Anti-Neuroinflammatory Agent, Restricticin B, from the Marine-Derived Fungus Penicillium janthinellum and Its Inhibitory Activity on the NO Production in BV-2 Microglia Cells

A new compound containing a triene, a tetrahydropyran ring and glycine ester functionalities, restricticin B (1), together with four known compounds (2–5) were obtained from the EtOAc extract of the marine-derived fungus Penicillium janthinellum. The planar structure of 1 was determined by detailed analyses of MS, 1D and 2D NMR data. The relative and absolute configurations of 1 were established via the analyses of NOESY spectroscopy data, the comparison of optical rotation values with those of reported restricticin derivatives and electronic circular dichroism (ECD). All the compounds were screened for their anti-neuroinflammatory effects in lipopolysaccharide (LPS)-induced BV-2 microglia cells. Restricticin B (1) and N-acetyl restricticin (2) exhibited anti-neuroinflammatory effects by suppressing the production of pro-inflammatory mediators in activated microglial cells.


Introduction
The novel class of potent antifungal agents, restricticin and restricticinol, were first isolated from the fermentation broth of Penicillium restrictum [1,2]. These compounds contain a tetrahydropyran ring and triene side chains in common and restricticin possesses a glycine unit linked to the tetrahydropyran ring [3]. The initial biological activity study of restricticin derivatives displayed that only restricticin exhibited potent antifungal activity against both yeast and filamentous fungi [1,4]. The very closely related compounds with a shorter polyene chain, lanomycin and lanomycinol, were isolated from Pycnidiophora dispersa in 1992 and chaunopyran A possessing a same polyene chain and 2-alkenyl-tetrahydropyran was reported in 2017 [5,6]. Lanomycin was active against some Candida species and dermatophytes and not active against Gram-positive, Gram-negative bacteria and Aspergillus fumigatus [7]. Restricticin and lanomycin were first reported as natural antifungal agents Aspergillus fumigatus [7]. Restricticin and lanomycin were first reported as natural antifungal agents to inhibit cytochrome P450 lanosterol demethylase [5,8]. Interestingly, restricticinol and lanomycinol did not show such antifungal activity [8]. Because of the considerable interest in their biological activity, several methods for the total synthesis of the antifungal agents have been described [8][9][10].
Microglia, macrophages and representatives of the innate immune system in the brain, have been implicated as active contributors to neuron damage and readily become activated in response to infection or injury [11,12]. Activated microglia upregulate pro-inflammatory and neurotoxic mediators including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and free radicals such as nitric oxide (NO) and superoxide [13]. Moreover, large numbers of activated microglia are present in the CNS tissue of patients with chronic neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease and prion disease [14,15]. Therefore, the suppression of pro-inflammatory and neurotoxic molecules in activated microglia would be an effective therapeutic approach to treat various neuronal diseases [16].
Over the past decade, the secondary metabolites from marine microbes have been researched as a promising source for medical applications and clinical development [17,18]. Among the marinederived microorganisms, marine fungi produce bioactive compounds with a broad range of bioactivities such as antibacterial, antiviral, antitumoral, antimalarial and anti-inflammatory [19]. As a part of our ongoing investigation for diverse secondary metabolites from marine microorganisms, we isolated the fungal 168CLC-17.1 strain from a sediment sample collected from Cu Lao Cham Island, Vietnam, and based on its nuclear ribosomal internal transcribed spacer (ITS) region sequence, it was identified as Penicillium janthinellum. Further chemical investigation of the ethyl acetate extracts of the fungal culture broth yielded a new restricticin derivative, named restricticin B (1), together with four known compounds, N-acetyl restricticin (2) [19], 3,3″-dihydroxy-6′-desmethyl terphenyllin (3) [20], fellutanine B (4) [21] and 10,23-dihydro-24,25-dehydro aflavinin (5) [22] (Figure 1). Here, we describe the chemical investigation, structural determination, and bioactivities of these compounds.

