Induction of Three New Secondary Metabolites by the Co-Culture of Endophytic Fungi Phomopsis asparagi DHS-48 and Phomopsis sp. DHS-11 Isolated from the Chinese Mangrove Plant Rhizophora mangle

Abstract

Co-cultivation is a powerful emerging tool for awakening biosynthetic gene clusters (BGCs) that remain transcriptionally silent under artificial culture conditions. It has recently been used increasingly extensively to study natural interactions and discover new bioactive metabolites. As a part of our project aiming at the discovery of structurally novel and biologically active natural products from mangrove endophytic fungi, an established co-culture of a strain of Phomopsis asparagi DHS-48 with another Phomopsis genus fungus DHS-11, both endophytes in mangrove Rhizophora mangle, proved to be very efficient to induce the production of new metabolites as well as to increase the yields of respective target metabolites. A detailed chemical investigation of the minor metabolites produced by the co-culture of these two titled fungal strains led to the isolation of six alkaloids (1–6), two sterols (7, 8), and six polyketides (9–14). In addition, all the compounds except 8 and 10, as well as three new metabolites phomopyrazine (1), phomosterol C (7), and phomopyrone E (9), were not present in discrete fungal cultures and only detected in the co-cultures. The structures were elucidated on the basis of spectroscopic analysis, and the absolute configurations were assumed by electronic circular dichroism (ECD) calculations. Subsequently, the cytotoxic, immunosuppressive, and acetylcholinesterase inhibitory properties of all the isolated metabolites were determined in vitro. Compound 8 exhibited moderate inhibitory activity against ConA-induced T and LPS-induced B murine splenic lymphocytes, with IC₅₀ values of 35.75 ± 1.09 and 47.65 ± 1.21 µM, respectively.

1. Introduction

Endophytic fungi from special ecological niches such as mangrove ecosystems are one of the most pivotal and promising sources of bioactive natural products, presumably owing to their intriguing structural skeleton and the promising pharmacological effect of their secondary metabolites, making them attractive repositories for structurally unique secondary metabolites endowed with numerous biological activities [1,2,3,4,5]. Until 2020, at least 1090 new structures have been reported from mangrove fungal endophytes, including polyketides, terpenes, alkaloids, and peptides representing the main chemotypes [6,7,8,9,10]. Nevertheless, owing to the rise in whole-genome sequences, most mangrove endophytic fungi were demonstrated to possess significantly more biosynthetic gene clusters (BGCs) than the number of compounds they produce previously expected [11,12]. The co-culturing of two or more different fungi within a confined vessel in a manner that approximates what they are forced to do in nature may trigger the expression of silent biosynthetic pathways and uncover unprecedented chemical diversity. These stimulated secondary metabolites are probably being used by those fungi to help fight for their survival [13].

In our efforts to identify new bioactive secondary metabolites from mangrove-derived fungi, we previously investigated the secondary metabolites of two strains of the fungal genus Phomopsis, namely P. asparagi DHS-48 and Phomopsis sp. DHS-11, from which three new dimeric xanthones phomoxanthones L-N have been isolated and the yields of respective target metabolites enhanced simultaneously [14].

To highlight the metabolite modifications that are caused by fungal interactions, an HPLC chromatogram with UV detection (HPLC-UV) and molecular networking were adapted to the analysis of the crude EtOAc extract of a 30-day solid rice medium as previously reported [14]. Comparing the HPLC profiles (Figure S1a) of the whole co-culture to those from the monocultures demonstrated that compounds 1, 3–5, 7, 9, 12, and 14 were induced metabolites that were not produced when the two fungi were cultured alone. These observations were further confirmed by the UPLC-ESI-MS/MS-based molecular networking (Figure S1b) generated through the online Global Natural Products Social Molecular Networking (GNPS) platform; nevertheless, some minor components cannot be detected by this technique. The chemical investigation of the minor metabolites obtained following the co-cultivation of these two titled fungal strains, including a new alkaloid (1), a new sterol (7), a new pyranone (9), as well as 11 known compounds such as cyclo(L-Tyr-L-Tyr) (2) [15], 5-methyl-uridine (3) [16], thymidine (4) [17], cyclo(L-Hyp-L-Ala) (5) [18], nicotine acid (6) [19], 3β-hydroxy-5,9-epoxy-(22E,24R)-ergosta-7,22-dien-6-dione (8) [20], 2-(2′-hydroxypropyl)-5-methyl-7-hydroxychromone (10) [21], 3-(2,6-dihydroxyphenyl)-4-hydroxy-6-methyl-isobenzofuran-1(3H)-one (11) [22], 3-(2-deoxy-β-erythro-pentofuranosyl)-6-hydroxy-2H-pyran-2-one (12) [23], (R)-mevalonolactone (13) [24], and bis(2-ethylhexyl) phthalate (14) [25], were isolated from the co-culture extracts of DHS-48 and DHS-11 (Figure 1). Herein, we report on the phylogenetic analysis of fungal strains DHS-48 and DHS-11, followed by the isolation, structure elucidation, and biological activities of these compounds.

