Diverse and Bioactive Lactones from the Sri Lankan Mangrove-Derived Fungus Talaromyces sp. SCSIO41445

Abstract

Three previously uncharacterized lactones, namely penicianstinoid L (1), talaromyketide J (2) and peniciisocoumarin K (3), along with twenty-eight known compounds (4–31), were yielded from the mangrove-derived fungus Talaromyces sp. SCSIO41445, collected from Mangrove Park (NARA), Sri Lanka. Their structures were established by HRESIMS and NMR spectroscopic analysis (including ¹H and ¹³C NMR, HSQC, and HMBC), with the stereostructures of 2 and 3 being confirmed by single-crystal X-ray crystallographic analysis. Furthermore, compounds 1–31 were evaluated in terms of their neuraminidase (NA) inhibitory activities. These bioassay results revealed that three lactones (11, 15, and 16) of them exerted NA inhibitory effects, with IC₅₀ values of 46.66 ± 2.31, 20.78 ± 1.89, and 34.14 ± 2.56 µM, respectively. Moreover, molecular docking analysis demonstrated the potential of these compounds to inhibit NA enzymes, revealing specific interactions between the compounds and target proteins.

1. Introduction

Mangrove ecosystems are among the most productive and ecologically significant habitats on Earth because of their location at the intersection of the terrestrial and marine environments. The extreme and fluctuating environmental conditions of mangroves characterized by variations in salinity, temperature, and oxygen availability have driven the evolution of unique microbial communities with exceptional metabolic capabilities [1]. These microbes, especially fungi that grow on mangroves, have become abundant and chemically diverse sources of marine natural products, including secondary metabolites that have strong biological effects [2,3].

Talaromyces sp. is widely distributed in sponges, plants, and soil and is categorized under the fungal phylum, ascomycete subphylum, ascomycetes, sporangia, and fungal family [4]. It is an important category of filamentous fungi that are often isolated from a variety of marine habitats, such as sediments and mangroves. This genus contains more than 208 known species [5], many of which are abundant producers of structurally diverse and biologically active secondary metabolites, including polyketides, meroterpenoids, alkaloids, anthraquinones, isocoumarins, and peptides [2]. Many bioactivities, such as antibacterial, antifungal, antiviral, cytotoxic, antioxidant, and enzyme-inhibitory properties, are displayed by these metabolites [6]. Neuraminidases (NAs) are hydrolytic enzymes linked to a range of human diseases. Beyond their established role in antiviral therapy, emerging research suggests NA inhibitors hold potential for cancer treatment. Specifically, in chemotherapy-resistant pancreatic cancer, NA inhibition may target key signaling pathways involved in tumor progression and metastasis. However, this field remains in early stages, requiring further study to fully assess its therapeutic potential and safety [7].

During our ongoing investigation into structurally unique and biologically active secondary metabolites from mangrove associated fungi, 31 metabolites including 25 lactones were identified from the extract of Talaromyces sp. SCSIO41445 (Figure 1), collected from the mangrove sediments Mangrove Park (NARA), Thaladuwa, Negombo, Sri Lanka. This study details the isolation procedures, structural elucidation, and bioactivity evaluations of all isolated compounds.

2. Results and Discussion

2.1. Structural Determination

Compound 1 was obtained as white solid, and the molecular formula was deduced to be C₃₂H₄₀O₁₃ based on HR-ESIMS m/z 633.2533 [M + H]⁺, implying 13 degrees of unsaturation. Analysis of the ¹H and ¹³C NMR data (

Table 1. The NMR data of 1 (500 and 125 MHz, TMS, δ in ppm, DMSO-d₆).