Results and Discussion
Compound 1 was isolated as a pale brown oil and its molecular formula was determined to be C26H35NO7 based on the HRESIMS ion at m/z 496

Results and Discussion
Compound 1 was isolated as a pale brown oil and its molecular formula was determined to be C 26  one methine carbon (δ C 32.1), one oxygenated and one nitrogenated methylene carbons (δ C 70.2 and 50.0, respectively), two methylene carbons (δ C 34.5 and 22.0), one methoxy carbon (δ C 55.0), and four methyl carbons (δ C 18.7, 12.6, 10.4, and 9.5). The planar structure of 1 was elucidated by the analysis of the 2D NMR data, including the COSY and HMBC spectra ( Figure 2). The COSY correlations of H-3/H-4/H-5/H-6/H-7/H 2 -8/H 2 -9/H 3 -10 revealed the presence of an unsaturated side chain. The presence of the tetrahydropyran ring was confirmed by the COSY correlations of H-1 /H-2 /H-3 /H-4 /H 2 -5 /H 3 -11 and the HMBC correlations from H-5 to C-1 , H 3 -12 to C-3 , and H 3 -11 to C-3 and C-5 . The HMBC correlations from H 3 -1 to C-1 , C-2, and C-3, H-1 to C-2 and C-3, and H-2 to C-2 indicated that the triene side chain is connected to the tetrahydropyran ring. The geometry of the side chain was determined as all E by the large 3 J H,H coupling constants for H-4/H-5 (14.5 ppm) and H-6/H-7 (15.0 ppm) and the NOESY correlations of H-3/H-5 and H 3 -1/H-4. Afterwards, a glycine moiety was confirmed by the HMBC correlations from H-2 and H 2 -14 to C-13. Detailed analysis of 1D and 2D NMR spectra of 1 revealed that the partial structure was closely similar to that of restricticin. The HMBC correlations from H-1" to C-2", C-3", C-6", and C-14, H-4" to C-2" and C-5", and H-7" to C-4" and C-5" established the α-pyrone moiety linked to the NH of the glycine unit.  Interestingly, the NMR data in one specific area showed a duplication of the proton and carbon signals ( Table 1). The 1 H NMR spectrum of 1 displayed a pair of olefinic singlet signals at H-1″ (major: δH 8.20, minor: δH 8.32) and doublet methylene signals at H-14 (major: δH 4.27, 4.36, minor: δH 4.29, 4.38). Moreover, a pair of signals for the exchangeable NH proton (major: δH 11.8, minor: δH 10.1) was detected in the 1 H NMR spectrum in CDCl3. The 13 C NMR spectrum also showed that two sets of the carbon signals at C-13 (δC 167.1 and 167.2), C-14 (δC 49.9 and 50.0), C-1″ (δC 161.5 and 162.7), C-2″ (δC 96.5 and 96.6), C-3″ (δC 181.7 and 184.1), C-4″ (δC 106.6 and 106.9) and C-5″ (δC 165.3 and 165.4). These phenomena were presumed to be involved in imine-enamine tautomerism involving the ketone group at C-3″. The form of 1a was probably explained as the E geometry of Δ 1″ , based on NMR evidence via tautomeric equilibrium, where an exchangeable amide proton is notably shifted downfield by a strong intramolecular hydrogen bond with a ketocarbonyl group (C-3″) (Scheme 1). Otherwise, the proton signal of 1b-NH in the more upfield region was assigned as the Z geometry of Δ 1″ due to the weaker hydrogen bond with a carbonyl group (C-6″) than 1a. For the same reason, the H-1″ of 1a and 1b also displayed the similar pattern.
To rationalize these unexpected observations, EXSY (exchange spectroscopy) experiments and the synthesis of the pyrone moiety were carried out following previous studies [23,24]. In the NOESY spectrum, EXSY correlations (equilibrium spots) of NH and H-1″ confirmed the presence of two isomers as an inseparable mixture ( Figure S9, Supporting Information). An extensive literature survey has indicated that 4-hydroxy-6-methyl-2-pyrone and glycine in 2-propanol in the presence of triethyl orthoformate led to 6 (Scheme 2), and the 1 H NMR spectra in previous studies displayed a pair of signals due to the existence of E and Z isomers [24,25]. Comparison of the NMR data of 1 and Interestingly, the NMR data in one specific area showed a duplication of the proton and carbon signals ( Table 1). The 1 H NMR spectrum of 1 displayed a pair of olefinic singlet signals at H-1" (major: δ H 8.20, minor: δ H 8.32) and doublet methylene signals at H-14 (major: δ H 4.27, 4.36, minor: δ H 4.29, 4.38). Moreover, a pair of signals for the exchangeable NH proton (major: δ H 11.8, minor: δ H 10.1) was detected in the 1 H NMR spectrum in CDCl 3 . The 13 C NMR spectrum also showed that two sets of the carbon signals at C-13 (δ C 167.1 and 167.2), C-14 (δ C 49.9 and 50.0), C-1" (δ C 161.5 and 162.7), C-2" (δ C 96.5 and 96.6), C-3" (δ C 181.7 and 184.1), C-4" (δ C 106.6 and 106.9) and C-5" (δ C 165.3 and 165.4). These phenomena were presumed to be involved in imine-enamine tautomerism involving the ketone group at C-3". The form of 1a was probably explained as the E geometry of ∆ 1" , based on NMR evidence via tautomeric equilibrium, where an exchangeable amide proton is notably shifted downfield by a strong intramolecular hydrogen bond with a ketocarbonyl group (C-3") (Scheme 1). Otherwise, the proton signal of 1b-NH in the more upfield region was assigned as the Z geometry of ∆ 1" due to the weaker hydrogen bond with a carbonyl group (C-6") than 1a. For the same reason, the H-1" of 1a and 1b also displayed the similar pattern.
