Talaroacids A–D and Talaromarane A, Diterpenoids with Anti-Inflammatory Activities from Mangrove Endophytic Fungus Talaromyces sp. JNQQJ-4

Abstract

Five new diterpenes including four diterpenes with 1,2,3,4,4a,5,6,8a-octalin skeleton talaroacids A–D (1–4) and an isopimarane diterpenoid talaromarane A (5) were isolated from the mangrove endophytic fungus Talaromyces sp. JNQQJ-4. Their structures and absolute configurations were determined by analysis of high-resolution electrospray ionization mass spectroscopy (HRESIMS), 1D/2D Nuclear Magnetic Resonance (NMR) spectra, single-crystal X-ray diffraction, quantum chemical calculation, and electronic circular dichroism (ECD). Talaromarane A (5) contains a rare 2-oxabicyclo [3.2.1] octan moiety in isopimarane diterpenoids. In bioassays, compounds 1, 2, 4, and 5 displayed significant anti-inflammatory activities with the IC₅₀ value from 4.59 to 21.60 μM.

1. Introduction

Diterpenoids are a class of terpenoids containing 20 carbons and consist of four isopentenyl groups, which are widely distributed in animals, plants, and microorganisms [1]. More than 100 basic diterpene skeletons have been found, which can be divided into linear, monocarbocyclic, bicarbocyclic, tricarbocyclic, tetracarbocyclic, and pentacarbocyclic [2]. Among diverse skeleton types of diterpenes, diterpenes with decalin skeleton mainly include labdane [3], clerodane [4], and other types of diterpenes [5]. These diterpenes have a variety of pharmacological activities including anticancer [6,7], anti-inflammatory [8,9,10], antiparasitical [11], antiviral [12], enzyme inhibition [13,14], immunosuppressive [15], anti-angiogenesis [16], and antidiabetic [17].

Mangrove ecosystems are usually located at the junction of land and ocean in tropical and subtropical regions and have abundant plant resources [18]. These mangrove plants, such as Kandelia obovata, were often used as traditional folk medicines [19]. In addition, due to the extreme environment of high salt, high temperature, local hypoxia, and periodic seawater immersion in mangroves, there are a variety of endophytic fungi resources [20]. Mangrove endophytic fungi can produce secondary metabolites with unique structures and remarkable biological activities, which capture the attention of numerous natural products and pharmacology researchers [21,22,23,24,25,26,27,28]. To date, more than 1300 new compounds have been identified from mangrove-derived fungi [29]. In our ongoing research search for bioactive compounds from mangrove endophytic fungi [30,31,32,33], the strain Talaromyces sp. JNQQJ-4 isolated from the leaf of Kandelia obovata was investigated. Four new diterpenes with 1,2,3,4,4a,5,6,8a-octalin skeleton talaroacids A–D (1–4) and a new isopimarane diterpenoid talaromarane A (5) were isolated from Talaromyces sp. JNQQJ-4 (Figure 1). In bioassay, compounds 1, 2, 4, and 5 indicated significant anti-inflammatory activities with IC₅₀ values from 4.59 to 21.60 µM. Herein, the isolation, structure elucidation, and biological assays of isolated diterpenoids are described.

2. Results and Discussion

2.1. Structure Identification

Talaroacid A (1) was obtained as a white powder, and had a molecular formula of C₂₀H₃₂O₃ with five degrees of unsaturation based on the HRESIMS (Figure S1) data. The ¹H NMR spectrum (

Table 1. ¹H NMR (600 MHz) data of compounds 1–5 (δ_H in ppm, J in Hz).

