New Sesquiterpenoids from the Mangrove-Derived Fungus Talaromyces sp. as Modulators of Nuclear Receptors

Abstract

Four new sesquiterpenoids, talaroterpenes A–D (1–4), were isolated from the mangrove-derived fungus Talaromyces sp. SCSIO 41412. The structures of compounds 1–4 were elucidated through comprehensive NMR and MS spectroscopic analyses. The absolute configurations of 1–4 were assigned based on single-crystal X-ray diffraction and calculated electronic circular dichroism analysis. Talaroterpenes A–D (1–4) were evaluated with their regulatory activities on nuclear receptors in HepG2 cells. Under the concentrations of 200 μM, 1, 3 and 4 exhibited varying degrees of activation on ABCA1 and PPARα, while 4 showed the strongest activities. Furthermore, 4 induced significant alterations in the expression of downstream target genes CLOCK and BMAL1 of RORα, and the in silico molecular docking analysis supported the direct binding interactions of 4 with RORα protein. This study revealed that talaroterpene D (4) was a new potential non-toxic modulator of nuclear receptors.

1. Introduction

Nuclear receptors (NRs) are an important class of drug targets because they are key regulators of many cardiovascular, metabolic and inflammatory diseases [1]. PPARs (peroxisome proliferator activated receptors), FXRs (farnesoid X receptors, FXRs), LXRs (liver X receptors) and RORs (retinal acid receptor-related orphan receptors) are important members of the nuclear receptor family, playing critical roles in regulating fat synthesis, cholesterol metabolism, fatty acid oxidation, inflammatory response and so on. In recent years, considerable research efforts have been devoted to the development of agonists or inhibitors targeting those receptors. Our previous review summarized 261 natural LXR/FXR modulators, originating from terrestrial plants and microorganisms, marine organisms and marine-derived microorganisms, in the recent two decades (2000–2020) [2]. Obeticolic acid is an FXR agonist that inhibits downstream CYP7A1 expression [3], suppresses bile acid synthesis, promotes bile secretion and so reduces intracellular bile acid concentration. It has been approved by the FDA for the treatment of primary biliary cirrhosis. Saroglitazar, a new PPAR agonist [4], which shows an evident effect on fat metabolism and insulin resistance, has been approved in India for two indications, including diabetes dyslipidemia and type 2 diabetes that cannot be controlled by statins.

Marine microorganisms produce metabolites with unique chemical structures that could target specific cellular physiological and biochemical processes, thus exhibiting significant biological activity and medicinal value [5,6,7]. In our previous studies, we discovered several LXRα agonists from marine microorganism-derived natural products [8,9,10,11], and some of them could promote lipid efflux by upregulating ABCA1 expression and reducing lipid synthesis, which could be considered as lead compounds for hypercholesterolemia [12].

Mangrove sediment-derived microbes, as a rich reservoir of natural product diversity, could be utilized in the research and development of new drugs [13], and many lead compounds have been obtained through our extensive research [14,15]. In our ongoing pursuit of pharmaceutically active molecules, especially natural modulators of nuclear receptors, regulatory activities on nuclear receptors were tested after the natural products were obtained from the mangrove sediment-derived microbes.

In this study, four new sesquiterpenoids, talaroterpenes A–D (1–4), were isolated from a mangrove sediment-derived fungus Talaromyces sp. SCSIO 41412 (Figure 1). Herein, the specifics of the isolation, structural elucidation and bioactive assessments of isolated compounds are reported.

2. Results and Discussion

2.1. Structural Determination

Compound 1 was obtained as colorless needles. Its molecular formula was determined to be C₁₅H₂₄O₄ based on the high-resolution electrospray ionization mass spectroscopy (HRESIMS) data, corresponding to 4 degrees of unsaturation. Analysis of the ¹H NMR data (

Table 1. ¹H (500 MHz) and ¹³C (125 MHz) NMR data of 1 and 2 in DMSO-d₆.