To rationalize these unexpected observations, EXSY (exchange spectroscopy) experiments and the synthesis of the pyrone moiety were carried out following previous studies [23,24]. In the NOESY spectrum, EXSY correlations (equilibrium spots) of NH and H-1" confirmed the presence of two isomers as an inseparable mixture ( Figure S9, Supporting Information). An extensive literature survey has indicated that 4-hydroxy-6-methyl-2-pyrone and glycine in 2-propanol in the presence of triethyl orthoformate led to 6 (Scheme 2), and the 1 H NMR spectra in previous studies displayed a pair of signals due to the existence of E and Z isomers [24,25]. Comparison of the NMR data of 1 and 6 indicated that they had the same phenomenon of imine-enamine tautomerism ( Figure S10, Supporting Information). Thus, the planar structure of 1 was assigned as shown in Figure 1.   (2), suggesting that 1 has the same absolute configuration with its derivatives as shown in Figure 3. Additionally, the ECD calculation of enantiomers (1: 1′S,2′R,3′S,4′S, ent-1: 1′R,2′S,3′R,4′R) was carried out at B3LYP/6-311 + G(d)1 level. The experimental CD spectrum showed a positive Cotton effect at 305 nm. Then, the theoretical ECD spectrum of 1a was in reasonable agreement with the measured CD spectrum, suggesting that the absolute configurations of 1 are defined as 1′S,2′R,3′S,4′S. Based on these results, the structure of 1 was assigned and named restricticin B.  (2), suggesting that 1 has the same absolute configuration with its derivatives as shown in Figure 3. Additionally, the ECD calculation of enantiomers (1: 1′S,2′R,3′S,4′S, ent-1: 1′R,2′S,3′R,4′R) was carried out at B3LYP/6-311 + G(d)1 level. The experimental CD spectrum showed a positive Cotton effect at 305 nm. Then, the theoretical ECD spectrum of 1a was in reasonable agreement with the measured CD spectrum, suggesting that the absolute configurations of 1 are defined as 1′S,2′R,3′S,4′S. Based on these results, the structure of 1 was assigned and named restricticin B.  (2), suggesting that 1 has the same absolute configuration with its derivatives as shown in Figure 3. Additionally, the ECD calculation of enantiomers (1: 1 S,2 R,3 S,4 S, ent-1: 1 R,2 S,3 R,4 R) was carried out at B3LYP/6-311 + G(d)1 level. The experimental CD spectrum showed a positive Cotton effect at 305 nm. Then, the theoretical ECD spectrum of 1a was in reasonable agreement with the measured CD spectrum, suggesting that the absolute configurations of 1 are defined as 1 S,2 R,3 S,4 S. Based on these results, the structure of 1 was assigned and named restricticin B.   , 1 and 2 showed no antibacterial activity and cytotoxicity against cancer cell lines. Along with the fact that restricticinol and lanomycinol have no antifungal activity in previous research, the results of this study suggested that the free amine of the glycine unit is important for the antifungal activity.
All the isolated compounds (1-5) were evaluated for inhibitory activity on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells and for their cytotoxicity. The cells were initially treated with 100 μΜ of each compounds and/or LPS (200 ng/mL) to measure the levels of NO and cytotoxicity using an MTT assay. As shown in Figure 5, all the compounds inhibited the LPS-induced NO production in BV-2 microglial cells. However, compounds 3 and 5 displayed weak and strong toxicity, respectively. Compounds 1, 2, and 4 decreased the production    , 1 and 2 showed no antibacterial activity and cytotoxicity against cancer cell lines. Along with the fact that restricticinol and lanomycinol have no antifungal activity in previous research, the results of this study suggested that the free amine of the glycine unit is important for the antifungal activity.