No.	1 b	2 a	3 b	4 a	5 a
1a	1.67, m	1.68, m	1.73, m	1.68, m	2.05, m
1b	1.20, overlap	1.22, overlap	1.26, m	1.19, m
2a	1.65, m	1.45, m	1.64, m	1.69, m	1.91, m
2b	1.39, overlap	1.21, overlap	1.48, overlap	1.21, m
3a	1.38, m	1.39, m	1.43, m	1.38, m	4.70, dd (3.5, 2.2)
3b	1.15, m	1.16, m	1.19, m	1.15, m
5	1.18, m	1.22, overlap	1.27, overlap	1.20, overlap
6a	1.57, m	1.58, m	1.74, overlap	1.58, m
6b	1.47, m	1.27, m	1.27, overlap	1.45, m
7	1.99, m	2.04, m	2.05, m	2.08, m	4.74, s
11a	3.23, d (17.4)	3.10, d (17.3)	3.03, d (16.7)	3.20, d (17.4)	1.76, m
11b	3.04, d (17.4)	2.99, d (17.3)	2.90, d (16.7)	3.00, d (17.4)	1.69, m
12a					1.96, m
12b					1.49, m
13a	2.84, d (14.6)	2.78, d (16.0)	5.65, s	1.88, d (7.5)
13b	2.71, d (14.6)	2.54 d (16.0)
14				1.82, m	5.88, s
15a	5.49, m	5.32, t (7.1)	2.23, t (7.1)	1.59, m	5.82, dd (17.5, 10.6)
15b				1.36, m
16a	4.18, m	4.15, d (7.1)	3.62, t (7.1)	3.68, m	5.04, dd (17.5, 1.0)
16b					4.99, dd (10.6, 1.0)
17	1.62, s	1.62, s	1.56, s	0.84, overlap	1.00, s
18	0.83, s	0.84, s	0.87, s	0.83, s	1.27, s
19	0.89, s	0.89, s	0.91, s	0.89, s	1.30, s
20	0.95, s	0.98, s	1.02, s	0.95, s
22					2.13, s

^a Measured in CDCl₃, ^b Measured in MeOH-d₄.

and Figure S2) indicated four methyl groups at δ_H 1.62 (s, H₃-17), 0.95 (s, H₃-20), 0.89 (s, H₃-19), and 0.83 (s, H₃-18); an olefinic proton signal at δ_H 5.49 (m, H-15). The ¹³C NMR (

Table 2. ¹³C NMR (150 MHz) data of compounds 1–5 (δ_C in ppm).

No.	1 b	2 a	3 b	4 a	5 a
1	36.3, CH2	36.2, CH2,	37.4, CH2	36.4, CH2,	14.4, CH2
2	18.9, CH2	18.9, CH2	19.9, CH2	19.1, CH2	21.8, CH2
3	41.6, CH2	41.6, CH2	42.9, CH2	41.6, CH2	80.2, CH
4	33.4, C	33.5, C	34.2, C	33.5, C	40.8, C
5	51.4, CH	51.4, CH	52.7, CH	51.4, CH	83.3, C
6	19.0, CH2	19.0 c, CH2	20.0, CH2	19.0, CH2	105.0, C
7	31.1, CH2	31.9, CH2	32.6, CH2	31.1, CH2	70.9, CH
8	132.1, C	132.0, C	134.1, C	133.6, C	135.4, C
9	135.8, C	136.7, C	137.6, C	135.2, C	73.2, C
10	39.1, C	39.1, C	39.7, C	39.1, C	55.9, C
11	32.7, CH2	32.7, CH2	34.7, CH2	32.8, CH2	27.2, CH2
12	178.0, C	177.0, C	176.9, C	171.2, C	29.6, CH2
13	35.9, CH2	43.3, CH2	129.2, CH	41.2, CH2	38.3, C
14	138.3, C	137.8, C	135.1, C	28.4, CH	135.9, CH
15	125.6, CH	123.2, CH	43.0, CH2	40.1, CH2	146.9, CH
16	59.1, CH2	59.5, CH2	61.8, CH2	61.2, CH2	111.7, CH2
17	22.9, CH3	16.8, CH3	17.2, CH3	19.4, CH3	24.3, CH3
18	21.8, CH3	21.8, CH3	22.1, CH3	21.8, CH3	24.4, CH3
19	33.3, CH3	33.3, CH3	33.7, CH3	33.3, CH3	22.2, CH3
20	19.9, CH3	20.2, CH3	20.4, CH3	20.2, CH3	172.7, C
21					168.9, C
22					21.4, CH3

^a Measured in CDCl₃, ^b Measured in MeOH-d₄, ^c overlap in ¹³C NMR.

and Figure S3) and HSQC spectra (Figure S4) data of 1 exhibited 20 carbon signals, including a carbonyl carbon, four methyls, four olefinic carbons (three non-hydrogenated carbons), eight methylenes (an oxygenated), a methine, and two quaternary carbons.

