Polyketide Derivatives from the Mangrove-Derived Fungus Penicillium sp. HDN15-312

Abstract

Four new polyketides, namely furantides A–B (1–2), talamin E (3) and arugosinacid A (4), and two known polyketides were obtained from the mangrove-derived fungus Penicillium sp. HDN15-312 using the One Strain Many Compounds (OSMAC) strategy. Their chemical structures, including configurations, were elucidated by detailed analysis of extensive NMR spectra, HRESIMS and ECD. The DPPH radicals scavenging activity of 3, with an IC₅₀ value of 6.79 µM, was better than vitamin C.

1. Introduction

ROS (reactive oxygen species) play a twofold role as both toxic and beneficial compounds to organism’s systems. At optimal concentrations, ROS contribute positively to a range of physiological functions. They are integral to maintaining cellular homeostasis through redox regulation, modulating cell signaling pathways, and bolstering immune responses. However, when ROS levels escalate, their reactivity can become detrimental, leading to a state of oxidative stress [1,2]. Excessive ROS accumulation causes oxidative damage to mitochondria, lipids, proteins, RNA, and DNA [3,4]. Furthermore, oxidative stress also plays a key role in mediating inflammation, cardiovascular diseases, and the development of multiple cancers [5,6,7,8,9]. Antioxidants function by inhibiting or delaying the oxidation of chemicals [10]. Antioxidants exert their protective effects through two principal modes of action: The initial mechanism is characterized by chain breaking, wherein primary antioxidants sacrifice an electron to neutralize free radicals. The subsequent mechanism involves the intervention of secondary antioxidants, which eliminate the reactive species initiators by deactivating the catalysts that trigger the chain reaction [3]. At present, the antioxidants on the market can be divided into synthetic antioxidants (such as BHA, BHT, PG, etc.) and natural antioxidants (such as tea polyphenols, phytic acid, etc.) according to their sources [11]. Most of the antioxidants from natural sources are derived from terrestrial plants; there are also some lead compounds of marine origin with antioxidant activity [10], but no marketed antioxidant from secondary metabolites of marine fungal origin.

Marine organisms have been proven to be rich sources of structurally novel and biologically active secondary metabolites [12,13,14]. In the ongoing process of our group’s research for natural products with excellent bioactivity from marine-derived microorganisms, mangrove-derived fungus Penicillium sp. HDN15-312 has piqued our interest. The OSMAC strategy was employed for cultivating Penicillium sp. HDN15-312, which led to producing compounds with significantly different UV absorption profiles in modified Fungus 2# liquid media under static conditions, compared to other conditions. Under the guidance of HPLC-UV and LC-MS, four new compounds (1–4) and two known compounds (5 and 6) were obtained (Figure 1). Compounds 1–6 were evaluated for scavenging activity against DPPH radicals. The DPPH radical scavenging activity of 3, with an IC₅₀ value of 6.79 µM, was better than that of a natural antioxidant, vitamin C. Herein, we cover the isolation, structure elucidation, and DPPH scavenging activities of 1–6 from the mangrove-derived fungus Penicillium sp. HDN15-312.

2. Results

The Penicillium sp. HDN15-312 was meticulously isolated from a mangrove forest sample collected in Sanya, Hainan. A comprehensive study of the metabolites produced by this fungal species was conducted across six distinct culture media. Notably, the ethyl acetate extract derived from Penicillium sp. HDN15-312, when cultured in the modified Fungus 2# liquid medium, exhibited markedly distinct UV absorption profiles. Following these intriguing findings, a large-scale fermentation process was initiated, utilizing the same modified Fungus 2# liquid medium to optimize the production of the desired metabolites. As a result, the ethyl acetate extract was fractionated by extensive column chromatography (silica gel, LH-20, and HPLC) to obtain four undescribed compounds, named furantides A–B (1–2), talamin E (3) and arugosinacid A (4) (Figure 1).

Furantide A (1) was isolated as a white solid. High-resolution electrospray ionization mass spectrometry (HRESIMS) data provided the molecular formula C₁₂H₁₄O₄ determined from the negative ion peak at m/z 223.0965 [M + H]⁺ (calcd for C₁₂H₁₅O₄, 223.0965), indicating six degrees of unsaturation. The data of the ¹H NMR, ¹³C NMR, (

Table 1. NMR data of 1 in CD₃OD-d₄ and 2 in DMSO-d₆ (δ in ppm, 400 MHz for ¹H and 100 MHz for ¹³C).