All the isolated compounds (1-5) were evaluated for inhibitory activity on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells and for their cytotoxicity. The cells were initially treated with 100 μΜ of each compounds and/or LPS (200 ng/mL) to measure the levels of NO and cytotoxicity using an MTT assay. As shown in Figure 5, all the compounds inhibited the LPS-induced NO production in BV-2 microglial cells. However, compounds 3 and 5 displayed weak and strong toxicity, respectively. Compounds 1, 2, and 4 decreased the production The structures of the four known compounds were determined as N-acetyl restricticin (2), 3,3"-dihydroxy-6 -desmethyl terphenyllin (3), fellutanine B (4) and 10,23-dihydro-24,25-dehydro aflavinin (5) by comparing their 1 H, 13 C NMR and MS data with those reported in the literature (Supporting Information).
Compounds 1 and 2 were tested for their antimicrobial activity using fungi (Penicillium italicum  However, 1 and 2 showed no antibacterial activity and cytotoxicity against cancer cell lines. Along with the fact that restricticinol and lanomycinol have no antifungal activity in previous research, the results of this study suggested that the free amine of the glycine unit is important for the antifungal activity. All the isolated compounds (1)(2)(3)(4)(5) were evaluated for inhibitory activity on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells and for their cytotoxicity. The cells were initially treated with 100 µM of each compounds and/or LPS (200 ng/mL) to measure the levels of NO and cytotoxicity using an MTT assay. As shown in Figure 5, all the compounds inhibited the LPS-induced NO production in BV-2 microglial cells. However, compounds 3 and 5 displayed weak and strong toxicity, respectively. Compounds 1, 2, and 4 decreased the production of NO without showing cytotoxicity at the treated concentrations. According to the results, restricticin derivatives have the most potent anti-inflammatory effect among the treated compounds without cytotoxicity. Therefore, restricticin B (1) and N-acetyl restricticin (2) were selected to further investigate the effects on the LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein expression, and the production of LPS-stimulated pro-inflammatory cytokines in BV-2 microglia cells.
of NO without showing cytotoxicity at the treated concentrations. According to the results, restricticin derivatives have the most potent anti-inflammatory effect among the treated compounds without cytotoxicity. Therefore, restricticin B (1) and N-acetyl restricticin (2) were selected to further investigate the effects on the LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein expression, and the production of LPS-stimulated proinflammatory cytokines in BV-2 microglia cells. Two restricticin class compounds (1 and 2) at the concentrations of 50 μM and 100 μM inhibited the NO production in BV-2 cells in a dose-dependent manner ( Figure 6A). Compound 1 showed a stronger inhibition of NO production than 2. As shown in Figure 6B,C, LPS treatment (200 ng/mL) significantly upregulated iNOS and COX-2 expression, and the mRNA and protein expression levels of iNOS and COX-2 were inhibited by restricticins in concentration-dependent manner. To investigate whether restricticins repress the production of pro-inflammatory cytokines such as TNFα, IL-1β and IL-6, RT-PCR was performed, and the results showed that restricticins reduced the secretion of pro-inflammatory cytokines. Interestingly, IL-1β was the most strongly inhibited by compound 1 at 100 μM. Consequently, these results indicated that restricticins have effective properties in neuroinflammation processes and suppress the expression of pro-inflammatory mediators at the transcriptional level. Two restricticin class compounds (1 and 2) at the concentrations of 50 µM and 100 µM inhibited the NO production in BV-2 cells in a dose-dependent manner ( Figure 6A). Compound 1 showed a stronger inhibition of NO production than 2. As shown in Figure 6B,C, LPS treatment (200 ng/mL) significantly upregulated iNOS and COX-2 expression, and the mRNA and protein expression levels of iNOS and COX-2 were inhibited by restricticins in concentration-dependent manner. To investigate whether restricticins repress the production of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6, RT-PCR was performed, and the results showed that restricticins reduced the secretion of pro-inflammatory cytokines. Interestingly, IL-1β was the most strongly inhibited by compound 1 at 100 µM. Consequently, these results indicated that restricticins have effective properties in neuroinflammation processes and suppress the expression of pro-inflammatory mediators at the transcriptional level.