The key HMBC correlations (Figure 2 and Figure S6) from H₂-7 to C-8 and C-9; from H₃-18/H₃-19 to C-3, C-4, and C-5; from H₃-20 to C-1, C-5, C-9, and C-10 together with the spin system of H₂-1/H₂-2/H₂-3 and H-5/H₂-6/H₂-7 indicated the presence of a 5,5,9-trimethyl-Δ^1,2-octalin moiety of 1. The HMBC correlations from H₂-11 to C-8, C-9, C-10, and C-11 (δ_C 178.0) revealed the branched chain of acetic acid located at C-9. Furthermore, the spin coupling system (Figure S5) of H-15/H₂-16 and the HMBC correlations from H₃-17 to C-13, C-14, and C-15; from H₂-16 to C-13; from H₂-13 to C-7, C-8, and C-9 indicated the fragment of 3-methylbut-2-en-1-ol was linked to C-8. Thus, the planar structure of 1 was established and shown. The NOESY correlations (Figure 3 and Figure S7) between H-15 and H₃-17 ensure the configuration of the Δ¹⁴ double bond as 14Z. Furthermore, the NOESY correlations of H₃-18/H₃-20 revealed they were positioned on the same face. In turn, the correlation of H-5/H₃-19 suggested they were at the opposite orientation. Based on the above information, the relative configuration of 1 was assigned to 5S* and 10S*. Finally, the absolute configuration of 1 was determined as 5S, 10S, and 14Z based on a comparison of experimental and calculated ECD spectra (Figure 4).

Talaroacid B (2) was obtained as a white powder. The HRESIMS data (Figure S8) suggested that 2 had the same molecular formula as that of 1. The NMR data (Table 1 and Table 2; Figures S9–S11) closely resembled those of 1, except for the chemical shift at C-15 (Δδ_c −2.4). The ¹H-¹H COSY spectra and (Figure S12) and HMBC spectra (Figure S13) also indicate that compounds 2 and 1 have similar planar structures. The NOESY correlation (Figure 3 and Figure S14) of H-15/H₂-13 indicated that the configuration of Δ¹⁴ double bond of 2 was 14E. Furthermore, the absolute configuration of 2 was determined as 5S, 10S, and 14E according to the NOESY correlations and ECD calculation (Figure 4).

Talaroacid C (3) was obtained as a white powder and shared the same molecular formula as that of 2 based on the HRESIMS data (Figure S15). Comparing the NMR data (Table 1 and Table 2; Figures S16–S18) of compounds 3 and 2 showed that 3 had a similar structure to 2. While, the ¹H-¹H COSY spectrum (Figure S19) and the HMBC correlations (Figure 2 and Figure S20) from H₂-15 to C-13 (δ_C 129.2), C-14 (δ_C 135.1), and C-17 (δ_C 17.2); from H-13 (δ_H 5.65) to C-7 (δ_C 32.6), C-8 (δ_C 134.1), and C-9 (δ_C 137.6) revealed that the Δ¹⁴ double bond in 2 has changed to Δ¹³ double bond in 3. Then, the configuration of Δ¹³ double bond of 3 was assigned as 13E based on the NOESY correlation of H-13/H₂-15 (Figure 3 and Figure S21). Finally, the analysis of NOESY correlations and ECD calculation (Figure 4) determined the absolute configuration of 3 as 5S, 10S, and 13E.

Talaroacid D (4), a white powder, had a molecular formula of C₂₀H₃₄O₃ and 4 degrees of unsaturation according to the HRESIMS data (Figure S22). The structure of 4 was similar to 2 by comparison of their NMR data (Table 1 and Table 2; Figures S23–S25). The main difference was the Δ¹⁴ double bond in 2 was reduced in 4. The deduction was further confirmed by the ¹H-¹H correlations (Figure 2 and Figure S26) of H₂-13/H-14(H₃-17)/H₂-15/H₂-16 and the HMBC correlations (Figure 2 and Figure S27) from H₃-17 to C-13 (δ_C 41.2), C-14 (δ_C 28.4), and C-15 (δ_C 40.1). Thus, the planar structure of 4 was established. According to the similar NOESY correlations (Figure 3 and Figure S28), the relative configuration of 4 was assigned to 5S* 10S*. Furthermore, to determine the relative configuration of C-14 in the side chain, the ¹³C NMR calculations of two possible structures (5S*,10S*,14S*)-4 and (5S*,10S*,14R*)-4 were performed using the gauge-including atomic orbital (GIAO) method at mPW1PW91-SCRF/6-311+G (d,p)/PCM (Chloroform). The results indicated that (5S*,10S*,14S*)-4 was a reasonable structure (Figure 5, Figures S36 and S37) with a better correlation coefficient (R² = 0.9985) and a high DP4+ probability score at 100% (all data). Finally, the absolute configuration of 4 was determined as 5S, 10S, and 14S based on the same experimental and calculated ECD spectra (Figure 4).