1
Pos	δC, Type	δH, (J in Hz)	Pos	δC, Type	δH, (J in Hz)
1	36.25 (CH2)	1.77 (m, 2H)	17	20.48 (CH3)	2.05 (s, 3H)
2	30.34 (CH2)	2.63 (m, 2H)	2′	191.25 (C)
3	173.54 (C)		3′	82.79 (C)
4	75.47 (C)		4′	164.37(C)
5	48.43 (C)		5′	75.72 (CH)	5.01 (q, J = 6.9 Hz, 1H)
6	33.29 (CH)	1.54 (dd, J = 14.1, 4.7 Hz, 1H)	6′	84.33 (C)
7	69.72 (CH)	5.11 (dd, J = 12.0, 4.7 Hz, 1H)	7′	65.84 (C)
8	59.51 (C)		8′	164.63 (C)
9	91.40 (C)		9′	15.84 (CH3)	1.31 (s, 3H)
10	140.24 (C)		10′	12.79 (CH3)	1.43 (d, J = 7.0 Hz, 3H)
11	76.84 (CH)	5.73 (s, 1H)	1″	166.14 (C)
12	11.69 (CH3)	1.45 (s, 3H)	2″	127.23 (C)
13	124.42 (CH2)	5.71 (s, 1H)	3″	138.99 (CH)	6.75 (dddd, J = 8.8, 7.3, 5.9,1.6 Hz, 1H)
14	26.34 (CH3)	1.09 (s, 3H)	4″	14.31 (CH3)	1.77(m, 3H)
15	26.55 (CH3)	1.12 (s, 3H)	5″	11.75 (CH3)	1.73 (m, 3H)
16	168.93 (C)		1‴	51.40 (CH3)	3.58 (s, 3H)

) indicated that compound 1 had six carbonyl carbons and four olefinic carbons, suggesting that 1 was a pentacyclic compound and had a meroterpenoid skeleton. In addition, the ¹H NMR data (Table 1) displayed one olefinic proton signal at δ_H 6.75 (1H, dddd, J = 8.8, 7.3, 5.9,1.6 Hz, H-3″), one terminal double-bond group at δ_H 5.71 (1H, s, H-13), three oxymethines at δ_H 5.73 (s, H-11), 5.11 (1H, dd, J = 12.0, 4.7 Hz, H-7), and 5.01 (1H, q, J = 6.9 Hz, H-5′), three methylenes at δ_H 1.77 (2H, m, H-1), 2.63 (2H, m, H-2) and 1.54 (1H, dd, J = 14.1, 4.7 Hz, H-6), seven methyl group at δ_H 1.45 (3H, s,12-CH₃), δ_H 1.09 (3H, s,14-CH₃), δ_H 1.12 (3H, s,15-CH₃), δ_H 1.31 (3H, s, 9′-CH₃), δ_H 1.43 (3H, d, J = 7.0 Hz, 10′-CH₃), δ_H 1.77 (3H, m, 4″-CH₃), δ_H 1.73 (3H, m, 5″-CH₃) and one methoxy group at δ_H 3.58 (3H, s, 1‴-OCH₃). In addition, a singlet at δ_H 2.05 (3H, H-17) corresponds to the acetyl methyl protons attached to the carbonyl group. The ¹H and ¹³C NMR spectroscopic data of compound 1 (Table 1) were similar to the known compound penicianstinoid I, containing multiple ester or lactone groups [8]. The obvious differences were presence of an additional carboxyl carbon at δ_C 168.93 (C-16), and a methyl group attached to this carbonyl carbon at δ_C 20.48 (17-CH₃). Compared with the reference compound [8], the hydroxyl group at C-11 is replaced by an O-acetyl substituent in penicianstinoid I, as confirmed by the presence of the carbonyl signal at C-16 and a methyl singlet assigned to 17-CH₃. The absence of the hydroxyl proton further confirms this acetylation. In addition, the HR-ESIMS data revealed that 1 contains one additional oxygen atom and two extra carbon and hydrogen atoms relative to penicianstinoid I, consistent with the bonding of the acetyl group. These were confirmed by the HMBC correlations of 17-CH₃ to C-11 and of H-11 to C-16. The whole structure was further determined by the 2D NMR data (Figure 2). Based on these results, the planar structure of 1 was elucidated, and 1 was unequivocally determined as penicianstinoid L.

Compound 2 was obtained as a white solid. Its molecular formula was determined to be C₁₅H₁₈O₅ based on HRESIMS analysis data at m/z 277.1080 [M − H]⁻. The formula requires seven degrees of unsaturation. Comprehensive analysis of its (1D and 2D NMR), ¹H NMR spectrum data and HSQC spectrum in DMSO-d₆ revealed the presence of signals (

Table 2. The NMR data of 2 and 3 (500 and 125 MHz, TMS, δ in ppm, DMSO-d₆).