2. Results and Discussion

2.1. Phylogenetic Analysis of Fungal Strains DHS-48 and DHS-11

The strains DHS-48 and DHS-11 were isolated from the Chinese mangrove plant Rhizophora mangle as endophytes and identified using the ITS region. BLAST search results indicated that DHS-48 and DHS-11 were found to have 100% identity to the type strain Phomopsis asparagi strain A0640 (KF498860) and Phomopsis sp. strain CBS:123 (OR801625), respectively. Based on the ITS gene sequences, a total of 60 Phomopsis-type strains originated from different ecosystems (including mangrove habitats, plants, soil, insects, and unknown origin) were retrieved from Genbank to construct the phylogenetic tree using unrooted neighbor-joining (NJ) algorithm. Phylogenetic and correlation analysis showed that the ITS sequence of DHS-48 clustered with other Phomopsis species from different ecological niches, e.g., DHS-11 from the same host, mangroves (3 strains), plants (5 strains), and unknown origin (10 strains), within a mono phylogenetic group in maximum parsimony with bootstrap support >75% (Figure 2). Amongst them, the ITS gene sequence of DHS-48 most closely resembled DHS-11 and formed a sister clade with 98% bootstrap support. Considering the impact of the taxonomic criteria and ecological impact, we selected Phomopsis asparagi DHS-48 and Phomopsis sp. DHS-11 belonging originally to the same habitat for the prioritization of co-cultivation to mimic the co-existing occurring interactions in naturally ecological situations to induce the production of new natural products derived from fungal interactions.

2.2. Structure Elucidation of New Compounds

Phomopyrazine (1) was obtained as a colorless amorphous powder. HRESIMS gave an [M + Na]⁺ ion peak at m/z 275.1002 (calcd for [M + Na]⁺ 275.1008), supporting a molecular formula of C₁₂H₁₆N₂O₄. The 1D NMR data (

Table 1. ¹H (400 MHz) and ¹³C (100 MHz) NMR data of 1 and 9 in CD₃OD.

Position	1			9
	δC Type	δH Mult (J in Hz)	HMBC (H to C)	δC Type	δH Mult (J in Hz)	HMBC (H to C)
1
2	152.6, C		C-5, 10, 11, 13	168.3, C
3	157.0, C		C-5, 10, 13, 14	101.2, C
4				169.1, C
5	141.5, CH	8.23, s	C-2, 3, 7, 10, 13	95.8, CH	6.43, s	C-3, 4, 6, 7
6	154.5, C		C-5, 7, 8	169.2, C
7	31.7, CH2	3.02, t, 7.5	C-5, 6, 8, 9	36.6, CH	2.86, m	C-6, 8, 9, 12
8	36.2, CH2	2.64, t, 7.5	C-6, 7, 9	38.2, CH2	1.90, m 1.73, m	C-6, 7, 9, 12
9	179.0, C		C-7, 8, 10, 11	60.3, CH2	3.53, m	C-7, 8
10	30.4, CH2	3.08, t, 7.5	C-2, 3, 11, 12	8.4, CH3	1.84, s	C-2, 3, 4, 5
11	35.3, CH2	2.66, t, 7.5	C-2, 10, 12	57.3, CH3	3.94, s	C-4
12	179.1, C			18.8, CH3	1.26, d, 7.0	C-6, 7, 8
13	28.2, CH2	2.87, q, 7.5	C-2, 3, 14
14	13.2, CH3	1.28, t, 7.5	C-3, 13

) of 1 in combination with distortionless enhancement by polarization transfer (DEPT) and heteronuclear single quantum coherence (HSQC) spectrum revealed the existences of a methyl [δ_H 1.28, (t, J = 7.5 Hz), δ_C 13.2, q, 14-CH₃], five methylenes [δ_H 3.02 (t, J = 7.5 Hz), δ_C 31.7, t, CH₂-7; δ_H 2.64 (t, J = 7.5 Hz), δ_C 36.2, t, CH₂-8; δ_H 3.08 (t, J = 7.5 Hz), δ_C 30.4, t, CH₂-10; δ_H 2.66 (t, J = 7.5 Hz), δ_C 35.3, t, CH₂-11; δ_H 2.87 (q, J = 7.5 Hz), δ_C 28.2, t, CH₂-13], an olefinic methine at δ_H (8.23, 1H, s, CH-5), and five quaternary carbons [including two carboxylic carbonyl at δ_C 179.0 (C-9) and δ_C 179.1 (C-12)]. The ¹H-¹H COSY correlations (Figure 3) suggested the presence of the fragments CH₂(7)-CH₂(8), CH₂(10)-CH₂(12), and CH₂(13)-CH₃(14) incorporating the HMBC correlations of H₂-7/C-5, C-6, and C-9; H-5/C-3 and C-13; and H₂-10/C-3 and C-4 and indicated the existence of the 2,6-pyrazinedipropanoic acid group. An additional ethyl moiety located at C-3 was corroborated by the HMBC correlations of H₂-13/C-2, C-3, and H₃-14/C-3. Thus, the structure of 1 was established and named as phomopyrazine.

