Penidihydrocitrinins A–C: New Polyketides from the Deep-Sea-Derived Penicillium citrinum W17 and Their Anti-Inflammatory and Anti-Osteoporotic Bioactivities

Three new polyketides (penidihydrocitrinins A–C, 1–3) and fourteen known compounds (4–17) were isolated from the deep-sea-derived Penicillium citrinum W17. Their structures were elucidated by comprehensive analyses of 1D and 2D NMR, HRESIMS, and ECD calculations. Compounds 1–17 were evaluated for their anti-inflammatory and anti-osteoporotic bioactivities. All isolates exhibited significant inhibitory effects on LPS-stimulated nitric oxide production in murine brain microglial BV-2 cells in a dose-response manner. Notably, compound 14 displayed the strongest effect with the IC50 value of 4.7 µM. Additionally, compounds 6, 7, and 8 significantly enhanced osteoblast mineralization, which was comparable to that of the positive control, purmorphamine. Furthermore, these three compounds also suppressed osteoclastogenesis in a dose-dependent manner under the concentrations of 2.5 μM, 5.0 μM, and 10 μM.


Introduction
Marine microbes are an ideal source to yield diverse secondary metabolites with unprecedented structures.As a matter of fact, about half of the new marine natural products are produced by marine microorganisms [1][2][3], especially those living in the deep sea under extremely tough environments such as low oxygen concentration, high salt, high hydrostatic pressure, and absence of light, which require various biochemical and physiological adaptations for survival [4,5].These adaptions are accompanied by adjustments of gene regulation, resulting in the formation of different metabolic pathways to give birth to a large number of new secondary metabolites [4,5].
Compounds 1-3 were three novel polyketide adducts of citrinin and diacetyl.They might be biosynthesized by citrinin (11) and diacetyl, a widely found secondary metabolite in microorganisms [21] (Scheme 1).Noteworthily, the reduced derivative of diacetyl, 2,3-butanediol (17), was also co-isolated from the same extract of the strain.Compound 3 was isolated as a colorless oil.The molecular formula of C17H22O7 was established based on its HRESIMS spectrum at m/z 337.1327 [M − H] − (calcd for C17H21O7, 337.1287).Its 1 H and 13 C NMR spectroscopic data were very similar to those of 2, except for the upshift of H-17 from δH 4.11 to δH 4.02 and H3-18 from δH 1.21 to 1.15 and the downshift of C-17 from δC 72.2 to 72.8.This implied that compound 3 could be an epimer of 2 with S-configuration at the C-17 position.The assumption was evidenced by the positive Cotton effect (CE) at λmax 287 nm (Δε +0.54) in 3, whereas a negative CE (Δε −0.34 at λmax 282 nm) in 2. Final confirmation was obtained by comparison of the calculated and experimental ECD spectra of 3, showing the calculated ECD spectrum of (1R,3R,4S,17S)-3 was in good accordance with that of the experimental curve (Figure 4).Accordingly, compound 3 was defined as (1R,3R,4S,17S)-3-hydroxy-2-butonyldihydrocitrinin, and named penidihydrocitrinin C.
Microglial activation plays a pivotal role in the pathogenesis of neurodegenerative diseases, orchestrating a complex interplay between inflammation and neuronal health Scheme 1.The proposed biosynthetic pathway of penidihydrocitrinins A-C (1-3).
Microglial activation plays a pivotal role in the pathogenesis of neurodegenerative diseases, orchestrating a complex interplay between inflammation and neuronal health [35].While microglial cells function as the guardians of the central nervous system, their dysregulated activation can lead to chronic neuroinflammation and exacerbation of neuronal damage, contributing to the progression of disorders such as multiple sclerosis and Alzheimer's and Parkinson's diseases [36].These diseases pose significant challenges to public health, which necessitate innovative therapeutic approaches.Thus, discovering new small molecules that can inhibit the dysregulated activation of microglial cells is essential for the targeted modulation of the immune response in the central nervous system, which can reduce inflammation and protect neurons from harm [37].A growing amount of evidence demonstrates that secondary metabolites derived from marine resources are potential therapeutic strategies for microglial-mediated neuroinflammation.