No.	1		2
	δH, (J in Hz)	δC, Type	δH, (J in Hz)	δC, Type
2		161.4, C		163.3, C
3	6.35, s	109.0, CH	6.28, s	108.4, CH
3a		140.4, C		137.4, C
4	4a: 3.07, dd (16.9, 5.2), 4b: 2.66, dd (16.9, 9.2)	29.6, CH2	4a: 2.97, dd (16.9, 4.5) 4b: 2.52, m	28.4, CH2
5	4.01, dd (9.2, 5.2)	75.2, CH	3.85, dt (7.3, 4.5)	73.8, CH
6		79.2, C		76.9, C
7		188.7, C		186.2, C
7a		145.5, C		144.4, C
8	1.31, s	18.4, CH3	1.18, s	18.7, CH3
1′	6.36, dd (15.8, 1.7)	120.3, CH	2.64, t (7.5)	29.6, CH2
2′	6.58, dd (15.8, 6.8)	133.6, CH	1.63, m	20.5, CH2
3′	1.91, dd (6.9, 1.7)	18.7, CH3	0.91, t (7.4)	13.6, CH3

) and HSQC spectra of 1 showed 12 carbon signals, including two methyls, one methylene, four methines, four quaternary carbons, and one carbonyl carbon (δ_C 188.7). The 1D NMR data of 1 (Table 1) were similar to aspergifuranone [15]. The difference was the replacement of a moiety of orsellinic acid at C-6 in aspergifuranone [15] by a hydroxyl group, which was supported by the NMR data (Figure 2) and a difference in molecular weight.

The side chain in 1 was identified as trans-unsaturated according to coupling constant of H-2′ (J = 15.8). In the ¹H NMR spectrum, H-5 (δ_H 4.01) showed ax/eq coupling (J = 5.2 Hz) to H-4a (δ_H 3.07) and ax/ax coupling (J = 9.2 Hz) to H-4b (δ_H 2.66). In the NOE spectrum, upon irradiation of H₃-8 (δ_H 1.31), a pronounced increase in the signal intensity from H-4b was observed (Figure S8), which implied that the methyl group and H-4b were oriented in the same spatial direction. In order to provide additional confirmation, we performed computational analysis of the NMR data, thereby determining the relative configurations. There were two relative configurations, named 1a (5S*, 6R*) and 1b (5S*, 6S*), theoretically. The two possible relative configurations of 1 were calculated by employing Time-Dependent Density Functional Theory (TDDFT) at the B3LYP/6-31+G(d)//B3LYP/6-311+G (d, p) levels. The result showed that 1a (5S*, 6R*) represented a striking predominance (100% probability). (

Table 2. DP4+ computational analysis of the relative configurations of 1 and 2.

DP4+	1		2
	1a	1b	2a	2b
1H	100.00%	0.00%	100.00%	0.00%
13C	100.00%	0.00%	100.00%	0.00%
1H + 13C	100.00%	0.00%	100.00%	0.00%

and Figure S8) The absolute configuration of 1 was identified by ECD calculations at the B3LYP/6-31+G(d) level. Analysis of the ECD curve of 1 permitted us to come to the conclusion that the negative Cotton effect around 217 nm and positive Cotton effect around 298 nm indicated the 5S and 6R absolute configuration of 1 (Figure 3).

Furantide B (2) was isolated as a white solid. HRESIMS data provided the molecular formula C₁₂H₁₆O₄, determined from the positive ion peak at m/z 225.1124 [M + H]⁺ (calcd for C₁₂H₁₇O₄, 225.1121). NMR analysis revealed that the trans-unsaturated side chain double bonds (δ_C 133.6, 120.3) present in compound 1 were reduced to alkanes (δ_C 20.5, 29.6) in compound 2. And, the absolute configuration of 2 was also the same as 1, which was determined by calculating NMR data and ECD (Table 2, Figure 3 and Figure S16).

Talamin E (3) was isolated as a pale yellow powder with the molecular formula of C₁₁H₁₀O₅ on the basis of HRESIMS data at m/z 223.0595 [M + H]⁺ (calcd for C₁₁H₁₁O₅, 223.0601), indicating seven degrees of unsaturation. The ¹H and ¹³C NMR data (

Table 3. NMR data of compound 3 and 5 in CD₃OD-d₄ (δ in ppm, 400 MHz for ¹H and 150 MHz for ¹³C).