Pos	2		Pos	3
	δC, Type	δH, (J in Hz)		δC, Type	δH, (J in Hz)
1	70.4 (C)		1	169.8 (C)
2	40.6 (CH2)	1.71 (m,1H), 1.57 (m,1H)	3	80.3 (CH)	4.64 (s, 1H)
3	18.9 (CH2)	1.64 (m,1H), 1.57 (m,1H)	4	31.5 (CH2)	2.98 (dd, J = 16.3, 3.3 Hz, 1H) 2.85 (dd, J = 16.2, 11.2 Hz, 1H)
4	29.2 (CH2)	1.96 (m,1H), 1.64 (m,1H)	4a	130.6 (C)
4a	79.0 (CH)	4.86 (d, J = 3.2 Hz, 1H)	5	117.1 (CH)	6.76 (d, J = 8.1 Hz, 1H)
6	170.8 (C)		6	118.2 (CH)	7.21 (d, J = 8.1 Hz, 1H)
6a	108.8 (C)		7	146.6 (C)
7	150.8 (C)		8	151.4 (C)
8	146.6 (C)		8a	108.3 (C)
9	117.7 (CH)	7.24 (d, J = 8.2 Hz, 1H)	9	56.0 (CH3)	3.78 (s, 3H)
10	119.8 (CH)	6.84 (d, J = 8.2 Hz, 1H)	1′	30.7(CH2)	1.71 (m, 1H), 1.95 (qd, J = 10.7, 4.5 Hz, 1H)
10a	132.1 (C)		2′	27.9 (CH2)	1.40 (dt, J = 12.8, 6.3 Hz, 1H) 1.71 (m, 1H)
11	47.0 (CH)	2.83 (d, J = 3.4 Hz, 1H)	3′	74.4 (CH)	3.10 (m,1H)
12	22.8 (CH3)	0.71 (s, 3H)	4′	69.7 (CH)	3.37 (m, 1H)
8-OCH3	56.0 (OCH3)	3.80 (s, 3H)	5′	19.5 (CH3)	1.05 (d, J = 6.2 Hz, 3H)

) corresponding to one oxygenated methine proton at δ_H 4.86 (1H, d, J = 3.2 Hz, H-4a), one methine at δ_H 2.83 (1H, d, J = 3.4 Hz, H-11), three methylene protons δ_H 1.71 (1H, m, H-2); 1.57 (1H, m, H-2), δ_H 1.64 (1H, m, H-3); 1.57 (1H, m, H-3) and δ_H 1.96 (1H, m, H-4); 1.64 (1H, m, H-4), two aromatic protons at δ_H 7.24 (1H, d, J = 8.2 Hz, H-9) and 6.84 (1H, d, J = 8.2 Hz, H-10), one methyl group at δ_H 0.71 (3H, s, 12-CH₃) and one methoxy moieties at δ_H 3.80 (3H, s, 8-OCH₃). The analysis of its ¹³C NMR data revealed the presence of fifteen carbon resonances comprising one carbonyl carbon (δ_C 170.8, C-6), three oxygenated carbons (δ_C 70.4, C-1; 79.0, C-4a; 108.8, C-6a), five aromatic or olefinic carbons (δ_C 146.6 (C-8), 150.8 (C-7), 117.7 (C-9), 119.8 (C-10), 132.1 (C-10a)), one methyl carbon (δ_C 22.8 (12-CH₃) and one methoxy group 56.0 (8-OCH₃)). According to the molecular formula, the COSY-related signal indicated a coupled system H₂-2/H₂-3/H₂-4 while H-4a/H-11 and H-9/H-10. The HMBC correlations (Figure 2) from H₂-2 to C-4 and C-11, as well as from H₂-3 to C-4a and C-1, established the connectivity of the lower ring system. Additionally, HMBC correlations from H-11 to C-6a, and C-10 confirmed linkage between the oxygenated and aromatic moieties through C-11. The methyl group 12-CH₃ was HMBC correlated with C-2 and C-11. The methoxy group 8-OCH₃ revealed a strong HMBC correlation with C-8, confirming its attachment at C-8. The combined COSY and HMBC data (Figure 2) supported the presence of a fused bicyclic oxygenated ring system attached to a substituted benzopyranone unit. The presence of a carbonyl carbon at C-6 suggested a lactone functionality, while the oxygenated carbons at C-1 and C-7 indicated hydroxyl substitution at these positions. Ultimately, the structure of 2 was further validated through single-crystal X-ray crystallographic analysis, with a perspective ORTEP plot depicted in Figure 3. Thus, based on these findings, the structure of 2 was unequivocally determined as talaromyketide J.