Phomosterol C (7) was obtained as a colorless amorphous powder, and its molecular formula was established as C₂₉H₄₄O₃ based on HRESIMS data (m/z 441.3363 [M + H]⁺, calcd for C₂₉H₄₅O₃ 441.3362) indicating eight indices of unsaturation. The ¹H NMR spectrum (

Table 2. ¹H (400 MHz) and ¹³C (100 MHz) NMR data of 7 and 8 in CD₃OD.

Position	7			8
	δC Type	δH Mult (J in Hz)	HMBC	δC Type	δH Mult (J in Hz)
1	26.6, CH2	Ha 2.29, dt, 14.5, 4.5	C-2, 3, 5, 9, 10, 19	26.6, CH2	Ha 2.20, dt, 1.5, 13.4
		Hb 1.50, m			Hb 1.39, m
2	31.0, CH2	Ha 1.87, m	C-1, 3, 4, 10	31.0, CH2	Ha 1.78, m
		Hb 1.46, m			Hb 1.36, m
3	67.8, CH	3.92, m	C-1, 2, 4, 5	67.8, CH	3.83, m
4	37.2, CH2	Ha 2.02, m		37.1, CH2	Ha 1.92, m
		Hb 1.63, m	C-2, 3, 5, 10		Hb 1.52, m
5	80.2, C		C-1, 3, 4, 6, 7, 19	80.2, C
6	200.2, C			200.1, C
7	120.9, CH	5.58, d, 2.0	C-5, 6, 8, 9, 14	120.9, CH	5,49, s
8	165.1, C			165.0, C
9	76.2, C			76.1, C
10	42.8, C			42.8, C
11	29.3, CH2	Ha 1.97, m Hb 1.76, m	C-8, 9, 10, 12, 13	29.1, CH2	Ha 1.70, m
					Hb 1.64, m
12	36.2, CH2	Ha 1.89, m Hb 1.72, m	C-9, 11, 13, 14, 18	36.2, CH2	Ha 1.80, m
					Hb 1.62, m
13	46.2, C			46.2, C
14	52.8, CH	2.75, ddd, 11.6, 9.8, 2.6	C-7, 8, 9, 12, 13, 15, 18	52.8, CH	2.66, dd, 11.5, 7.6
15	23.4, CH2	Ha 1.60, m Hb 1.52, m	C-19, 25, 24	23.4, CH2	Ha 1.52, m Hb 1.42, m
16	28.5, CH2	Ha 1.87, m Hb 1.30, m	C-13, 14, 15, 17, 20	29.3, CH2	Ha 1.89, m Hb 1.36, m
17	58.2, CH	1.46, m	C-13, 14, 15, 16, 20, 21, 22	57.4, CH	1.34, m
18	12.7, CH3	0.68, s	C-12, 13, 14, 17	20.6, CH3	0.90, s
19	20.6, CH3	0.99, s	C-1, 5, 9, 10	12.6, CH3	0.57, s
20	35.9, CH	2.41, m	C-13, 16, 17, 21, 22, 23	41.7, CH	1.97, m
21	21.2, CH3	0.98, d, 6.8	C-17, 20, 22	21.6, CH3	0.96, d, 6.6
22	132.5, CH	4.94, d, 9.6	C-17, 20, 21, 23, 24, 29	133.6, CH	5.13, dd, 15.2, 8.2
23	137.3, C		C-20, 22, 24, 25, 28	136.7, CH	5.19, dd, 15.2, 7.6
24	51.8, CH	1.68, m	C-22, 23, 25, 26, 27, 28, 29	44.4, CH	1.76, m
25	32.0, CH	1.55, m	C-23, 24, 26, 27, 28	34.4, CH	1.39, m
26	22.2, CH3	0.8, d, 6.6	C-24, 25, 27	20.5, CH3	0.77, d, 6.8
27	20.6, CH3	0.87, d, 6.6	C-24, 25, 26	20.1, CH3	0.75, d, 6.8
28	17.5, CH3	0.97, d, 6.8	C-23, 24, 25	18.2, CH3	0.85, d, 6.8
29	13.4, CH3	1.53, s	C-22, 23, 24