Talaromarane A (5) was obtained as a colorless crystal with the molecular formula of C₂₂H₃₀O₈ and 8 degrees of unsaturation according to the HRESIMS data (Figure S29). The ¹H NMR spectrum (Table 1 and Figure S30) revealed two hydroxyl proton signals at δ_H 4.94 (s, OH-5), and 3.90 (s, OH-7); four methyls at δ_H 2.13 (s, H₃-22), 1.30 (s, H₃-18), 1.27 (s, H₃-19), and 1.00 (s, H₃-17); four olefinic proton signals at δ_H 5.88 (s, H-14), 5.82 (dd, J = 17.5, 10.6 Hz, H-15), 5.04 (dd, J = 17.5, 1.0 Hz, H-16a), and 4.99 (dd, J = 10.6, 1.0 Hz, H-16b). Analysis ¹³C NMR (Table 2 and Figure S31) and HSQC data (Figure S32) to obtain 22 carbons including four methyls, five methylenes (one olefinic), four methines (two olefinic and an oxygenated), six non-hydrogenated carbons (two carbonyl carbons, three oxygenated and an olefinic), and three quaternary carbons. These data suggested that 5 belongs to an isopimarane diterpene [30]. The ¹H-¹H COSY correlations (Figure 2 and Figure S33) of H₂-1/H₂-2/H-3, H₂-11/H₂-12, and H-15/H₂-16 together with the HMBC correlations from H-1 to C-10 and C-20; H₃-18/19 to C-3, C-4 and C-5; H-7 to C-5, C-6, C-8, C-9, and C-14; H₃-17 to C-12, C-13, and C-14; H-15 to C-14 and from H-11 to C-9 and C-10 to establish a typical tricyclic isopimarane diterpene skeleton. The acetyl group was located at C-3 based on the HMBC correlations (Figure 2 and Figure S34) from H₃-22 (δ_H 2.13) and H-3 (δ_H 4.70) to C-21 (δ_C 168.9). The deshielding chemical shift at C-7 (δ_H/δ_C 4.74/70.9) indicates that a hydroxyl group was located at C-7 in 5. Moreover, the HMBC correlations from H-7 (δ_H 4.74) to non-hydrogenated carbons C-5 (δ_C 83.3), C-6 (δ_C 105.0), and C-9 (δ_C 73.2) revealed they were replaced by hydroxy groups, respectively. The HMBC correlations from H-1 to C-20 (δ_C 172.7) and the remaining unsaturation together with the deshielding chemical shift at non-hydrogenated carbon C-6 (δ_C 105.0) indicate that an oxygen bridge between C-7 and C-20. Thus, the plate structure of 5 was established.

The NOESY correlations (Figure S35) of H-3/H-6/H-15/H₃-18 indicated that these protons were in the same orientation. However, due to the absence of key NOE correlations for OH-5, OH-6, and OH-9 in the NOE spectrum (CDCl₃), the relative configurations of 5 were difficult to determine. Luckily, the single crystal of 5 was successfully obtained by slow volatilization in MeOH. Finally, the absolute configuration of 5 was unambiguously determined as 3R, 5R, 6R, 7R, 9R, 10S, and 13R using single crystal X-ray diffraction analysis with a flack parameter of −0.22 (8) (Figure 6). In addition, ECD calculation also verifies the conclusion above (Figure 4).

2.2. Anti-Inflammatory Activities

On RAW264.7 cells test all compound’s cytotoxicity and anti-inflammatory activities (

Table 3. Inhibitory Effects against NO Production of Compounds 1–5 in LPS-Induced RAW264.7 Cells.