Compound 3 was isolated as a yellowish powder. Its molecular formula was established as C₁₅H₂₀O₆, corresponding to seven degrees of unsaturation, as determined by HR-ESIMS m/z 297.1330 [M + H]⁺. In the ¹H NMR data of Compound 3 (Table 2), the proton signals and the coupling constants at δ_H 7.21 (d, J = 8.1 Hz, H-6) and 6.76 (d, J = 8.1 Hz, H-5), indicating a 1,2,3,4-tetrasubstituted benzene ring. An oxymethine proton at δ_H 4.64 (s, H-3), two oxymethylene at δ_H 3.1 (m, H-3′) and δ_H 3.3 (m, H-3′), three methylene groups at δ_H 2.98 (dd, J = 16.3, 3.3 Hz, H-4); 2.85 (dd, J = 16.2, 11.2 Hz, H-4), 1.71 (m, H-1′); 1.95 (qd, J = 10.7, 4.5 Hz, H-1′) and 1.40 (dt, J = 12.8, 6.3 Hz, H-2′); 1.71 (m, H-2′). Additionally, a methoxy singlet appeared at δ_H 3.78 (s, H-9). The ¹³C NMR and HSQC data revealed 15 carbon signals (Table 2), including one lactone carbonyl carbon at δ_C 169.8 (C-1), six aromatic carbons at δ_C 130.6 (C-4a), 117.1 (C-5), 118.2 (C-6), 146.6 (C-7), 151.4 (C-8), and 108.3 (C-8a) and one methoxy carbon at δ_C 56.0 (C-9). The spectrum also showed an oxymethine δ_C 80.3 (C-3), three aliphatic methylene carbons at δ_C 31.5 (C-4), 30.7 (C-1′), and 27.9 (C-2′). Key HMBC correlations from H-5 to C-4, C-8a, and C-7, and from H-6 to C-4a, C-8, and C-7, verified the substitution pattern of the aromatic ring. The methoxy proton (H-9) showed a three-bond HMBC correlation to C-7 (δ_C 146.6), confirming its attachment at C-7. The oxymethine proton H-3 (δ_H 4.64) exhibited COSY correlations to C-1′ and C-4, supporting the lactone linkage. HMBC correlations from the methylene protons H-4 to C-8a and C-5 indicated the intact fused ring system. The aliphatic side chain was characterized by COSY and HMBC correlations. The methylene protons H-1′ showed COSY with H-2′ and H-3. H-3′ showed further COSY correlations with H-2′ while H-2′ correlated with H-1′ and H-4′ corelated with H-5′ supporting a 1′,2′,3′,4′,5′-dihydroxypentyl chain moiety attached at C-3. The ¹H–¹H COSY, HMBC and HSQC data collectively supported the full structural elucidation of compound 3 (Figure 2). Moreover, the structure of 3 was further validated through single-crystal X-ray crystallographic analysis, with a perspective ORTEP plot depicted in Figure 4. Finally, the 3 was named as peniciisocoumarin K.

Meanwhile, the other twenty eight known compounds were identified as penicimarin H (4) [9] and M (5) [10], peniciisocoumarin F (6) [11], aspergillumarin A (7) and B (8) [12], penicimarin G (9) [9], talaromarin B (10) [13], penicilloxalone B (11) [12], penicimarin N (12) [14], I (13) [9], and J (14) [15], penicianstinoid A (15) [16], and D (16) [17], austinolide (17) [18], austinol (18) [10], austin (19) [19], dehydroaustinol (20) [20], 11β-acetoxyisoaustnone (21) [21], austinoneol A (22) [18], asperterpenoid A (23) [22], penicichrysogene A (24) [23], purpactin A (25) [24], questin (26) [25], peniciisocoumarin A (27) [16], talaroyene A (28) [26], 4-hydroxy-3(3-methyl-2 butenyl)-benzoic acid (29) [27], 4-hydroxybenzaldehyde (30) [28], hexadecanoic acid (31) [29], respectively, by comparing their NMR data (Supplementary Materials) to previous reports.