) showed the presence of a series of characteristic signals for three methyl singlets (δ_H 0.68, s, 18-CH₃; 0.99, s, 19-CH₃; 1.53, s, 29-CH₃), four methyl doublets [δ_H 0.98 (d, J = 6.8 Hz), 21-CH₃; δ_H 0.80 (d, J = 6.6 Hz), 26-CH₃; δ_H 0.87 (d, J = 6.6 Hz), 27-CH₃; 0.98 (d, J = 6.8 Hz), 28-CH₃], an oxymethine (δ_H 3.92, m, CH-3), and two olefinic proton [δ_H 5.58 (d, J = 2.0 Hz), CH-7; δ_H 4.94 (d, J = 9.6 Hz), CH-22]. The ¹³C NMR and HSQC spectral data of 7 exhibited 29 carbon signals, including seven methyls, seven methylenes, eight methines (two olefinic and one oxygenated), and seven non-hydrogenated carbons (one carbonyl, two olefinic, and two oxygenated). These observed data suggested that 7 was an ergostane-type pentacyclic steroid, and closely resembled those of the co-isolated 3β-hydroxy-5,9-epoxy-(22E,24R)-ergosta-7,22 dien-6-one (8) previously isolated from the endophytic fungus Chaetomium sp. M453 [20]. The main difference between the two compounds is the presence of a methyl group (δ_H 1.53, s, δ_C 13.4, q, 29-CH₃) located at C-23 (δ_C 137.3, s) in 7 instead of olefinic methine (CH-23) in 8. Confirming evidence was obtained from the ¹H-¹H COSY correlation (Figure 3) of H₃-21/H-20/H-22 and H₃-28/H-24/H-25/H₃-26/H₃-27, and HMBC correlations from H₃-29 to C-22 (δ_C 132.5, d), C-23, and C-24 (δ_C 51.8, d) (Figure 2). The relative stereochemistry of 7 was determined by the analysis of the NOESY spectrum (Figure 4). Diagnostic correlations positioned H_b-1, H₃-19, H-7, H-14 and H₃-18, and H-20 on the α-face and H_a-1, H-3, H-17, and H₃-28 on the β-face of 7, whereas the configuration of the double bond at Δ22 was deduced to be E by the comparison of the chemical shifts with those of the same positions of 8 and the observed NOE correlations (Figure 4) between H₃-29/H₃-26 and H-22/H₃-28/H₃-27. The absolute configuration was assigned by the comparison of the experimental and simulated electronic circular dichroism (ECD) spectra generated by the time-dependent density functional theory (TDDFT) calculations at the B3LYP/6-31+G(d,p) level using the Gaussian 09 program. The calculated and experimental curves showed a high degree of coincidence (Figure 5). Therefore, the structure of 7 was elucidated as 3β-hydroxy-3S,5R,9R,10R,13R,14S,17R,20R,22E,24R-5,9-epoxy-23-methyl-ergosta-7,22 dien-6-one.

Phomopyrone E (9) was obtained as a colorless amorphous powder. The molecular formula of 9 was established as C₁₁H₁₆O₄ based on the HR-ESI-MS peak at m/z 235.0941 [M + Na]⁺ (calcd for C₁₁H₁₆O₄ Na 235.0941), requiring four degrees of unsaturation. The ¹H and ¹³C NMR data of 9 (Table 1) and the ¹H-¹H COSY spectrum (Figure 3) revealed the presence of one methoxy group (δ_H 3.94, s, δ_C 57.3, q, OCH₃-11), two methyl groups (δ_H 1.84, s, δ_C 8.4, q, CH₃-10; δ_H 1.26 (d, J = 7.0 Hz), δ_C 18.8, q, 12-CH₃), two methylene groups (δ_H 1.90, m, H-8a, 1.73, m, H-8b, δ_C 36.6, t, CH₂-8; δ_H 3.55, m, δ_C 60.3, t, CH₂-9, oxygenated), a methine group (δ_H 2.86, m, δ_C 36.6, d, CH-7), and an olefinic methine (δ_H 6.43 brs, δ_C 95.8, d, CH-5, aromatic). The typical ¹³C NMR data at δ_C 168.3 (C-2), 101.2 (C-3), 169.1 (C-4), 95.8 (C-5), and 169.2 (C-6) suggested 9 was an α-pyrone. The COSY correlations between H₃-12/H-7, H-7/H₂-8, and H₂-8/H₂-9, as well as the HMBC correlations from H-7 to C-5 and C-6 (δ_C 169.2), and from H₂-8 and H₃-12 to C-6 indicated the presence of a 4-hydroxybutan-2-yl group at C-6 of the pyrone core. Furthermore, key HMBC correlations from H₃-10 to C-2, C-3, and C-4, and from H₃-11 to C-4 allowed the connection of CH₃-10 and OCH₃-11 at C-3 and C-4 positions, respectively. The proposed planer structure is identical to phomopyronol [26] isolated from the medicinal plant Erythrina crista-galli. Interestingly, the measured optical rotation value ${\left[\mathsf{\alpha }\right]}_D^{20}$ + 60 (c 0.0001, MeOH) of 9 reported in this work does not correspond with those reported for phomopyronol ${\left[\mathsf{\alpha }\right]}_D$ − 11 (c 0.5, MeOH). The absolute configuration of C-7 was assigned to be R in 9 by the comparison of its calculated and experimental ECD spectrum (Figure 5). As a result, the structure of 9 was determined and considered to be a new dextro optical isomer of the known (−)-phomopyronol.