Therefore, all isolates were tested for nitrite secretion in lipopolysaccharide (LPS)induced BV-2 microglial cells.As a result, they all demonstrated a dose-dependent suppression of nitrite secretion induced by LPS, displaying inhibitory actions at concentrations of 3.0 µM, 10 µM, and 20 µM (Figure 5).Moreover, none exhibited cytotoxicity effects against BV-2 cells at 20 µM under the microscope.We further compared the inhibition of these compounds on nitrite production at a concentration of 10 µM (Figure 6), and the results showed that the inhibitory rates of compounds 1-7 on nitrite production were 31.1-53.5% which indicates that substitution of the C-1 position on isochroman weakened the anti-inflammatory activity of the compounds, suggesting that this substitution is related to anti-inflammatory activity.Compound 8 only inhibited nitrite production by 26.4% in LPS-stimulated BV-2 cells, demonstrating that the dimeric structure further weakened the anti-inflammatory effect.Meanwhile, the anti-inflammatory activity of compound 12, emodin, was consistent with a previous report [38].Notably, compound 14 (GKK1032 B) displayed the most potent nitrite inhibitory activity with an inhibitory ratio of 73.0 ± 1.6% at 10 µM (nitrite concentration: 12.2 ± 0.4 µM), compared to the LPS-treated group (nitrite concentration: 30.9 ± 0.4 µM) (Figures 5 and 6).Furthermore, this compound displayed an IC 50 value of 4.7 µM.Although GKK1032 analogues were reported to exhibit antibacterial activities [39], it is the first time that they had anti-neuroinflammatory activity.These findings demonstrate the effects of marine-derived compounds in modulating microglial activation, suggesting their potential as therapeutic candidates for neuroinflammatory conditions and neurodegenerative diseases.
which can reduce inflammation and protect neurons from harm [37].A growing amount of evidence demonstrates that secondary metabolites derived from marine resources are potential therapeutic strategies for microglial-mediated neuroinflammation.
Therefore, all isolates were tested for nitrite secretion in lipopolysaccharide (LPS)induced BV-2 microglial cells.As a result, they all demonstrated a dose-dependent suppression of nitrite secretion induced by LPS, displaying inhibitory actions at concentrations of 3.0 µM, 10 µM, and 20 µM (Figure 5).Moreover, none exhibited cytotoxicity effects against BV-2 cells at 20 µM under the microscope.We further compared the inhibition of these compounds on nitrite production at a concentration of 10 µM (Figure 6), and the results showed that the inhibitory rates of compounds 1-7 on nitrite production were 31.1-53.5% which indicates that substitution of the C-1 position on isochroman weakened the anti-inflammatory activity of the compounds, suggesting that this substitution is related to anti-inflammatory activity.Compound 8 only inhibited nitrite production by 26.4% in LPS-stimulated BV-2 cells, demonstrating that the dimeric structure further weakened the anti-inflammatory effect.Meanwhile, the anti-inflammatory activity of compound 12, emodin, was consistent with a previous report [38].Notably, compound 14 (GKK1032 B) displayed the most potent nitrite inhibitory activity with an inhibitory ratio of 73.0 ± 1.6% at 10 µM (nitrite concentration: 12.2 ± 0.4 µM), compared to the LPS-treated group (nitrite concentration: 30.9 ± 0.4 µM) (Figures 5 and 6).Furthermore, this compound displayed an IC50 value of 4.7 µM.Although GKK1032 analogues were reported to exhibit antibacterial activities [39], it is the first time that they had anti-neuroinflammatory activity.These