2.2. Bioactivity Assay

All these compounds were subjected to screening for their inhibitory effects neuraminidase (NA) in vitro [30]. Initially, we carried out comprehensive screening for the enzyme inhibitory activities of NA at a concentration of 50 μg/mL. This initial screening was crucial as it enabled us to identify the promising compounds for further research. The compounds chosen for IC₅₀ value determination were those exhibiting an inhibition rate exceeding 80% and therefore determined the IC₅₀ value for each of them. The standard curve of these compounds was established by measuring the inhibition rate of different concentrations, and the equation of the standard curve was derived. Subsequently the concentration of the compound with an inhibition rate of 80%, known as the IC₅₀ value, was determined. Ultimately, 11, 15, and 16 showed anti-neuraminidase properties, with IC₅₀ values of 46.66 ± 2.31, 20.78 ± 1.89, and 34.14 ± 2.56 μM, respectively, compared with the positive control, with an IC₅₀ value of 20 μM (oseltamivir acid). Previously, only 15 have been reported to exhibit growth inhibition activity against newly hatched larvae of Helicoverpa armigera Hubner and to show activity against Caenorhabditis elegans [16]. No studies reporting positive biological activities have been published for 11 and 16. The neuraminidase inhibitory activity described herein represents the first discovery for all three compounds. The purpose of this evaluation was to identify potential candidates for further research and development, with the ultimate goal of harnessing their potential inhibitory properties to treat various medical conditions.

Molecular docking analysis was conducted to gain insights into the potential molecular interactions between 11, 15, 16 and neuraminidase (NA), providing a plausible explanation for their observed inhibitory activities. The docking simulations revealed that 11, 15, 16 were favorably accommodated within the binding cleft of NA protein (PDB code: 6GY5), with calculated binding free energies of −9.7, −8.5 and −10.2 kcal/mol, respectively. Compound 11 primarily formed hydrogen bond with SER-405, LEU-549 and VAL-408 at distances ranging from 2.7 Å to 3.4 Å. Compound 15 interacted with the residue SER-361, VAL-362, VAL-551 and VAL-504, via a single hydrogen bond at distances ranging from 1.1 Å to 3.6 Å. Compound 16 established hydrogen bonds with VAL-551 and SER-361 at a distance of 3.1 Å (Figure 5).

3. Materials and Methods

3.1. Standardized Experimental Procedures

Nuclear magnetic resonance (NMR) spectra were recorded on a Quantum-I Plus 500 MHz spectrometer (Q-one Instrument Co., Ltd., Wuhan, China) operating at 500 MHz for ¹H NMR and 125 MHz for ¹³C NMR, and on a Bruker AVANCE III HD 700 MHz spectrometer (Bruker Switzerland AG, Fällanden, Switzerland) operating at 700 MHz for ¹H NMR and 175 MHz for ¹³C NMR. Tetramethylsilane (TMS) was used as the internal standard. Ultraviolet (UV) spectra were obtained on a Shimadzu UV-2600 PC spectrophotometer (Shimadzu, Beijing, China), and infrared (IR) spectra were recorded using a Shimadzu IR Affinity-1 spectrometer. Circular dichroism (CD) spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics, Leatherhead, Surrey, UK). High-resolution electrospray ionization mass spectrometry (HRESIMS) data were acquired on a Bruker maXis Q-TOF mass spectrometer (Bruker BioSpin International AG, Fällanden, Switzerland). Semipreparative high-performance liquid chromatography (HPLC) was conducted on a Hitachi Primaide system equipped with a diode-array detector (DAD) (Hitachi, Tokyo, Japan), utilizing ODS columns (ChromCore 120 C18, 10 × 250 mm, 5 µm; YMC-Pack ODS-A, 10 × 250 mm, 5 µm; COSMOSIL πNAP, 10 × 250 mm; COSMOSIL 5C18-AR-II, 10 × 250 mm). Column chromatography was performed on silica gel with particle sizes ranging from 100–200 mesh grade and 200–300 mesh grade and compound spot visualization was achieved by thin-layer chromatography (TLC) using silica gel GF254 plates (0.4–0.5 mm) from Qingdao Marine Chemical Factory in Qingdao, China under UV light at 254 nm. All solvents used were of analytical grade and were supplied by Tianjin Fuyu Chemical and Industry Factory (Tianjin, China). A neuraminidase inhibitor screening kit was utilized for assessing anti-neuraminidase activity.

3.2. Fungal Material

The fungal strain Talaromyces sp. SCSIO41445 was isolated from mangrove sediments collected from the Mangrove Park (NARA), Thaladuwa, Negombo, Sri Lanka. It was stored in the Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China. The BLAST (version 2.17.0) analysis of the Internal Transcribed Spacer (ITS) region of the rDNA indicated that the strain belongs to Talaromyces sp., showing 98% sequence similarity to previously reported Talaromyces sp. The sequence was deposited in GenBank with the accession number SUB15876641 LKD9 PX735977.