2.3. Bioactivities of Isolated Compounds

All of the isolated compounds (1–14) were evaluated for their cytotoxic, immunosuppressive, and acetylcholinesterase (AChE) inhibitory activities. The results indicated that compounds 7, 8, and 14 showed slight in vitro cytotoxicity against human liver cells HepG2 (IC₅₀ values ranging from 65.97 to 73.37 μM) and cervical cancer cells Hela (IC₅₀ values ranging from 72.02 to 87.30 μM) (

Table 3. Cytotoxicity of compounds 1–14.

Compound	IC50 (µM) a
	HepG2	Hela
7	65.97 ± 2.56	72.34 ± 2.03
8	77.41 ± 4.12	72.02 ± 2.89
14	73.37 ± 2.25	87.30 ± 0.74
1–6, 9–13	-	-
Adriamycin b	\	0.88 ± 0.71
Fluorouracil c	179.03 ± 25.82	\

^a data are presented as mean ± SD from three separate experiments. ^b Hela cell positive control. ^c HepG2 cell positive control. ‘-’ stands for no inhibitory at 10 µg/mL. ‘\’stands for not tested.

). Compounds 1 and 8–10 exhibited weak to moderate immunosuppressive activity, of which 8 was most potent against the proliferation of ConA-induced (T-cell) and LPS-induced (B-Cell) murine splenic lymphocytes with the IC₅₀ values of 35.75 ± 1.09 and 47.65 ± 1.21 µM, respectively (

Table 4. Immunosuppressive activity of compounds 1–14.

Compound	IC50 (µM) a
	ConA-Induced T-Cell Proliferation	LPS-Induced B-Cell Proliferation
1	125.1 ± 1.12	133.87 ± 3.43
2–7	-	-
8	35.75 ± 1.09	47.65 ± 1.21
9	108.21 ± 1.32	112.76 ± 2.11
10	111.01 ± 1.02	123.84 ± 1.25
11–14	-	-
cyclosporin A b	4.39 ± 0.02	25.11 ± 0.43

^a data are presented as mean ± SD from three separate experiments. ^b positive control. ‘-’ stands for no inhibitory effect at 200 µM.

). Nonetheless, only compound 11 showed weak inhibition of AChE with an IC₅₀ value of 86.11 ± 1.56 μM (

Table 5. Acetylcholinesterase inhibitory activity of compounds 1–14.

Compound	IC50 (µM) a
11	86.11 ± 1.56
1–10, 12–14	-
Donepezil b	0.25 ± 0.76

^a data are presented as mean ± SD from three separate experiments. ^b positive control. ‘-’ stands for no inhibitory effect at 200 µM.

).

3. Materials and Methods

3.1. General Procedures

The optical rotations were acquired using the ATR-W2 HHW5 digital Abbe refractometer (Shanghai Physico-optical Instrument Factory, Shanghai, China). The UV spectra were obtained by using a Shimadzu UV-2600 PC spectrophotometer (Shimadzu Corporation, Tokyo, Japan), while the ECD spectra were measured on a JASCO J-715 spectra polarimeter (Japan Spectroscopic, Tokyo, Japan). All the LC/MS data were collected by the LCMS-IT-TOF instrument (Shimadzu Corporation, Tokyo, Japan) with an ESI source. The ¹H, ¹³C, and 2D NMR spectra were acquired on a Bruker AV 400 NMR spectrometer (Bruker Corporation, Fällanden, Switzerland) using TMS as an internal standard. TLC and column chromatography (CC) were executed on silica gel (200–400 mesh, Qingdao Marine Chemical Inc., Qingdao, China) or a Sephadex-LH-20 (18–110 µm, Merck, Darmstadt, Germany), respectively. Semi-preparative HPLC was obtained on an Agilent Technologies 1200LC with a C18 column (Agilent Technologies, California, United States, 10 mm × 250 mm). High-speed centrifugation was performed at a TGL-16B Anting centrifugal machine (Anting Scientific Instrument Factory, Shanghai, China). The constant temperature water bath was in HH-2 thermostat water baths (Hervey Biotechnology Corporation, Jinan, China). All the crude extracts were eluted with a flow rate of 0.8 mL·min⁻¹ over a 50 min gradient (solvents: A, H₂O; B, MeOH), as follows: 0–5 min, 25% B; 5–15 min, 25–30% B; 15–30 min, 30–55% B; 30–40 min, 55–75% B; 40–50 min, 70–90% B; and 50–60 min, 90–100% (Figure S1).