findings demonstrate the effects of marine-derived compounds in modulating microglial activation, suggesting their potential as therapeutic candidates for neuroinflammatory conditions and neurodegenerative diseases.Osteoporosis, a disease associated with aging, is characterized by excessive activation of osteoclasts or reduction of osteoblasts.Among women aged 65 or older, approximately 25% are affected by osteoporosis, with accelerated bone loss occurring after menopause.Osteoporosis, a disease associated with aging, is characterized by excessive activation of osteoclasts or reduction of osteoblasts.Among women aged 65 or older, approximately 25% are affected by osteoporosis, with accelerated bone loss occurring after menopause.Therefore, promoting osteoblast differentiation and suppressing osteoclastogenesis are effective strategies for treating osteoporosis [40].BMSCs are able to differentiate into osteoblasts, chondroblasts, and adipocytes [41], and bone regeneration achieved via osteogenic induction of MSCs could provide a rational therapeutic strategy for preventing age-related osteoporosis [42].Accordingly, all 17 isolates were tested for both osteoblast and osteoclast activities.Firstly, compounds 1-17 were subjected to anti-proliferative tests on BMSCs.At the concentration of 10 µM, none showed cytotoxicity (Figure 7).Osteoporosis, a disease associated with aging, is characterized by excessive activation of osteoclasts or reduction of osteoblasts.Among women aged 65 or older, approximately 25% are affected by osteoporosis, with accelerated bone loss occurring after menopause.Therefore, promoting osteoblast differentiation and suppressing osteoclastogenesis are effective strategies for treating osteoporosis [40].BMSCs are able to differentiate into osteoblasts, chondroblasts, and adipocytes [41], and bone regeneration achieved via osteogenic induction of MSCs could provide a rational therapeutic strategy for preventing age-related osteoporosis [42].Accordingly, all 17 isolates were tested for both osteoblast and osteoclast activities.Firstly, compounds 1-17 were subjected to anti-proliferative tests on BMSCs.At the concentration of 10 µM, none showed cytotoxicity (Figure 7).Intracellular calcium deposition at a later stage served as a significant evaluation indicator for osteogenic activity.Interestingly, compounds 6, 7, and 8 exerted a noticeable enhancing effect on osteoblast mineralization within BMSCs (Figure 8).Intracellular calcium deposition at a later stage served as a significant evaluation indicator for osteogenic activity.Interestingly, compounds 6, 7, and 8 exerted a noticeable enhancing effect on osteoblast mineralization within BMSCs (Figure 8).
Furthermore, bioactive compounds 6, 7, and 8 also exhibited a distinct inhibitory effect on osteoclast activity, as evidenced by a significant reduction in tartrate-resistant acid phosphatase (TRAP)-positive cells (Figure 9).These findings indicate that compounds 6, 7, and 8 not only facilitated osteoblast mineralization but also exerted a substantial dosedependent inhibitory effect on RANKL-induced osteoclasts.Furthermore, bioactive compounds 6, 7, and 8 also exhibited a distinct inhibitory effect on osteoclast activity, as evidenced by a significant reduction in tartrate-resistant acid phosphatase (TRAP)-positive cells (Figure 9).These findings indicate that compounds 6, 7, and 8 not only facilitated osteoblast mineralization but also exerted a substantial dose-dependent inhibitory effect on RANKL-induced osteoclasts.25 ng/mL RANKL (or not).After five days, these cells were fixed and stained with TRAP.(B) TRAPpositive multinucleated (there or more) cells were regarded as mature osteoclasts.Osteoclast numbers were quantified and analyzed (n = 3).* p < 0.05, **** p < 0.0001 vs. the RANKL+DMSO group.(C) BMM viability was measured by the CCK-8 assay.The culture medium and the solvent (0.1% DMSO) were regarded as Vehicle and DMSO groups, respectively.