3.3. Fermentation and Extraction

The fungal strain Talaromyces sp. was cultivated on MB agar plates at a temperature of 25 °C for a duration of five days. The fungal strain was statically cultivated using MA medium and then was cultured in 200 mL seed medium (malt extract (15 g), sea salt (10 g), and distilled water (1000 mL)) in 1 L Erlenmeyer flasks at 28 °C for 72 h on a rotary shaker (180 rpm). A large-scale fermentation was incubated at 26 °C for 28 days using a rice medium (200 g rice, 2% sea salt, 230 mL H₂O) in the 1 L flask (×100) under static conditions. After 28 days, the rice medium was soaked in EtOAc (600 mL per flask), cut into small fragments, and sonicated for 20 min. The resulting mixture was then transferred to fermentation vats and subjected to three successive extractions with EtOAc. The combined extracts were concentrated under reduced pressure to yield 79.37 g of crude extract.

3.4. Isolation and Purification

The crude extract (79.37 g) was subjected to a silica gel vacuum liquid chromatography using a step gradient elution of petroleum ether (PE)-dichloromethane (DCM) (ν:ν 1:0, 1:1, 0:1), DCM-methyl alcohol (CH₃OH) (ν:ν 100:1, 100:3, 50:3, 10:1, 1:1, 0:1), to yield 9 fractions (Frs. 1-9) in the light of TLC profiles. Then the Fr. 2–Fr. 4 combined as a one fraction (Fr. 2) and divided into 14 subfractions (Fr. 2-1–Fr. 2-14) by ODS silica gel eluting with CH₃OH/H₂O (5–100%).

Fr. 2-2 was separated by semipreparative HPLC (45% CH₃OH/H₂O, 2.5 mL/min) to gain 30 (19.8. mg, t_R 10.0 min). Fr. 2-4 was further purified by semipreparative HPLC (30% CH₃CN/H₂O, 3 mL/min) to afford 2 (3.8 mg, t_R 19.40 min). Compound 5 (30.4 mg, t_R 28.30 min) was further purified from Fr. 2-5 by semipreparative HPLC (45% CH₃OH/H₂O, 2.5 mL/min). Compounds 7 (50.6 mg, t_R 42 min) and 8 (16 mg, t_R 62 min) were purified from Fr. 2-6 by semipreparative HPLC (45% CH₃OH/H₂O, 2.5 mL/min). Fr. 2-8 was purified by semipreparative HPLC (58% CH₃OH/H₂O, 2.5 mL/min) to gain four compounds (15, 16, 17, 19). Compounds 15 (8 mg, t_R 20.30 min), 16 (6.7 mg, t_R 18.30 min), compound 17 (5.3 mg, t_R 27.30 min) and 19 (8 mg, t_R 20.30 min). Compound 25 (4.5 mg, t_R 36.0 min) was further purified from Fr. 2-11 by semipreparative HPLC (65% CH₃OH/H₂O, 2.5 mL/min).

Fr. 3 divided into 23 subfractions (Fr. 3-1–Fr. 3-23) by ODS silica gel eluting with CH₃OH/H₂O (5–100%). Fr. 3-8 was separated by semipreparative HPLC (45% CH₃OH/H₂O, 2.5 mL/min) to gain 12 (17.6 mg, t_R 24.30 min) and 13 (11.4 mg, t_R 37 min). Compound 10 (17.1 mg, t_R 29.30 min) was further purified from Fr. 3-8-2 by semipreparative HPLC (50% CH₃OH/H₂O, 2.5 mL/min). Fr. 3-9 was purified by semipreparative HPLC (48% CH₃OH/H₂O, 0.06% formic acid, 2.5 mL/min) to gain 20 (4.6 mg, t_R 29.30 min) and 29 (3.9 mg, t_R 41 min). Compound 18 (5.9 mg, t_R 23 min) was gain from further purified Fr. 3-9-3 by semipreparative HPLC (50% CH₃OH/H₂O, 2.5 mL/min). Fr. 3-15 was further purified by semipreparative HPLC (57% CH₃OH/H₂O, 2.5 mL/min) to gain 21 (3.9 mg, t_R 30 min), 1 (7.7 mg, t_R 36 min), 26 (3.7 mg, t_R 42 min) while repurify Fr. 3-15-8 afford 22 (3.5 mg, t_R 36 min).

Compound 14 (15.6 mg, t_R 19 min) was further purified from Fr. 3-6-2 by semipreparative HPLC (45% CH₃OH/H₂O/Fr. 3-15 0.06% formic acid, 2.5 mL/min). Fr. 3-7 was purified by semipreparative HPLC (35% CH₃CN/H₂O, 3 mL/min) to gain 11 (10.7 mg, t_R 19 min) and 23 (9 mg, t_R 28 min) was purified by semipreparative HPLC (85% CH₃CN/H₂O/0.08% formic acid, 3 mL/min) from Fr. 3-21.