Pos.	1		2
	δC Type	δH (J in Hz)	δC Type	δH (J in Hz)
1	39.3, CH2	2.15, overlapped 2.06, m	39.3, CH2	2.16, overlapped 2.06, m
2	74.7, CH	3.03, overlapped	74.7, CH	3.02, overlapped
3	75.5, CH	3.03, overlapped	75.5, CH	3.02, overlapped
4	47.3, CH	1.00, m	47.4, CH	0.98, m
5	38.0, C		37.9, C
6	42.3, CH2	1.61, overlapped 0.89, m	40.5, CH2	1.67, m 0.83, m
7	32.4, CH	1.74, m	32.7, CH	1.72, overlapped
8	28.5, CH2	1.92, m 1.61, overlapped	29.7, CH2	1.89, m 1.72, overlapped
9	120.1, CH	5.31, d (5.1)	120.2, CH	5.30, d (5.0)
10	141.0, C		140.9, C
11	44.4, CH	2.15, overlapped	43.8, CH	2.16, overlapped
12	177.0, C		176.9, C
13	13.9, CH3	1.01, d (7.0)	13.9, CH3	1.03, d (6.9)
14	19.1, CH3	0.88, s	19.1, CH3	0.89, overlapped
15	10.8, CH3	0.86, d (6.7)	10.8, CH3	0.89, overlapped

) revealed the presence of one olefinic proton (δ_H 5.31 (1H, d, J = 5.1 Hz, H-9)), five sp³ methines with two of them being attached to oxygen atoms (δ_H 3.03 (1H, overlapped, H-2), 3.03 (1H, overlapped, H-3), 1.00 (1H, m, H-4), 1.74 (1H, dd, J = 7.0, 2.5 Hz, H-7), 2.15 (1H, overlapped, H-11)), three methylenes (δ_H 2.15 (1H, overlapped, H-1a), 2.06 (1H, overlapped, H-1b), 1.61 (1H, overlapped, H-6a), 0.89 (1H, m, H-6b), 1.61 (1H, overlapped, H-8a), 1.92 (1H, m, H-8b)) and three methyls (δ_H 1.01 (3H, d, J = 7.0 Hz, H₃-13), 0.88 (3H, s, H₃-14), 0.86 (3H, d, J = 6.7 Hz, H₃-15)). In addition to the aforementioned 12 proton linked carbon signals, the ¹³C NMR and HSQC data also showed three additional carbon signals, including one carboxylic carbonyl (δ_C 177.0 (C-12)), one olefinic carbon (δ_C 141.0 (C-10)) and one quaternary carbon (δ_C 38.0 (C-5)). The NMR data were similar to those of the sesquiterpenoid, artefreynic acid A [16]. Combined with the two spin systems H₂-1/H-2/H-3/H-4/H₃-15 and H₂-6/H-7/H₂-8/H-9 observed in the ¹H-¹H COSY spectrum, as well as the HMBC correlations of H₂-1/C-5, C-9, C-10 and H₃-14/C-4, C-5, C-6 (Figure 2A), 1 was shown to be a sesquiterpenoid derivative with a 6/6 bicyclic skeleton, with the two methyl groups attached to C-4 and C-5, respectively. The HMBC correlation of H-7, H₃-13/C-12 and ¹H-¹H COSY correlation of H-7/H-11/H₃-13 confirmed the presence of a branched carboxylic acid moiety attached at C-7. Considering the chemical shifts of CH-2 (δ_H/C 3.03/74.7) and CH-3 (δ_H/C 3.03/75.5) as well as the molecular formula, it was deduced that the two hydroxyl groups were attached to C-2 and C-3, respectively. Based on these data, the planar structure of 1 was established as shown in Figure 1. The relative configuration of the rings in 1 was presumed to be rel-(2S, 3S, 4R, 5R, 7S) based on the NOESY correlations of H-2/H-4, H₃-15/H-3, H₃-15/H₃-14 and H₃-14/H-7 (Figure 2B). However, the peaks of H-2 and H-3 overlapped heavily and the configuration of the CH-11 side chain could not be unambiguously determined. Finally, the absolute configuration of 1 was established as 2S, 3S, 4R, 5R, 7S, 13R by X-ray diffraction analysis (Figure 3), and the compound was named talaroterpene A (1).