Fungal Identification, Fermentation, and Extraction
The fungal strain W17 was isolated from a deep-sea sediment sample of the western Pacific Ocean at the depth of 5278 m.It was identified to be Penicillium citrinum as the 18S rRNA gene sequence alignment (OR398934) demonstrated that it showed great similarity (99.8%) to Penicillium citrinum NRRL 1841 (GenBank accession number NR_121224.1).The strain was preserved at the Key Laboratory of Marine Genetic Resources, Third Institute of Oceanography, Ministry of Natural Resources (Xiamen, China).The microbial strain was cultivated on a PDA plate medium at 25 • C for 3 days and the colony was inoculated into 250 mL Erlenmeyer flasks containing 50 mL PDB medium.Then, it was cultured in a rotary shaker (130 rpm) at 25 • C for 3 days as spore medium.After 3 days, the spore solution was inoculated in 120 Erlenmeyer flasks (1 L) with each containing 400 mL tap water, 80 g potato power, 8 g glucose, and 6 g marine salt.The fermentation was performed in a 130 rpm rotary shaker at 25 • C.After 7 days, the fermentation broth was extracted with EtOAc three times and concentrated under reduced pressure to give a crude extract (36.5 g).

ECD Calculation
As reported previously [16], the conformational analysis was first conducted via random searching stochastically using the MMFF94 force field.All conformers were consecutively optimized at the PM6 and HF/6-31G(d) levels.Dominative conformers were further optimized at the B3LYP/6-31G(d) level in the gas phase.The optimized conformers possessed no imaginary frequencies and were true local minima.ECD calculations were conducted at the B3LYP/6-311G(d,p) level in MeOH with the IEFPCM model using timedependent density functional theory (TD-DFT).The ECD spectrum was simulated by overlapping Gaussian functions for each transition.

BV-2 Cell Culture and Compound Treatment
BV-2 cell culture and compound treatment were carried out as previously reported [43,44].Briefly, BV-2 microglial cells were cultured in DMEM supplemented with 10% fetal bovine serum (Thermofisher, Shanghai, China) and antibiotics (100 units/mL of penicillin and 100 µg/mL of streptomycin) in a humidified 5% CO 2 incubator at 37 • C. Cells were seeded into 24-well plates at a density of 2 × 10 4 cells per well and allowed to adhere overnight.On the subsequent day, the cells were treated with freshly prepared culture medium containing the specified concentrations of the investigated compounds for a duration of 30 min before exposure to LPS (1 µg/mL).A control group was treated with a vehicle solution (DMSO, 0.1%).

Quantification of Nitrite Levels
The concentration of nitrite present in the culture medium was assessed using the Griess Reagent Kit (Thermo Fisher, Shanghai, China).Briefly, 75 µL of cell culture supernatants was mixed with an equal volume of the Griess Reagent Kit and allowed to react for 30 min at room temperature.The absorbance of the resulting diazonium compound was measured at a wavelength of 560 nm.The concentration of nitrite production was calculated based on the nitrite standard solution.

Cell Extraction and Culture
The bone mesenchymal stem cells (BMSCs) and bone marrow monocytes (BMMs) were flushed from the femur of C57BL/6J mice aged 3 weeks and 6-week-old C57BL/6 mice, respectively, with the methods as previously described [16].In brief, the BMSCs were carefully removed from the animals and cultured in α-MEM and induced with supplemented complete α-MEM (10% v/v FBS, 1% v/v penicillin/streptomycin (P/S)).The BMSCs were induced with 2 mM β-glycerophosphate and 50 µg/mL ascorbate, of which half were changed twice a week.The BMMs were cultured in complete α-MEM (10% v/v FBS, 1% v/v P/S, and 25 ng/mL M-CSF).

Figure 3 .
Figure 3.The calculated and experimental ECD spectra of compounds 1-3.Figure 3. The calculated and experimental ECD spectra of compounds 1-3.

Figure 3 .
Figure 3.The calculated and experimental ECD spectra of compounds 1-3.Figure 3. The calculated and experimental ECD spectra of compounds 1-3.

14 Figure 4 .
Figure 4.The key COSY, HMBC, and NOESY correlations of compounds 2 and 3. Compound 3 was isolated as a colorless oil.The molecular formula of C17H22O7 was established based on its HRESIMS spectrum at m/z 337.1327 [M − H] − (calcd for C17H21O7, 337.1287).Its 1 H and 13 C NMR spectroscopic data were very similar to those of 2, except for the upshift of H-17 from δH 4.11 to δH 4.02 and H3-18 from δH 1.21 to 1.15 and the downshift of C-17 from δC 72.2 to 72.8.This implied that compound 3 could be an epimer of 2 with S-configuration at the C-17 position.The assumption was evidenced by the positive