3.2. Fungal Material

Endophytic fungi Phomopsis asparagi and Phomopsis sp. were isolated from the fresh root of the mangrove plant Rhizophora mangle collected in the Dong Zhai Gang-Mangrove Garden on Hainan Island, China, in October 2015. The fungi were identified as Phomopsis asparagi (strain no. DHS-48) and Phomopsis sp. (strain no. DHS-11) by ITS gene sequence (GenBank Accession No. MT126606 and No. OR801625). Two voucher strains were deposited at one of the authors’ laboratories (J.X.).

3.3. Phylogenetic Analysis

The available homologs were searched in the GenBank database (http://ncbi.nlm.nih.gov, accessed on 12 February 2024) using the BLASTN algorithm (http://www.ncbi.nlm.nih.gov/BLAST, accessed on 12 February 2024). Multiple alignments were made using the CLUSTAL_X tool in MEGA version 7.0. [27]. A phylogenetic tree based on the neighbor-joining method (NJ) was used to infer the evolutionary history of the fungi under Kimura’s two-parameter model [28], and the bootstrapping was carried out using 1000 replications. Tree visualization was carried out via the Interactive Tree of Life (iTOL) web service [29].

3.4. Interaction between Phomopsis asparagi, Phomopsis sp., and Co-Cultivation

A morphological investigation of the co-culture interaction between Phomopsis asparagi and Phomopsis sp. was conducted on a 90 mm PDA plate using different inoculation amounts. The circular pieces of actively growing mycelium agar from each fungus (1 cm, 0.5 cm, and 0.2 cm in diameter) were placed 5 cm from each other on a new agar plate. The plates were sealed with parafilm and incubated at 28 °C for 15 days, and the diameter of the mycelium was measured every day. Growth characteristics such as overgrowth, contact inhibition, and distance inhibition of the fungal organisms were visually observed.

3.5. Preparation of Phomopsis asparagi, Phomopsis sp., Co-Cultivation, Large-Scale Fermentation, and Extracts

The two fungi were independently cultivated on PDA at 28 °C for 14 days. After that, the two fungi colonies were simultaneously inoculated into an autoclaved rice solid-substrate medium in Erlenmeyer flasks (130 × 1 L), each containing 100 g of rice and 100 mL of 0.3% saline water, and fermented at 28 °C for 30 days. Following the fermentation process, the co-cultured fermentation mixes were extracted three times with EtOAc, and the filtrate was then distilled under reduced pressure to obtain 30 g of crude extract.