No.	3		5
	δH, (J in Hz)	δC, Type	δH, (J in Hz)	δC, Type
2		164.6, C		165.0, C
3		115.2, C		115.7, C
4		183.5, C		183.5, C
4a		104.2, C		104.8, C
5		154.9, C		159.6, C
6	6.23, s	99.2, CH	6.47, s	96.3, CH
7		153.7, C		158.5, C
8		125.7, C		129.6, C
8a		146.7, C		150.7, C
9	1.98, d (0.8)	9.2, CH3	1.99, s	9.1, CH3
10	2.44, d (0.8)	18.4, CH3	2.45, s	18.5, CH3
11			3.91, s	61.8, CH3
12			3.81, s	56.9, CH3

) showed two singlet methyls, eight olefinic carbons (δ_C 99.2, 104.2, 115.2, 125.7, 146.7, 153.7, 154.9 and 164.6) and one carbonyl carbon (δ_C 183.5). Discreet analysis of the NMR data indicated that 3 and talamin B [16] (5) shared the same skeleton. The key distinction was the substitution of two methoxy groups at the C-7, C-8 position respectively in 5 with hydroxyl groups, which was corroborated by the absence of HSQC correlation for the methyl group and the molecular formula (Table 3 and Figure 4). Thus, compound 3 was determined as 5,7,8-dihydroxy-2,3-dimethylchromone, referred to as talamin E.

Arugosinacid A (4), isolated as yellow powder, had a molecular formula of C₁₆H₁₂O₇ from the negative ion peak at m/z [M − H]⁻ 315.0505 (calcd for C₁₆H₁₁O₇, 315.0501) in the HRESIMS spectrum, indicating 11 degrees of unsaturation. Analysis of ¹H NMR, ¹³C NMR and HSQC data (

Table 4. NMR data of compound 4 in DMSO-d₆ (δ in ppm, 400 MHz for ¹H and 150 MHz for ¹³C).

No.	4
	δH, (J in Hz)	δC, Type
1		161.2, C
2	6.67, dd (8.2, 1.1)	110.5, CH
3	7.50, t (8.2)	137.9, CH
4	6.62, dd (8.2, 1.1)	109.9, CH
5		154.9, C
6	6.19, s	102.3, CH
7a		136.9, C
7	7.60, d (1.5)	115.3, CH
8		134.8, C
9	7.56, d (1.6)	118.7, CH
10		156.9, C
10a		126.1, C
11		194.7, C
11a		113.5, C
12		166.2, C
13	3.45, s	56.6, CH3

and Figure 4) revealed the presence of 16 carbons, including one methoxy group, twelve aromatic carbons, one methine carbon, and two possible carbonyl carbons (δ_C 194.8, 166.2), which shared a similar skeletal structure with arugosin K [17]. The differences were the absence of isopentenyl at C-2 supported by HSQC correlation from H-2 (δ_H 6.67) to C-2 (δ_C 110.5) and ¹H-¹H COSY of H-2 to H-3 (δ_H 7.50), along with the replacement of methyl at C-8 (δ_C 134.8) by a carboxyl group (δ_C 166.2), which was manifested in HMBC correlation from H-7 (δ_H 7.60) and H-9 (δ_H 7.56) to C-12 (δ_C 166.2). Based on these findings, the planar structure was successfully elucidated. It can be known from the literature [18] that the planar structure of this compound has appeared in an article on synthesis, but the stereostructure has not been characterized.

To further determine the absolute configuration of 4, the optimized conformations of (6S)-4 were obtained at the B3LYP/6-31+G(d) level and used for ECD calculations. The agreement of the experimental and calculated ECD curves (Figure 5) indicated the 6S absolute configuration of 4.

Compound 5 was isolated as a pale yellow powder. The ¹H and ¹³C NMR data (Table 3) showed two singlet methyls (δ_C 9.1, 18.5), two methoxy groups (δ_C 56.9, 61.8), eight olefinic carbons (δ_C 96.3, 104.8, 115.7, 129.6, 150.7, 158.5, 159.6, 165.0) and one carbonyl carbon (δ_C 183.5). Upon careful analysis of the ¹H NMR, ¹³C NMR, and HMBC spectrum (Table 3 and Table S1 and Figures S36–S39), it was found that compound 5 was identical to the known talamin B [16]. Therefore, compound 5 was identified as talamin B.

Compound 6 was isolated as a white solid and was identified astalaminoid C [19] through meticulous comparison of its physical and spectroscopic characteristics with those documented in the existing literature (Table S2, Figures S40 and S41).