Compound 2 was obtained as colorless needles and its molecular formula was determined as C₁₅H₂₄O₄ by HRESIMS data, indicating the same degree of unsaturation as 1. Analysis of the 1D NMR data (Table 1) showed that 2 was similar to 1. Careful analysis of the 2D NMR data of 2 and 1 (Figure 2) revealed that they were diastereomers with the same planar structure. Based on the NOESY correlations of H-2/H-4, H₃-15/H-3, H₃-15/H₃-14 and H₃-14/H-7, the relative configuration of 2 was presumed to be rel-(2S, 3S, 4R, 5R, 7S), which was the same as that of 1, indicating that the difference might lie in the configuration of the CH-11 side chain. The absolute configuration of 2 was finally established as 2S, 3S, 4R, 5R, 7S, 13S by X-ray diffraction analysis (Figure 3), and 2 was named talaroterpene B.

Compound 3 was obtained as colorless crystals. Its molecular formula was determined as C₁₅H₂₂O₄ by HRESIMS data, indicating 5 degrees of unsaturation, which was one higher than that of 1/2. Analysis of the 1D NMR data of 3 (

Table 2. ¹H (500 MHz) and ¹³C (125 MHz) NMR data of 3 and 4 in DMSO-d₆.

Pos.	3		4
	δC Type	δH (J in Hz)	δC Type	δH (J in Hz)
1	39.3, CH2	2.19, dd (13.3, 4.8) 2.11, d (10.3)	30.4, CH2	2.22, m 2.10, dt (14.0, 4.0)
2	74.8, CH	3.04, overlapped	36.0, CH2	1.88, m 1.13, m
3	75.5, CH	3.04, overlapped	69.6, CH	3.29, m
4	47.3, CH	1.05, overlapped	49.7, CH	0.96, m
5	38.5, C		38.3, C
6	43.4, CH2	1.70, d (12.5) 1.05, overlapped	40.1, CH2	1.65, dd (12.9, 5.0) 1.06, m
7	31.2, CH	2.06, m	38.2, CH	2.59, m
8	31.1, CH2	2.64, m 1.81, m	74.6, CH	3.97, m
9	120.3, CH	5.36, d (5.4)	117.3, CH	5.45, s
10	140.9, C		149.1, C
11	146.2, C		153.7, C
12	168.4, C		69.6, CH2	4.35, d (13.4) 4.10, d (13.4)
13	121.9, CH2	6.04, s 5.51, s	103.8, CH2	5.00, d (1.25) 4.90, s
14	19.1, CH3	0.94, s	17.7, CH3	0.90, s
15	10.8, CH3	0.89, d (6.7)	11.2, CH3	0.93, d (4.7)

) revealed similarities with 1/2, except for the presence of two additional olefinic carbon–hydrogen signals (δ_H/C 6.04, 5.51/103.8; δ_C 146.2) and the absence of one methyl and one methine signal, suggesting that the -CH-CH₃ moiety of the side chain had been oxidized to a double bond. The HMBC correlations of H₂-13/C-7, 11, 12 (Figure 2A) of 3 confirmed this hypothesis. Based on the NOESY correlations of H-2/H-4, H₃-15/H-3, H₃-15/H₃-14 and H₃-14/H-7, the relative configuration of 3 was presumed to be rel-(2S, 3S, 4R, 5R, 7S) which was the same as that of 1/2, suggesting that they shared the same relative configuration. Finally, the absolute configuration of 3 was confirmed as 2S, 3S, 4R, 5R, 7S by X-ray diffraction analysis, and 3 was named talaroterpene C.