Fr. 4 divided into 5 subfractions (Fr. 4-1–Fr. 4-5) by semipreparative HPLC (55% CH₃OH/H₂O, 2.5 mL/min). Fr. 4-5 was purified by semipreparative HPLC (35% CH₃CN/H₂O, 2.5 mL/min) to gain 6 (15.8 mg, t_R 14.30 min) while 4 (4.4 mg, t_R 19.30 min) purified by semipreparative HPLC (22% CH₃CN/H₂O, 2.7 mL/min) from Fr. 4-1. Compound 3 (6.6 mg, t_R 24.30 min) was purified from Fr. 4-2 by semipreparative HPLC (35% CH₃OH/H₂O, 2.5 mL/min).

The TLC profiled Fr. 7 to Fr. 9 were merged (Fr. 5) and then was divided into 28 subfractions (Fr. 5-1–Fr. 5-28) by ODS silica gel eluting with CH₃OH/H₂O (5–100%). Based on this, Fr. 5-10 was directly separated by semipreparative HPLC (40% CH₃OH/H₂O, 2.5 mL/min) to obtained 9 (16.3 mg, t_R 23.10 min). Compound 28 (14.3 mg, t_R 22.30 min) was further purified from Fr. 5-7 by semipreparative HPLC (30% CH₃OH/CF₃COOH 0.1%, 2.7 mL/min). Fr. 5-12 was purified by semipreparative HPLC (40% CH₃OH/H₂O, 0.01% CF₃COOH, 2.5 mL/min) to gain 27 (5 mg, t_R 23.30 min) while 24 (9.5 mg, t_R 45 min) was gain from further purified Fr. 5-16 by semipreparative HPLC 45% CH₃OH/H₂O, 0.06% formic acid, 2.5 mL/min).

Fr. 1 was divided into 8 subfractions (Fr. 1-1–Fr. 1-8) by ODS silica gel eluting with CH₃OH/H₂O (5–100%). Compound 31 (28 mg, t_R 62 min) was further purified from Fr. 1-8 by semipreparative HPLC (90% CH₃OH/H₂O, 3 mL/min).

3.5. Spectroscopic Data of Compounds

Penicianstinoid L (1): Brown solid; −2.4 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 213 (−0.37), 203 (−0.38) nm; IR (film) ν_max 3392, 1797, 1774, 1757, 1712, 1647, 1446, 1379, 1251,1209, 1153, 1128, 1072, 1047, 829, 732, 594, 542 cm⁻¹; ¹H and ¹³C NMR data; HRESIMS m/z 633.2533 [M + H]⁺ (calcd. for C₃₂H₄₁O₁₃, 633.2542).

Talaromyketide J (2): White solid; ${\left[\alpha \right]}_D^{25}$−89.4 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 333 (−0.48), 280 (−1.31), 256 (−0.34), 245 (−0.43), 222 (0.24), 214 (0.21), 212 (0.21) nm; IR (film) ν_max 3838, 3539, 2943, 2360, 1660, 1438, 1379, 1294, 1253, 1147, 1128, 1058, 999, 908, 825, 813, 771, 696, 677, 542, 524, 503 cm⁻¹; ¹H and ¹³C NMR data; HRESIMS m/z 277.1080 [M − H]⁻ (calcd. for C₁₅H₁₇O₅, 278.1081).

Peniciisocoumarin K (3): Yellowish powder; −7.7 (c 0.1, CH₃OH); UV (CH₃OH) λ_max (log ε) 330 (−1.20), 282 (−1.78), 255 (−1.00), 245 (−1.07), 222 (−0.47), 214 (−0.51), 211 (−0.51) nm; IR (film) ν_max 3342,1643, 1016, 675, 597, 551 cm⁻¹; ¹H and ¹³C NMR data; HRESIMS m/z 297.1330 [M + H]⁺ (calcd. for C₁₅H₂₁O₆, 297.1333).

3.6. Bioassays

For testing the inhibitory effects of NA (neuraminidase), we employed a NA inhibitor screening kit as per its manufacturer’s instructions, according to previous work [7]. The neuraminidase (NA) inhibition assay was performed using the Neuraminidase Inhibitor Screening Kit (Fluorometric) manufactured by Beyotime Biotechnology (Shanghai, China), with the catalog number P0309M. All experimental procedures were carried out strictly in accordance with the manufacturer’s instructions (Instruction Version: 2017.08.29). The inhibitory activity against NA was assessed using a commercial neuraminidase inhibitor screening kit according to the manufacturer’s instructions. The kit components included NA assay buffer, neuraminidase, a fluorogenic NA substrate, Milli-Q water, and the instruction manual.