3.6. Isolation of Compounds

Using stepped gradient elution with CH₂Cl₂-MeOH (0–100%) on silica gel column chromatography (CC), the crude extracts were separated into nine fractions (Fr. 1–Fr. 9). The fraction Fr. 3 was subjected to open silica gel CC using gradient elution with CH₂Cl₂-MeOH (100:0–1:1, v/v) to obtain 6 fractions (Fr. 3.1–Fr. 3.6). Fr. 4 was subjected to open silica gel CC using gradient elution with CH₂Cl₂-MeOH (100:0–1:2, v/v) to obtain 7 fractions (Fr. 4.1–Fr. 4.7). The subfraction Fr. 4.3 was applied to ODS CC with the gradient elution of MeOH/H₂O mixtures (v/v, 1:4, 3:7, 2:3, 1:1, 3:2, 7:3, 4:1, 0:1) and obtained five subfractions (Fr. 4.3.1–Fr. 4.3.5). Then, Fr. 4.3.4 were purified by semi-preparative reversed-phase HPLC using the isocratic elution of MeOH-H₂O (60:40, v/v, 2 mL/min, UV λ_max 210 nm) to afford compound 9 (6 mg, t_R = 45 min). Fr. 5 was subjected to open silica gel CC using gradient elution with CH₂Cl₂-MeOH (100:2–1:2, v/v) to obtain 5 fractions (Fr. 5.1–Fr. 5.4). Fr. 5.2 was subjected to open silica gel CC using gradient elution with CH₂Cl₂-MeOH (100:2–1:2, v/v) to obtain 5 fractions (Fr. 5.2.1–Fr. 5.2.5). Then, Fr. 5.2.2 were purified by semi-preparative reversed-phase HPLC using the isocratic elution of MeOH-H₂O (60:40, v/v, 2 mL/min, UV λ_max 210 nm) to afford compound 10 (5 mg, t_R = 24 min). Fr. 6 was subjected to open silica gel CC using gradient elution with CH₂Cl₂-MeOH (100:3–1:2, v/v) to yield 5 fractions (Fr. 6.1–Fr. 6.4). Fr. 6.2 was purified by semi-preparative reversed-phase HPLC using isocratic elution MeOH-H₂O (70:30, v/v, 2 mL/min, UV λ_max 210 nm) to afford compound 13 (5 mg, t_R = 38 min). Fr. 7 was subjected to open silica gel CC using gradient elution with CH₂Cl₂-MeOH (100:2–1:2, v/v) to obtain 5 fractions (Fr. 7.1–Fr. 7.5). Fr. 7.2 was chromatographed on a Sephadex LH-20 CC by eluting with MeOH to yield three fractions (Fr. 7.2.1–Fr. 7.2.3). Then, Fr. 7.2.2 were purified by semi-preparative reversed-phase HPLC using the isocratic elution of MeOH-H₂O (60:40, v/v, 2 mL/min, UV λ_max 210 nm) to afford compound 1 (6 mg, t_R = 35 min). Fr. 8 was applied to ODS CC with the gradient elution of MeOH/H₂O mixtures (v/v, 1:4, 3:7, 2:3, 1:1, 3:2, 7:3, 4:1, 0:1) and obtained five subfractions (Fr. 8.1–Fr. 8.5). Fr. 8.2 was chromatographed on a Sephadex LH-20 CC by eluting with MeOH to yield three fractions (Fr. 8.2.1–Fr. 8.2.3). Then, Fr. 8.2.2 were purified by semi-preparative reversed-phase HPLC using the isocratic elution of MeOH-H₂O (70:30, v/v, 2 mL/min, UV λ_max 210 nm) to afford compound 11 (6 mg, t_R = 32 min) and compound 6 (6 mg, t_R = 39 min). Fr. 8.5 was purified by HPLC using the isocratic elution of (MeOH/H₂O, 70:30, v/v; 2 mL/min, UV λ_max 210 nm) to yield compound 8 (6 mg, t_R = 14 min) and compound 7 (6 mg, t_R = 37 min). Fr. 9 was subjected to open silica gel CC using gradient elution with CH₂Cl₂-MeOH (100:4–1:2, v/v) to obtain 5 fractions (Fr. 9.1–Fr. 9.5). The subfraction Fr. 9.3 was applied to ODS CC with the gradient elution of MeOH/H₂O mixtures (v/v, 1:4, 3:7, 2:3, 1:1, 3:2, 7:3, 4:1, 0:1) and obtained five subfractions (Fr. 9.3.1–Fr. 9.3.5). Then, Fr. 9.3.2 were purified by semi-preparative reversed-phase HPLC using the isocratic elution of MeOH-H₂O (20:80, v/v, 2 mL/min, UV λ_max 210 nm) to afford compound 12 (5 mg, t_R = 62 min), compound 3 (5 mg, t_R = 37 min), compound 2 (5 mg, t_R = 25 min), and compound 4 (8 mg, t_R = 40 min).

Fr. 9.3.3 were purified by semi-preparative reversed-phase HPLC using the isocratic elution of MeOH-H₂O (20:80, v/v, 2 mL/min, UV λ_max 210 nm) to afford compound 5 (5 mg, t_R = 46 min) and compound 14 (5 mg, t_R = 28 min).

Phomopyrazine (1): colorless amorphous residue (MeOH); [α]²⁰_D 0 (c 0.0001, MeOH); UV (MeOH) λ_max 209, 278, and 304 nm (the absorptions due to aromatic rings); HRESIMS m/z 275.1002 [M + Na]⁺ (calcd for C₁₂H₁₆N₂O₄ Na 275.1002), m/z 251.1037 [M − H]⁻ (calcd for C₁₂H₁₅N₂O₄ 251.1037).

Phomosterol C (7): colorless amorphous residue (MeOH); [α]²⁰_D + 10 (c 0.0001, MeOH); UV (MeOH) λ_max 202, 259, and 263 nm (the absorptions due to aromatic rings); HRESIMS m/z 441.3363 [M + H]⁺ (calcd for C₂₉H₄₅O₃ 441.3362).