Compound 4 was obtained as a colorless oil, and its molecular formula was found to be two oxygen atoms less than that of 3. Analysis of the NMR data (Table 2) showed that 4 was similar to 3, except for the appreciable differences in the presence of CH₂-2, CH-8, CH₂-12 and a tetrahydrofuran ring in 4 versus the presence of CH-2, CH₂-8 and CO-12 in 3. The ¹H-¹H COSY correlations of H₂-1/H₂-2/H-3/H-4/H₃-15 indicated that 4 was one hydroxy group less at C-2 compared to 3. The HMBC correlations (Figure 2A) of H₂-12/C-7, C-8, C-11 and H₂-13/C-7, C-11, C-12 of 4 confirmed the formation of a tetrahydrofuran ring. The relative configuration of the rings in 4 was determined to be rel-(3R, 4R, 5R, 7S, 8S) based on the NOESY correlations (Figure 2B) of H₃-15/H-3, H₃-15/H₃-14, H₃-14/H-7, H₃-14/H-8 and H-7/H-8. Based on the calculated electronic circular dichroism (ECD) for the 3R, 4R, 5R, 7S, 8S and 3S, 4S, 5S, 7R, 8R configuration (Figure 4), the experimental ECD spectrum of 4 showed a good match with the calculated one with the 3R, 4R, 5R, 7S, 8S configuration. Thus, 4 was named talaroterpene D.

2.2. Bioactivity Assay

For this work, compounds 1–4 were evaluated with their regulatory activities on nuclear receptors in HepG2 cells [10]. Firstly, CCK8 assay was taken to evaluate the toxicity against HepG2 cells (Figure 5). Compounds 2 and 4 are non-toxic to cells at concentrations of 200 μM and below. Compounds 1 and 3 showed weak cytotoxic effects on cells at concentration of 200 μM, with IC₅₀ > 500 μM.

It was observed that talaroterpene D (4), with a non-toxic concentration of 200 μM, notably stimulated LXRα and its downstream target gene ABCA1, and significantly suppressed the expression of FXR and downstream CYP7A1. Compounds 1 and 3 (200 μM) could also stimulate expression of ABCA1, but there were no obvious activities with respect to LXRα, FXR and CYP7A1. Additionally, compounds 1, 3 and 4 (200 μM) exhibited varying degrees of activation on PPARα. Talaroterpene D (4) also showed activation on downstream target genes CPT1α and ACOX1. Furthermore, talaroterpene D (4) showed minimal impact on RORα itself; however, it induced significant alterations in the expression of downstream target genes CLOCK and BMAL1 of RORα, with a concentration of 200 μM, possibly due to direct binding interactions with RORα (Figure 6).

To further elucidate the binding mode of compounds 1–4 with RORα, a homology model of RORα (PDB code: 1N83) was selected for in silico molecular docking analysis [17]. The docking results demonstrated that compound 4 fits comfortably within the binding pocket of RORα (Figure 7A), yielding a binding score of −8.613. The 2D binding models of 4 with RORα (Figure 7B) revealed that the hydroxy group forms hydrogen bonds with the active-site residue TYR380. Similarly, the hydroxyl groups of compounds 1–3 form hydrogen bond interactions with TYR380 in the RORα (Figures S41–S43). The above results indicate that the presence of the hydroxyl group is beneficial for enhancing the binding interactions between the compounds and RORα. Compounds 1–3 all contain a carboxyl group, which under normal physiological pH conditions, typically exists in an ionized form, leading to a decrease in lipophilicity, thus hindering drug absorption and ultimately resulting in reduced biological activity, suggesting that the presence of the carboxyl group is not favorable for enhancing the RORα activity. Additionally, 3 experiences spatial conflict between the alkene group on the side chain and the RORα active site, leading to a reduction in its interactions with RORα. In contrast, compound 4, which also contains an alkene group, has a larger gap between the cyclic alkene and the RORα active site due to the cyclization, thereby eliminating the spatial conflict and enhancing its interactions with RORα. For future structural optimization and modification, it is promising to explore the possibility of cyclizing the carboxyl-containing side chains, which may further improve the biological activity of talaroterpenes.

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotations were determined using an Anton Paar MPC 500 polarimeter (Anton, Graz, Austria). UV and IR spectra were recorded using a Shimadzu UV-2600 PC spectrometer (Shimadzu, Beijing, China) and an IR Affinity-1 spectrometer (Shimadzu), respectively. A Quantum-I Plus 500 Hz NMR spectrometer (Q-one Instrument Co., Ltd., Wuhan, China) operating at 500 MHz for ¹H and 125 MHz for ¹³C was used. HRESIMS were acquired on a Bruker maXis Q-TOF mass spectrometer (Bruker BioSpin International AG, Fällanden, Switzerland). HPLC was performed on the Hitachi Primaide (Hitachi, Tokyo, Japan) with a DAD detector, using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 μm). X-ray diffraction was performed on an XtalLAB PRO diffractometer (Rigaku, Akishima-shi, Japan) with Cu Kα radiation.