BV-2 Microglial Cell Culture and Treatment
The BV-2 murine microglial cells were cultured in DMEM (Dulbecco's modified Eagle medium) supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin (100 units/mL) at 37 • C in a humidified 5% CO 2 incubator. The cell was seeded at a density of 2.5 × 10 5 cells/mL and pretreated with the same concentration of samples and followed by LPS incubation (200 ng/mL) [27].

Cell Viability and Nitrite Assay
The BV-2 microglial cells seeded at a density of 2.5 × 10 5 cells/well were pretreated with 50~100 µM of compounds 1-5 for 1 h, followed by LPS (200 ng/mL) for 24 h. Twenty microliters of MTT solution (2 mg/mL, conc.) was added to each well in 24 wells. After 1 h, the supernatant was sucked and dissolved the formazan crystals in viable cells from DMSO. Optical density was measured at 550 nm using a microplate reader and values were determined in comparison to control cells. For the nitrite assay, the BV-2 microglial cells seeded at a density of 2.5 × 10 5 cells/well were pretreated with 50~100 µM of compounds 1-5 for 1 h, followed by LPS (200 ng/mL) for 24 h. One hundred microliters of supernatants was collected and transferred to new microplate. After transfer, the supernatants were mixed with an equal volume of Griess reagent for 10 min at room temperature in the dark. The amount of nitrite in each sample was measured by using a range of sodium nitrite dilutions as standard solutions. Absorbance was determined at 540 nm using a microplate reader [27].

Total RNA Extraction and Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Total RNA extraction of BV-2 microglial cells was prepared using Trizol reagent following the manufacturer's instructions. RNA (2.5 µg) was reverse-transcribed using ReverTra Ace-α kit and PCR amplification was carried out using specific primers [28].

Western Blot Analysis
Treated BV-2 cells (5 × 10 5 cells/well) were washed twice with PBS and lysed for 10 min using RIPA lysis buffer at 4 • C to obtain the total cell lysate. The cells were centrifuged at 14,000 rpm at 4 • C and collected and separately stored for further analysis. DC Protein Assay kit was used for the protein concentration of isolated compounds. Equal amounts of protein (35 µg for cells) were separated by 10% sodium dodecyl sulfate-polyacrylamide electrophoresis, and the resolved proteins were transferred to polyvinylidene difluoride membranes. The membranes were incubated for 1 h with 5% skim-milk in TBS buffer to block nonspecific binding. The blots were visualized by a PowerOpti-ECL kit obtained from the detection system (Animal Genetics Inc., Tallahassee, FL, USA) [28].

Fungal Strain and Fermentation
The strain 168CLC-17.1 was isolated from a sediment sample, and collected at Cu Lao Cham Island, Quang Nam, Vietnam in August 2016. The strain was identified by DNA amplification and the sequencing of the ITS region (GenBank accession number AB293968.1) and named as Penicillium janthinellum 168CLC-17.1. The cultivation and fermentation of the fungal strain have been previously described [29].

Isolation of Compounds 1-5
The mycelia and rice media were extracted with EtOAc and then concentrated to yield a crude extract (6 g

Calculation of ECD Spectra
Conformational searches were performed by CONFLEX version 8.0 program (CONFLEX Corporation, Tokyo, Japan) with a molecular mechanics force field (MMFF) within a window of 3.0 kcal/mol. The conformers were further optimized by the B3LYP/6-311G (d,p). The theoretical calculations of the ECD data were conducted using the time-dependent density functional theory (TD-DFT) method at the B3LYP/6-311G (d,p), which were performed with Gaussian 16 software (Gaussian Inc., Wallingford, CT, USA). The calculated ECD spectra data were averaged based on their Boltzmann populations.

Conclusions
Two restrciticin derivatives (1 and 2), along with three known compounds (3)(4)(5) were isolated from the rice medium cultures of the marine-derived fungus Penicillium janthinellum 168CLC-17.1. The structures of the isolated compounds were determined by the analysis of their NMR and mass spectrometric data. The absolute configuration of restricticin B (1) was established by NOESY correlations, comparison of specific rotation values with those of restricticin derivatives reported and electronic circular dichroism (ECD). Interestingly, the new restricticin 1 possessed an α-pyrone ring connected to the NH of glycine moiety and showed signal duplication as a 3:2 mixture of isomers. Two isolated restricticins exhibited inhibitory activity on NO production in LPS-stimulated BV-2 microglial cells. Moreover, the restricticins suppressed iNOS and COX-2 expression (both at the protein and mRNA levels), and also inhibited the LPS-induced production of pro-inflammatory cytokines. Additionally, compound 1, possessing α-pyrone moiety and linked to NH. showed stronger activity than 2 containing the N-acetyl group. To the best of our knowledge, this is the first report on the anti-neuroinflammatory activity of restrciticins and compounds 3-5.