Compounds	1	2	3	4	5	Quercetin
IC50 (μM)	15.78	4.59	>50	21.60	13.38	11.33

). The results indicated compound 2 had better anti-inflammatory activities than positive control quercetin (IC₅₀ = 11.33 μM) with IC₅₀ values of 4.59 μM. Compounds 1, 4, and 5 showed moderate anti-inflammatory activities with IC₅₀ values of 15.78, 21.60, and 13.38 μM, respectively. None of the compounds were cytotoxic to RAW264.7 cells at the tested concentrations.

3. Materials and Methods

3.1. General Experiment Procedures

The optical rotations were recorded by using an MCP300 (Anton Paar, Shanghai, China). UV spectrum was obtained using a Shimadzu UV-2600 spectrophotometer (Shimadzu, Kyoto, Japan). The CD spectra were obtained from a J-810 spectropolarimeter in MeOH (JASCO, Tokyo, Japan). The IR data were performed on a Shimadzu IRTrace-100 spectrometer (Shimadzu, Tokyo, Japan) in KBr discs. All NMR data were measured on a Bruker Advance 600 MHz spectrometer at room temperature using the signals of residual solvent protons (CDCl₃: δ_H/δ_C 7.26/77.1; CD₃OD: δ_H/δ_C 3.31/49.2). The HRESIMS data were recorded by using a ThermoFisher LTQ-Orbitrap-LC-MS spectrometer (Palo Alto, CA, USA). Semi-preparative HPLC (Ultimate 3000 BioRS, Thermo Scientific, Waltham, MA, USA) was conducted using a semipreparative column (5 μm, 10 × 250 mm, Ultimate XB-C₁₈, Welch Materials, Inc., Shanghai, China). A Rigaku XtaLAB Pro diffractometer (Rigaku, Tokyo, Japan) was used to obtain the crystallographic data of 5 (Cu Kα radiation). Column chromatography (CC) was performed using silica gel (200–300 mesh, Qingdao Marine Chemical, Qingdao, China) and Sephadex LH-20 (Sigma-aldrich, Saint Louis, MO, USA).

3.2. ECD and NMR Calculations

The ECD calculation was carried out using described previously [30]. The conformers were subjected to geometric optimization at the level of B3LYP/6-31+G (d,p) and the optimized conformers were calculated on the TD-DFT method using the B3LYP/6-311+G (d,p). All NMR calculations were performed using the GIAO method at mPW1PW91-SCRF/6-311+G (d,p)/PCM (Chloroform) [34].

3.3. Plant and Fungal Material

The healthy leaves of Kandelia obovata were collected in Jinniu Island Mangrove Nature Reserve in Guangzhou province of China in July 2023. The plant was identified by Dr. Yayue Liu, Guangdong Ocean University, and voucher sp. (JNQJ202306) is stored at Sun Yat-sen University. The strain Talaromyces sp. JNQQJ-4 was isolated from the healthy leaves of Kandelia obovate. The specific separation process was as follows: the fresh leaf tissue of Kandelia candel was transferred to 3% sodium hypochlorite solution and 75% ethanol solution with sterilized tweezers, and washed with sterile water. The leaf tissue was cut into regular small pieces (about 0.2 × 0.6 cm) and cultured on an autoclaved Bengal Rose agar plate incubated at 28 °C for 3 days. After the colony appeared, the mycelia were picked and inoculated on PDA medium. Repeat the above steps until a pure single colony is obtained on PDA plate. Fungal species were identified using DNA amplification and ITS sequence analysis previously [35]. The strain sequence data were reserved for the GenBank with accession number PP660349, and BLAST analysis revealed that it was 100% homologous to the sequence of Talaromyces sp. (MK450749.1). This strain was preserved at Sun Yat-sen University, China.