Phomopyrone E (9): colorless amorphous residue (MeOH); [α]²⁰_D + 60 (c 0.0001, MeOH); UV (MeOH) λ_max 213, 299, 303 nm (the absorptions due to aromatic rings); HRESIMS m/z 235.0941 [M + Na]⁺ (calcd for C₁₁H₁₆O₄ Na 235.0941).

3.7. Theory and Calculation Details

Detailed Monte Carlo conformational analyses were performed utilizing Spartan’s 14 software (v1.1.4) using the Merck molecular force field (MMFF). The conformers exceeding a Boltzmann population of 0.4% were selected for electronic circular dichroism (ECD) calculations as presented in Tables S1–S4. Subsequently, these conformers underwent initial optimization at the B3LYP/6-31G(d) level in the gas phase, complemented by the polarizable conductor calculation model based on the polarizable continuum model (PCM). The stable conformations identified at the B3LYP/6-31G(d) level were then used in magnetic shielding constants. The theoretical calculation of ECD was conducted in MeOH using the time-dependent density functional theory (TD-DFT) at the B3LYP/6-31+g (d,p) level for all the conformers of compounds 7 and 9. The ECD spectra were generated with the aid of the SpecDis 1.6 program (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California, San Diego, CA, USA) through the conversion of dipole-length rotational strengths into band shapes modeled by Gaussian functions with a standard deviation of 0.3 eV.

3.8. Cytotoxicity Assay

The liver cancer cell line, HepG2, and the cervical cancer cell line, Hela, were obtained from the Type Culture Collection of the Chinese Academy of Sciences in Shanghai, China. The cells were cultivated using an RPMI-1640 culture medium. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to evaluate cytotoxicity against the HepG2 and HeLa cells, sourced from Sigma-Aldrich, St. Louis, MO, USA, and it was employed as described previously [30]. Additionally, adriamycin (from Shanghai Macklin Biochemical Co., Ltd., with a purity of 99.8%) (Shanghai, China) and 5-fluorouracil (5-FU) (from Beijing Solarbio Science and Technology Co., Ltd., with a purity of 99.8%) (Beijing, China) served as the positive controls.

3.9. Splenocyte Proliferation Assay

Spleen cells were collected from BALB/c mice under aseptic conditions, plated in a 96-well plate at a concentration of 1 × 10⁷ cells/mL per well, and activated by Con A (5 μg/mL) or LPS (10 μg/mL) in the presence of various concentrations of compounds or cyclosporine A (CsA) at 37 °C and 5% CO₂ for 48 h. Then, 20 μL CCK-8 was added to each well 4 h before the end of the incubation. The absorbance at OD₄₅₀ was measured on ThermoFisher Scientific Multiskan™ FC Microplate Photometer (ThermoScientific, Waltham, MA, USA), and the IC₅₀ value was calculated from the correlation curve between the compound concentration and the OD₄₅₀.

3.10. Acetylcholinesterase Inhibitory Activity Studies

In total, 20 μL (1.2 mM) of acetylthiocholine (ATCH, from Shanghai Macklin Biochemical Co., Ltd., with a purity of 99.8%) (Shanghai, China) as the enzyme reaction substrate was added to a 96-well plate, then 20 μL of the tested compounds at different concentrations (10 μM–200 μM) and 20 μL (0.025 U/mL) of acetylcholinesterase solution (from Shanghai Macklin Biochemical Co., Ltd., with a purity of 99.8%) (Shanghai, China), and finally 100 μL of PBS phosphate buffer. After 30 min of incubation at 37 °C, 20 μL of 4% sodium dodecyl sulfate (SDS, from Shanghai Macklin Biochemical Co., Ltd., with a purity of 99.8%) (Shanghai, China) was added to terminate the reaction, and finally, 20 μL of 0.6 mM DTNB colorimetric solution was added (from Shanghai Macklin Biochemical Co., Ltd., with a purity of 99.8%) (Shanghai, China), and using a microplate reader, the intensity of the developed color was measured at 450 nm.

3.11. Statistical Analysis

All the cell data are presented as the mean and standard deviation of the means (S.D.), and a one-way analysis of variance (ANOVA) was used to evaluate the statistical significance of the differences between the groups by GraphPad Prism 10.2.0.

4. Conclusions

In summary, the co-cultivation of Phomopsis asparagi DHS-48 and another Phomopsis genus fungus DHS-11 endophytes within the same mangrove host plant Rhizophora mangle demonstrated to be effective to stimulate biosynthetic gene clusters with the potential to produce bioactive compounds that remain dormant under the axenic monocultures, leading to the production of an array of alkaloids, sterols, and polyketides, with some having cytotoxic, immunosuppressive, and AChE inhibitory properties.