3.2. Fungal Material

A fungal strain identified as Talaromyces sp. SCSIO 41412 was isolated from a sediment sample obtained from the Gaoqiao Mangrove in Zhanjiang City, Guangdong Province, China, in August 2021. The taxonomic assignment of this fungus was based on analysis of the internally transcribed spacer (ITS) region of the ribosomal DNA (rDNA), and the ITS sequence has been deposited in GenBank under accession number PP001498. The fungal strain was stored on malt extract agar (15 g malt extract, 18 g agar, 10 g sea salt and 1 L water) at 4 °C, and deposited in the CAS Key Laboratory of Tropical Marine Bioresources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.

3.3. Fermentation, Extraction and Isolation

The fungal strain Talaromyces sp. SCSIO 41412 was cultured in 200 mL of seed medium (15 g malt extract, 10 g sea salt and 1 L water) on a rotary shaker (180 rpm) at 28 °C for 3 days. This seed culture was then used to inoculate a large-scale fermentation, which was incubated statically at 26 °C for 28 days using a rice medium (200 g rice, 2% sea salt and 230 mL water) in 47 Erlenmeyer flasks. The entire fermented culture was extracted with ethyl acetate (EtOAc) three times, yielding a total extract of 326.1 g. The EtOAc extract was chromatographed over a silica gel column eluted with CH₂Cl₂/petroleum ether (0:1, 1:1, 1:0) and CH₃OH/CH₂Cl₂ (1:99, 2:98, 3:97, 5:95, 10:90, 20:80, 50:50) to yield ten fractions (Frs.1–10). Fr.4 was subjected to ODS silica gel chromatography and eluted with CH₃OH/H₂O (5–100%) to yield 14 subfractions. Fr.4-3 was further separated by semi-preparative HPLC (50% CH₃CN/H₂O, 3 mL/min, YMC-pack ODS-A, 10 × 250 mm, 5 μm) to afford Fr.4-3-3, which was then purified by HPLC (68% CH₃OH/H₂O, 2.5 mL/min) to yield 4 (6.8 mg, t_R = 15.5 min). Fr.6 was subjected to ODS silica gel chromatography and eluted with CH₃OH/H₂O (5–100%) to yield 10 subfractions. Fr.6-4 was separated by semi-preparative HPLC (33% CH₃CN/H₂O, 3.0 mL/min, YMC-pack ODS-A, 10 × 250 mm, 5 μm) to afford Fr.6-4-1, which was further separated by semi-preparative HPLC (50% CH₃CN/H₂O, 3.0 mL/min, YMC-pack ODS-A, 10 × 250 mm, 5 μm) to yield 2 (9.9 mg, t_R = 16.0 min), 1 (119.8 mg, t_R = 16.2 min) and 3 (49.1 mg, t_R = 16.9 min).

3.4. Spectroscopic Data of Compounds

Talaroterpene A (1): colorless needles; ${\left[\alpha \right]}_{ \mathrm{D}}^{25}$ +14.5 (c 0.1, CH₃OH); ECD (0.3 mg/mL, CH₃OH) λ_max (Δε) 224 (−2.36); UV (CH₃OH) λ_max (log ε) 200 (3.88), 320 (2.73) nm; IR ν_max 3398, 1697, 1558, 1541, 1456, 1047 cm⁻¹; ¹H and ¹³C NMR, Table 1; HRESIMS m/z 286.2014 [M + NH₄]⁺ (calcd for C₁₅H₂₈NO₄⁺, 286.2013).