3.4. Fermentation, Extraction and Purification

The fungal strain was seeded to sixty 1 L Erlenmeyer flasks with 70 g raw rice and 30 mL 0.3% seawater and incubated at 25 °C for 28 days. The solid rice media were extracted with ethyl acetate and concentrated to obtain 45.9 g of crude extract. Five fractions (Fr.A-Fr.E) were isolated from the extract using silica gel CC (200–300 mesh) eluting with petroleum ether/ethyl acetate gradient (1:0~0:1). Fractions B was purified using CC on silica gel (CH₂Cl₂/MeOH, 80:1) and Sephadex LH-20 (CH₂Cl₂/MeOH, 1:1) to produce subfractions B₁–B₃. Fr. B₂ was purified using semipreparative HPLC (CH₃CN/H₂O/Trifluoroacetic acid, 60:40:0.05, 1.5 mL/min) to obtain compounds 1 (5.3 mg, t_R = 13.5 min), 2 (4.8 mg, t_R = 14.5 min), 3 (4.5 mg, t_R = 17.0 min), and 4 (3.6 mg, t_R = 19.0 min). Fr. C₁–C₄ was obtained by separating fractions C using CC on a silica gel (CH₂Cl₂/MeOH, 75:1). Then, compound 5 (3.3 mg, t_R = 14.2 min) was obtained using semipreparative HPLC (CH₃CN /H₂O, 70:30, 1.5 mL/min) from Fr. C₁.

Talaroacid A (1): white powder.; ${\left[\alpha \right]}_D^{25}$ = 10.4 (c 0.23, MeOH); UV (MeOH) λ_max (log ε): 201 (2.26) nm; ECD (c 0.33 mM, MeOH) λ_max (Δε) 201 (30.5), 218 (20.3) nm; IR (KBr) ν_max: 3326, 2935, 1706, 1445, 1385 and 1180 cm⁻¹; ¹H NMR (500 MHz, MeOH-d₄) data, Table 1; ¹³C NMR (125 MHz, MeOH-d₄) data, Table 2; HRESIMS m/z 343.2246 [M + Na]⁺ (calcd: C₂₀H₃₂O₃Na, 343.2244).

Talaroacid B (2): white powder.; ${\left[\alpha \right]}_D^{25}$ = 8.8 (c 0.25, MeOH); UV (MeOH) λ_max (log ε): 201 (1.56) nm; ECD (c 0.33 mM, MeOH) λ_max (Δε) 203 (27.5) nm; IR (KBr) ν_max: 3328, 2940, 1712, 1447, 1390 and 1175 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) data, Table 1; ¹³C NMR (125 MHz, CDCl₃) data, Table 2; HRESIMS m/z 343.2238 [M + Na]⁺ (calcd: C₂₀H₃₂O₃Na, 343.2244).

Talaroacid C (3): white powder.; ${\left[\alpha \right]}_D^{25}$ = 12.3 (c 0.28, MeOH); UV (MeOH) λ_max (log ε): 201 (1.88) nm; ECD (c 0.33 mM, MeOH) λ_max (Δε) 208 (19.5), 225 (25.3) nm; IR (KBr) ν_max: 3327, 2938, 1716, 1450, 1388 and 1171 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) data, Table 1; ¹³C NMR (125 MHz, MeOH-d₄) data, Table 2; HRESIMS m/z 343.2246 [M + Na]⁺ (calcd: C₂₀H₃₂O₃Na, 343.2244).

Talaroacid D (4): white powder.; ${\left[\alpha \right]}_D^{25}$ = 5.3 (c 0.28, MeOH); UV (MeOH) λ_max (log ε): 201 (1.23) nm; ECD (c 0.33 mM, MeOH) λ_max (Δε) 201 (1.5), 225 (1.8) nm; IR (KBr) ν_max: 3325, 2936, 1714, 1448, 1389 and 1173 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) data, Table 1; ¹³C NMR (125 MHz, CDCl₃) data, Table 2; HRESIMS m/z 345.2401 [M + Na]⁺ (calcd: C₂₀H₃₄O₃Na, 345.2400).

Talaromarane A (5): colorless crystal; ${\left[\alpha \right]}_D^{25}$ = 8.3 (c 0.30, MeOH); UV (MeOH) λ_max (log ε): 201 (1.80) nm; ECD (c 0.35 mM, MeOH) λ_max (Δε) 210 (8.0); IR (KBr) ν_max: 3422, 2928, 1637 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) data, Table 1; ¹³C NMR (125 MHz, CDCl₃) data, Table 2; HRESIMS m/z 421.1869 [M − H]⁻ (calcd: C₂₂H₂₉O₇, 421.1868).

3.5. Crystallographic Data for Talaromarane A

The X-ray diffraction data of talaromarane A (5) were measured using a Rigaku XtaLAB Pro diffractometer with CuKα radiation (λ = 1.54184 Å). The structure of 5 was resolved using SHELXT methods and refined by full-matrix least-squares difference Fourier techniques on an OLEX2 interface program. The crystallographic data of 5 were preserved at the Cambridge Crystallographic Data Centre.