Compounds 1–6 were evaluated for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical eliminating activity. Consequently, 3, 4, and 6 had DPPH radical eliminating activities, with IC₅₀ values of 6.79 to 56.92 µM (

Table 5. DPPH radical scavenging activity of compounds 1–6 (IC₅₀, µM).

Compd.	1	2	3	4	5	6	Vitamin C
IC50 (µM)	>200	>200	6.79	56.92	>200	32.11	12.15

). Among them, 3 displayed significantly better values than those of the positive control Vitamin C (IC₅₀ = 12.15 µM). Analysis of compounds 3 and 5 shows that the hydroxyl group is the basis for antioxidant activity, and the hydroxyl group at position C-8 of 3 may be essential for bioactivity.

3. Materials and Methods

3.1. General Experimental Procedures

The HRESIMS data were measured on a Thermo Scientific LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). UV spectra were recorded on a Hitachi 5430 spectrophotometer (Hitachi Ltd., Tokyo, Japan). NMR spectra were collected on Bruker AVANCE NEO 400 MHz spectrometer and JEOL JNM-ECZ600R/S1 spectrometer with tetramethylsilane (TMS) as an internal standard. Column chromatography (CC) was performed with silica gel (300–400 mesh, Qingdao Marine Chemical Inc., Qingdao, China) and Sephadex LH-20 (Amersham Biosciences, Buckinghamshire, UK). MPLC was performed on a Waters 1526. HPLC spectra were recorded on a Hitachi Primaide HPLC using an ODS column (HPLC (YMC-Pack ODS-A, 10 × 250 mm, 5 µm, 3 mL/min)) (YMC Co., Ltd., NJ, USA).

3.2. Fungi Material and Fermentation

Penicillium sp. HDN15-312 (GenBank No.MK41874) was isolated from a sample of a mangrove forest in Sanya, Hainan. It was deposited at Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, People’s Republic of China. This strain is a facultative anaerobe, producing an aerial mycelium that is beige to light yellow, a substrate mycelium that is green, and straight to curved hyphae with long, straight spore chains.

3.3. OSMAC Study, Fermentation, and Extraction

Based on the OSMAC strategy, six different liquid media, labeled as Fungus 2# liquid media (glucose 1%, maltose 2%, mannitol 2%, monosodium glutamate 1%, KH₂PO₄ 0.05%, MgSO₄·7H₂O 0.03%, corn steep liquor 0.1% and yeast extract 0.3%, seawater), modified Fungus 2# liquid media (soluble starch 4%, yeast extract 0.1%, MgSO₄·7H₂O 0.03%, monosodium glutamate 0.2%, sucrose 4%, KH₂PO₄ 0.05%, maltose 3%, soybean flour 0.05%, peptone 0.2%, seawater), SDA liquid media (peptone 1%, NaCl 0.5%, glucose 4%, freshwater), oligotrophic liquid media (soluble starch 1%, peptone 0.1%, seawater), PDB liquid media (glucose 20 g/L, potato extract 200 g/L, seawater), and glycerol liquid media (glycerin 2%, peptone 0.2%, yeast powder 0.4%, seawater) were utilized to culture the strain HDN15-312 under static or shaking conditions. The products of the strain HDN15-312 cultured in modified Fungus 2# liquid media under static conditions were different from the products under other conditions. Therefore, the modified Fungus 2# liquid media under static conditions was selected as the optimal condition for large-scale fermentation. The fungus was cultured in 1000 mL Erlenmeyer flasks, each containing 300 mL of modified Fungus 2# liquid media, at 28 °C for 30 days after adjusting its pH to 6.5 in natural seawater (collected from JiaoZhou Bay, Qingdao, China). A total of 15 L of broth was extracted using EtOAc (3 × 15 L) to obtain an extract weighing 50 g.

3.4. Isolation and Purification of Compounds

The crude extract was applied over a VLC column and eluted with mixtures of Petroleum ether-EtOAc to give nine fractions (Fr.1–Fr.9). Fr.2–Fr.5 were combined as Fr.A, which was separated by MPLC using an ODS column to obtain ten subfractions (Fr.A.1–Fr.A.10). Fr.A.6 was purified by semi-preparative HPLC to obtain 1 (3 mg, t_R = 15 min) and 2 (2 mg, t_R = 16 min). Fr.A.7 was purified by semi-preparative HPLC to obtain 3 (2.5 mg, t_R = 11 min). Fr.6–Fr.8 were combined as Fr.B, which was separated by HPLC using an ODS column to obtain ten subfractions (Fr.B.1–Fr.B.8). Fr.B.6 was purified by semi-preparative HPLC to obtain 4 (3 mg, t_R = 15 min) and 5 (5 mg, t_R = 19 min). Fr.B.3 was purified by semi-preparative HPLC to obtain 6 (3 mg, t_R = 11 min).