Talaroterpene B (2): colorless needles; ${\left[\alpha \right]}_{ \mathrm{D}}^{25}$ +18.8 (c 0.1, CH₃OH); ECD (0.3 mg/mL, CH₃OH) λ_max (Δε) 202 (−2.35); UV (CH₃OH) λ_max (log ε) 200 (3.81) nm; IR ν_max 3375, 2968, 2914, 1703, 1550, 1448, 1373, 1251, 1199, 1041, 1024 cm⁻¹; ¹H and ¹³C NMR, Table 1; HRESIMS m/z 286.2018 [M + NH₄]⁺ (calcd for C₁₅H₂₈NO₄⁺, 286.2013).

Talar terpene C (3): colorless block crystal; ${\left[\alpha \right]}_{ \mathrm{D}}^{25}$ +23.5 (c 0.1, CH₃OH); ECD (0.3 mg/mL, CH₃OH) λ_max (Δε) 224 (−2.94); UV (CH₃OH) λ_max (log ε) 200 (3.89) nm; IR ν_max 3338, 1681, 1541, 1373, 1253, 1041 cm⁻¹; ¹H and ¹³C NMR, Table 2; HRESIMS m/z 265.1451 [M-H]⁻ (calcd for C₁₅H₂₁O₄⁻, 265.1445).

Talaroterpene D (4): colorless oil; ${\left[\alpha \right]}_{ \mathrm{D}}^{25}$ +3.0 (c 0.1, CH₃OH); ECD (0.3 mg/mL, CH₃OH) λ_max (Δε) 203 (−27.70); UV (CH₃OH) λ_max (log ε) 200 (3.90) nm; IR ν_max 3392, 1653, 1373, 1026 cm⁻¹; ¹H and ¹³C NMR, Table 2; HRESIMS m/z 235.1694 [M + H]⁺ (calcd for C₁₅H₂₃O₂⁺, 235.1693).

3.5. X-ray Crystallographic Analysis

The X-ray diffraction data for 1–3 were collected using an XtaLAB PRO diffractometer with Cu Kα radiation, with the crystals grown from methanol by slow evaporation. The crystal structures were solved using SHELXS97, expanded through difference Fourier techniques, and then refined by full-matrix least-squares methods. All non-hydrogen atoms were refined anisotropically, and the hydrogen atoms were fixed at calculated positions. The crystallographic data for 1–3 have been deposited in the Cambridge Crystallographic Data Centre.

Crystal Data for Talaroterpene A (1): C₁₅H₂₄O₄, M = 268.34, monoclinic, space group P2₁, a = 10.23266 (8) Å, b = 6.72249 (4) Å, c = 11.56363(9) Å, β = 115.5824(10)°, V = 717.467 (11) ų, Z = 2, T = 100.00 (10) K, μ(Cu Kα) = 0.719 mm⁻¹, D_calc = 1.242 g/cm³, 13,788 reflections measured (8.478° ≤ 2Θ ≤ 148.13°), 2843 unique (R_int = 0.0420, R_sigma = 0.0236) which were used in all calculations. The final R₁ was 0.0280 (I > 2σ(I)) and wR₂ was 0.0724. The Flack parameter was 0.06 (7) (CCDC 2372513).

Crystal Data for Talaroterpene B (2): C₁₅H₂₄O₄, M = 268.34, monoclinic, space group P2₁, a = 9.09200 (10) Å, b = 7.93080 (10) Å, c = 9.95020 (10) Å, β = 96.4630 (10)°, V = 712.918 (14) ų, Z = 2, T = 100.00 (10) K, μ (Cu Kα) = 0.724 mm⁻¹, D_calc = 1.250 g/cm³, 10,853 reflections measured (8.944° ≤ 2Θ ≤ 148.192°), 2834 unique (R_int = 0.0223, R_sigma = 0.0158) which were used in all calculations. The final R₁ was 0.0289 (I > 2σ(I)) and wR₂ was 0.0787. The Flack parameter was 0.09 (5) (CDCC 2372514).

