to yield 1b (0.8 mg from 1 and 0.7 from 2), respectively. 1b: 1

Cell-Based Lipid Accumulation Assay
HepG2 cells, seeded in a 96 wells plate at the concentration of 1 × 10 5 cells/well, were cultured in high glucose DMEM medium containing 10% fetal bovine serum (FBS) at 37 • C and 5% CO 2 . After reaching 90% confluence, cells were incubated with the indicated concentration of compounds (10 µM) or with simvastatin (10 µM) in high glucose DMEM containing OA (100 µM) for 24 h. The blank group was incubated with serum-free high glucose DMEM alone. Oil red O staining was performed as previous reported [30] and the intracellular contents of total cholesterol and triglyceride were determined by kits according to manufacturer's instructions.

Assay of Cytotoxicity, Antiviral Activity and NF-κB Inhibitory Activity
These biological evaluations were carried out as previously reported [27,31,32].

Statistical Analysis
The data of lipid lowering effect were expressed as mean ± SEM, representing at least three different experiments with n = 8 in each test. SPSS 17.0 software (SPSS, Chicago, IL, USA) was used for statistical analysis. Differences were assessed by an unpaired t-test. A probability level (p) of 0.05 was considered significant.

Conclusions
In summary, seven new tanzawaic acid derivatives, along with seven known compounds were isolated from Penicillium steckii HDN13-279. The absolute configurations of all the compounds (including the known compounds of which the absolute configurations were not confirmed in the literature) were determined by NMR, X-ray diffraction, CD analyses, as well as chemical derivatization. In addition, for the first time, we evaluated the lowering effects against oleic acid (OA)-elicited lipid accumulation in HepG2 liver cells of this kinds of compounds, and five (2, 3, 6, 10 and 12) showed pharmaceutical potential with lipid-lowering activity.  1-6, 11).