Molecular formula C₂₂H₃₀O₈, formula weight 422.46, orthorhombic, space group = P2₁2₁2₁, unit cell: a = 8.71630 (10) Å α = 90°, b = 11.06960(10) Å β = 90°, c = 21.1646(2) Å γ = 90°, V = 2042.09(4) ų, ρ_calcg = 1.374 cm³, Z = 4, T = 99.98(10) K, μ (CuKα) = 0.868 mm⁻¹, F (000) = 904.0. A total of 16096 reflections (8.356° ≤ 2Θ ≤ 148.688°) were measured with 4108 independent reflections (R_int = 0.0453, R_sigma = 0.0337). Final R indexes [I ≥ 2σ (I)]: R₁ = 0.0320, wR₂ = 0.0841. Final R indexes [all data]: R₁ = 0.0336, wR₂ = 0.0841. Largest diff. peak and hole = 0.24 and −0.18 eÅ⁻³. Flack parameter = −0.22 (8). Crystallographic data for the structure reported in this paper were deposited in the Cambridge Crystallographic Data Centre (Accession No. CCDC 2351536).

3.6. Anti-Inflammatory Assay

Standard Anti-inflammatory assays employing RAW264.7 cell lines were carried out as described previously [30]. All compounds were tested for cytotoxic activity before anti-inflammatory testing. The RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, NY, USA) at 37 °C with 5% CO₂ humidified incubator. Quercetin (Sigma, Burlington, VT, USA) or compound was dissolved in DMSO to prepare mother liquor (10 mm/mL). Cytotoxic activity was tested by MTT assay. The cells were pretreated with different concentrations of quercetin or compounds (5, 10, 20, 30, 40, and 50 µM) for 24 h, then 10 µL of MTT (0.5 mg/mL) was added to each well and cultured for 4 h to test the absorbance at 540 nm. The concentration of DMSO was 0.2% of the medium culture. The NO content was determined by the Griess method to evaluate the anti-inflammatory activity of the compounds. Firstly, 500 μL cells (3 × 10⁶ cells/mL) were seeded in 24-well plates and cultured overnight. Different concentrations of quercetin or compounds (5, 10, 20, 30, 40, and 50 µM) pretreated with LPS were added and cultured for 24 h, and the absorbance of final products was measured at 540 nm. None compounds displayed cytotoxic on RAW264.7 cell at 50 µM. Quercetin was the positive control.

3.7. Solubility and the Stability

Compounds 1–5 were dissolved in chloroform, and no change in compounds 1–5 was found by TLC detection after overnight storage. It was shown that compounds 1–5 were stable under normal conditions.

4. Conclusions

In conclusion, four new diterpenes with 1,2,3,4,4a,5,6,8a-octalin skeleton talaroacids A-D (1–4) and a new isopimarane diterpenoid talaromarane A (5) were isolated from the mangrove endophytic fungus Talaromyces sp. JNQQJ-4. It is noteworthy that 5 contains a rare 2-oxabicyclo [3.2.1] octan moiety in isopimarane. Moreover, compound 2 exhibited promising NO inhibitory activity with IC₅₀ values of 4.59 μM. In addition, the better activity of compounds 1–2 than 3–4 indicated that the Δ¹⁴ double bond in the side chain makes a contribution to NO inhibitory activity. Nitric oxide (NO) is a signaling molecule produced by inducible nitric oxide synthase (iNOS), playing an important regulatory role in the occurrence and development of inflammation [36]. It is closely related to many major inflammation-induced diseases, such as autoimmune diseases, arthritis, cardiovascular diseases, and diabetes [37]. Inhibiting the production of NO can reduce inflammatory responses and prevent subsequent diseases [38]. Therefore, NO inhibitors were considered a promising direction for anti-inflammatory drug research [39]. Recently, several diterpenes with decalin skeleton have been reported to have significant NO inhibitory activity [40,41,42]. Among diterpenes, tinopanoid M, a clerodane diterpenoid isolated from Tinospora crispa, exerts good anti-inflammatory effects by reducing the expression of various pro-inflammatory factors and modulating multiple inflammatory pathways [41]. Thus, talaroacid B (2) might be worthy of further study as a potential anti-inflammatory lead compound.