(1)

Preparation of the standard curve:

• A measure of 70 µL of NA assay buffer was dispensed into each well of a 96-well fluorescent microplate.
• Neuraminidase (0, 1, 2, 5, 7.5, or 10 µL) was added to the respective wells.
• Milli-Q water (0–20 µL) was supplemented to adjust the total volume in each well to 90 µL.

(2)

Preparation of sample testing:

• A measure of 70 µL of NA assay buffer was dispensed into each well.
• A measure of 10 µL of neuraminidase was added to each well.
• A measure of 0–10 µL of the tested NA inhibitor sample was added.
• Milli-Q water (0–10 µL) was added to bring the total volume in each well to 90 µL.

(3)

Detection procedure:

• The loaded 96-well plate was shaken for approximately 1 min to mix thoroughly.
• The plate was incubated at 37 °C for 2 min to allow full interaction between the inhibitor and neuraminidase (standard-curve wells were incubated simultaneously).
• A measure of 10 µL of the fluorogenic NA substrate was added to each well.
• The plate was shaken again for about 1 min.
• After incubation at 37 °C for 30 min, fluorescence was measured with excitation at 322 nm and emission at 450 nm.

To comprehensively evaluate the 31 compounds described in this study, a stock solution of each compound was prepared at a concentration of 0.5 mg/mL. The screening was performed in two stages: primary screening followed by IC₅₀ determination. In the primary screening, each compound was tested at the maximum concentration (by adding 10 µL of sample in step (2) c.), and the inhibition rate was calculated based on the standard curve. Compounds showing inhibition rates higher than 80% were selected for further dose–response studies. For IC₅₀ determination, serial dilutions (corresponding to 1, 2, 5, 7.5, and 10 µL additions of sample in step (2) c.) were tested to establish the concentration-inhibition curve.

3.7. Molecular Docking Analysis

The AutoDock Tools (ADT 1.5.6) suite was employed to perform docking analyses as previously described. The crystal structure of NA (PDB id: 6GY5) [31] was obtained from the Protein Data Bank (http://www.rcsb.org; https://www.rcsb.org/structure/6GY5 (accessed on 1 December 2025)) were utilized after removing all water molecules and organic small molecules, while the ligand structures were generated in ChemBioOffice 20.0 (PerkinElmer Informatics, Waltham, MA, USA), followed by MM2 calculations to minimize the conformation energy. The default settings and calculations were applied for other docking parameters, while PyMol software version 2.4.0 (Schrödinger, New York, NY, USA) was employed for analyzing the docking results.

3.8. X-Ray Crystallographic Analysis

Compounds 2 and 3 were obtained as yellow crystals through the process of slow evaporation at room temperature in a mixture of MeOH and H₂O (1:1). The crystal’s information was collected using Cu Kα radiation on an XtalLAB PRO single-crystal diffractometer. The X-ray crystal structure of 2 and 3 was determined using SHELXS-97, expanded by difference Fourier techniques, and refined through full-matrix least-square calculation. Crystallographic data of 2 and 3 have been deposited at the Cambridge Crystallographic Data Centre (deposition number: 2524630 and 2524625). These data can be acquired free of charge by containing CCDC at 12 Union Road, Cambridge CB21EZ, UK.

4. Conclusions

Three new compounds, penicianstinoid L (1), talaromyketide J (2) and peniciisocoumarin K (3), were isolated from mangrove-derived fungus Talaromyces sp. SCSIO41445 from Mangrove Park (NARA), Sri Lanka. Twenty-eight additional compounds were also identified from this fungus. The planar structures and absolute configurations of these compounds were determined through comprehensive spectroscopic analysis using single-crystal X-ray crystallographic analysis, which were then compared with the existing data in the literature. Several of the isolated compounds displayed enzyme inhibitory activity against neuraminidase (NA). Compounds 11, 15, and 16 showed anti-neuraminidase activity with IC₅₀ values of 46.66 ± 2.31, 20.78 ± 1.89, and 34.14 ± 2.56, respectively. In this study, three compounds exhibited enzyme activities, which provided valuable information for further development of NA inhibitors.