Furantide A (1): pale yellow powder, ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$ +45.6 (c 0.05 MeOH); UV (MeOH) λ_max 217 (1.8), 322 (1.3) nm; IR (KBr) ν_max 3298, 2932, 1671, 1446 cm⁻¹; ¹H and ¹³C NMR data, Table 1; HRESIMS m/z 223.0965 [M + H]⁺ (calcd for C₁₂H₁₅O₄, 223.0965).

Furantide B (2): pale yellow powder, ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$ +81.4 (c 0.1 MeOH); UV (MeOH) λ_max 206 (0.9), 234 (0.8), 297 (1.9) nm; IR (KBr) ν_max 2967, 2873, 1671, 1528, 1447 cm⁻¹; ¹H and ¹³C NMR data, Table 1; HRESIMS m/z 225.1124 [M + H]⁺ (calcd for C₁₂H₁₇O₄, 225.1121).

Talamin E (3): yellow powder, ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$ +56.2 (c 0.05 MeOH); UV (MeOH) λ_max 226 (0.3), 260 (0.6), 344 (0.1) nm; IR (KBr) ν_max 1655, 1576, 1523, 1436, 1198 cm⁻¹; ¹H and ¹³C NMR data, Table 2; HRESIMS m/z 223.0595 [M + H]⁺ (calcd for C₁₁H₁₁O₅, 223.0601).

Arugosinacid A (4): yellow powder, ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$ −37.5 (c 0.05 MeOH); UV (MeOH) λ_max 216 (0.5), 263 (0.4), 344 (0.1) nm; IR (KBr) ν_max 3736.19, 2932.84, 1682.38, 1586.14, 1206.97 cm⁻¹; ¹H and ¹³C NMR data, Table 2; HRESIMS m/z 315.0505 [M − H]⁻ (calcd for C₁₆H₁₁O₇, 315.0501).

3.5. Computation Section

Conformational searches were conducted using Spartan’14 [20], based on the MMFF (Merck Molecular Force Field). Compounds 1, 2, and 4 were further optimized with DFT calculations at the B3LYP/6-31+G(d) level, utilizing the Gaussian 09 program [21]. TDDFT calculations were performed on the two lowest-energy conformations for 1 and 2 (>5% population). ECD spectra were gained using the SpecDis program [22] in conjunction with a Gaussian [21] band shape with 0.2 eV width for 1, 0.4 eV width for 2, and 0.1 eV width for 4 from dipole-length rotational strengths. The calculated spectra were shifted by −10 nm for 1, −20 nm for 2, +5 nm for 4 to facilitate comparison to the experimental data. We acknowledge the support of the High-Performance Biological Supercomputing Center at the Ocean University of China for this research.

3.6. Assay of DPPH Activity

Based on the method of Sharma [23], with some amendments, the eliminating activity against DPPH radicals was enforced. The compounds 1–6 and vitamin C were dissolved in absolute ethanol and diluted into 5 gradients. These were vortexed to mix well, protected from light at room temperature for 30 min, and the absorbance was read at 515 nm as a positive control.

The ability to scavenge the DPPH was calculated according to the equation:

DPPH free radical scavenging rate D VC% = [(A_blank − A_control) ÷ A_blank] × 100%

DPPH free radical scavenging rate D _sample% = [[A_blank − (A_sample − A_control)] ÷ A_blank] × 100%

A_control: the absorbance of the DPPH solution;

A_blank: the absorbance of ethanol.

4. Conclusions

In conclusion, four polyketides (1–4) were isolated and determined from Penicillium sp. HDN15-312, which was isolated from a sample of a mangrove forest in Egret Park in Sanya, Hainan. Compounds 1 and 2 stood as the unique pair of naturally occurring substances characterized by their distinctive 6-methyl-cyclohexanone-furan ring structure. The bioactivity screening showed that compounds 3, 4 and 6 showed potent DPPH radical scavenging capacities. The DPPH radicals scavenging activity of 3, with an IC₅₀ value of 6.79 µM, was better than vitamin C. Our research highlights the potential for sifting and exploiting therapeutic molecules from marine fungi.