Crystal Data for Talaroterpene C (3): C₁₅H₂₂O₄, M = 266.32, orthorhombic, space group P2₁2₁2₁, a = 7.24180 (10) Å, b = 11.42500 (10) Å, c = 16.6083 (2) Å, V = 1374.13 (3) ų, Z = 4, T = 99.99 (10) K, μ(Cu Kα) = 0.751 mm⁻¹, D_calc = 1.287 g/cm³, 12,963 reflections measured (9.396° ≤ 2Θ ≤ 148.8°), 2750 unique (R_int = 0.0569, R_sigma = 0.0327) which were used in all calculations. The final R₁ was 0.0433 (I > 2σ(I)) and wR₂ was 0.1198. The Flack parameter was 0.09 (13) (CDCC 2372529).

3.6. ECD Computation Section

Compound 4 was subjected to conformational searching in Spartan’14 (v1.1.4, Wavefunction, Irvine, CA, USA) using the Molecular Merck force field. The stable conformers of 99% (the relative energy within 2 kcal/mol) were then optimized in methanol solvent at the B3LYP/6-31G (d) level of theory using Gaussian 09 (D.01, Pittsburgh, PA, USA). The optimized low-energy conformations were further analyzed using TDDFT with a polarizable continuum model at the B3LYP/6-311G (d, p) level [18]. The calculated ECD spectra were generated from GaussView 6.0 and Origin 2021, with a half-bandwidth of 0.3 eV, wavelength-corrected by the calculated UV curve, and Boltzmann-weighted to obtain the final calculated ECD spectra.

3.7. Cell Culture

HepG2 cells were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences, and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, New York, NY, USA) supplemented with 10% fetal bovine serum (FBS, ExCell Bio, Suzhou, China) at 37 °C under 5% CO₂. Mother solutions of 20 mM 1–4 were dissolved in dimethyl sulfoxide (DMSO, Tianjin Fuyu Chemical and Industry Factory, Tianjin, China) and then diluted to 200 μM with the cell-culture medium. The control group was treated with a medium containing 1‰ DMSO.

3.8. Cytotoxic Bioassay

Cell viability was assessed using the CCK-8 assay (Dojindo, Kumamoto, Japan). Initially, cells were plated at a density of 3000 cells/well in 96-well plates and exposed to varying concentrations of compounds or a solvent control. Following a 24 h incubation period, CCK-8 reagent was introduced, and the absorbance of triplicate samples was measured at 450 nm using an Envision 2104 multilabel reader (PerkinElmer, Waltham, MA, USA). Dose-response curves were generated to determine the IC₅₀ values with Prism 5.0 software (GraphPad, San Diego, CA, USA).

3.9. RT-qPCR

Total RNA was extracted using the RNAprep Kit (RE-03014, FOREGENE, Chengdu, China) and reverse transcribed to cDNA using RT Master Mix (RR037A, Takara, Shiga, Japan). RT-qPCR was performed using SYBR Green Master Mix (A6002, Promega, Madison, WI, USA). The data were normalized to the housekeeping gene GAPDH. Primers used are listed in Table S1.

3.10. Statistical Analysis

Prism 9 software (GraphPad, San Diego, CA, USA) was used to perform statistical tests. Groups (n = 6) were compared by one-way analysis of variance (ANOVA). Nonparametric data were log-transformed for statistical analysis, or when this failed to normalize the data, Mann–Whitney tests were used. p < 0.05 was significant (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared to the control group).

4. Conclusions

In summary, this study identified four new sesquiterpenoid compounds, talaroterpenes A–D (1–4), isolated from the mangrove-derived fungus Talaromyces sp. SCSIO 41412. Through detailed structural characterization, the absolute configurations of these compounds were elucidated. Those new sesquiterpenoids were evaluated with their regulatory activities on nuclear receptors in HepG2 cells. Under the non-toxic concentration of 200 μM, 1, 3 and 4 exhibited varying degrees of activation on ABCA1 (downstream target of LXRα) and PPARα, while 4 showed the strongest activities. Furthermore, 4 induced significant alterations in the expression of downstream target genes CLOCK and BMAL1 of RORα, and the in silico molecular docking analysis supported the direct binding interactions of 4 with RORα protein. This study revealed that the sesquiterpenoid talaroterpene D (4), as a new potential non-toxic modulator of nuclear receptors, holds promise as a lead compound for the development of candidate drugs for metabolic or cardiovascular diseases.