Next Article in Journal
Jellyfish Venom Peptides Targeting Human Potassium Channels Identified through Ligand Screening: Morphometric and Molecular Identification of the Species and Antibiotic Potential
Previous Article in Journal
Macrolactin XY, a Macrolactin Antibiotic from Marine-Derived Bacillus subtilis sp. 18
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Marine Drugs
Volume 22
Issue 8
10.3390/md22080332
marinedrugs-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Communication

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

by
Jingwan Wu
1,
Jingjing Ye
1,
Juren Cen
2,
Yuanjie Chen
2 and
Jing Xu
1,2,*
1
Collaborative Innovation Center of Ecological Civilization, School of Chemistry and Chemical Engineering, Hainan University, Haikou 570228, China
2
School of Life and Health Sciences, Hainan University, Haikou 570228, China
*
Author to whom correspondence should be addressed.
Mar. Drugs 2024, 22(8), 332; https://doi.org/10.3390/md22080332
Submission received: 13 June 2024 / Revised: 3 July 2024 / Accepted: 22 July 2024 / Published: 24 July 2024
(This article belongs to the Section Marine Pharmacology)
Browse Figures
Versions Notes

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.

Graphical Abstract

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) of 1 in combination with distortionless enhancement by polarization transfer (DEPT) and heteronuclear single quantum coherence (HSQC) spectrum revealed the existences of a methyl [δH 1.28, (t, J = 7.5 Hz), δC 13.2, q, 14-CH3], five methylenes [δH 3.02 (t, J = 7.5 Hz), δC 31.7, t, CH2-7; δH 2.64 (t, J = 7.5 Hz), δC 36.2, t, CH2-8; δH 3.08 (t, J = 7.5 Hz), δC 30.4, t, CH2-10; δH 2.66 (t, J = 7.5 Hz), δC 35.3, t, CH2-11; δH 2.87 (q, J = 7.5 Hz), δC 28.2, t, CH2-13], an olefinic methine at δH (8.23, 1H, s, CH-5), and five quaternary carbons [including two carboxylic carbonyl at δC 179.0 (C-9) and δC 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) showed the presence of a series of characteristic signals for three methyl singlets (δH 0.68, s, 18-CH3; 0.99, s, 19-CH3; 1.53, s, 29-CH3), four methyl doublets [δH 0.98 (d, J = 6.8 Hz), 21-CH3; δH 0.80 (d, J = 6.6 Hz), 26-CH3; δH 0.87 (d, J = 6.6 Hz), 27-CH3; 0.98 (d, J = 6.8 Hz), 28-CH3], an oxymethine (δH 3.92, m, CH-3), and two olefinic proton [δH 5.58 (d, J = 2.0 Hz), CH-7; δH 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 (δH 1.53, s, δC 13.4, q, 29-CH3) located at C-23 (δC 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 (δC 132.5, d), C-23, and C-24 (δC 51.8, d) (Figure 2). The relative stereochemistry of 7 was determined by the analysis of the NOESY spectrum (Figure 4). Diagnostic correlations positioned Hb-1, H3-19, H-7, H-14 and H3-18, and H-20 on the α-face and Ha-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 (δH 3.94, s, δC 57.3, q, OCH3-11), two methyl groups (δH 1.84, s, δC 8.4, q, CH3-10; δH 1.26 (d, J = 7.0 Hz), δC 18.8, q, 12-CH3), two methylene groups (δH 1.90, m, H-8a, 1.73, m, H-8b, δC 36.6, t, CH2-8; δH 3.55, m, δC 60.3, t, CH2-9, oxygenated), a methine group (δH 2.86, m, δC 36.6, d, CH-7), and an olefinic methine (δH 6.43 brs, δC 95.8, d, CH-5, aromatic). The typical 13C NMR data at δC 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 (δC 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 [ α ] D 20 + 60 (c 0.0001, MeOH) of 9 reported in this work does not correspond with those reported for phomopyronol [ α ] 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). 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). Nonetheless, only compound 11 showed weak inhibition of AChE with an IC50 value of 86.11 ± 1.56 μM (Table 5).

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 λmax 210 nm) to afford compound 9 (6 mg, tR = 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 λmax 210 nm) to afford compound 10 (5 mg, tR = 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 λmax 210 nm) to afford compound 13 (5 mg, tR = 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 λmax 210 nm) to afford compound 1 (6 mg, tR = 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 λmax 210 nm) to afford compound 11 (6 mg, tR = 32 min) and compound 6 (6 mg, tR = 39 min). Fr. 8.5 was purified by HPLC using the isocratic elution of (MeOH/H2O, 70:30, v/v; 2 mL/min, UV λmax 210 nm) to yield compound 8 (6 mg, tR = 14 min) and compound 7 (6 mg, tR = 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 λmax 210 nm) to afford compound 12 (5 mg, tR = 62 min), compound 3 (5 mg, tR = 37 min), compound 2 (5 mg, tR = 25 min), and compound 4 (8 mg, tR = 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 λmax 210 nm) to afford compound 5 (5 mg, tR = 46 min) and compound 14 (5 mg, tR = 28 min).
Phomopyrazine (1): colorless amorphous residue (MeOH); [α]20D 0 (c 0.0001, MeOH); UV (MeOH) λmax 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]20D + 10 (c 0.0001, MeOH); UV (MeOH) λmax 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]20D + 60 (c 0.0001, MeOH); UV (MeOH) λmax 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.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/md22080332/s1, Figure S1: Chemical profiles of EtOAc extracts deriving from the whole co-culture of DHS-48 and DHS-11 and the monocultures of DHS-48 and DHS-11. Figure S2: 1H-NMR of (1). Figure S3: 13C-NMR of (1). Figure S4: DEPT of (1). Figure S5: 1H-1H COSY of (1). Figure S6: HSQC of (1). Figure S7: HMBC of (1). Figure S8: NOSEY of (1). Figure S9: HR-ESI-MS of (1). Figure S10: 1H-NMR of (2). Figure S11: 13C-NMR of (2). Figure S12: HR-ESI-MS of (2). Figure S13: 1H-NMR of (3). Figure S14: 13C-NMR of (3). Figure S15: HR-ESI-MS of (3). Figure S16: 1H-NMR of (4). Figure S17: 13C-NMR of (4). Figure S18: HR-ESI-MS of (4). Figure S19: 1H-NMR of (5). Figure S20: 13C-NMR of (5). Figure S21: HR-ESI-MS of (5). Figure S22: 1H-NMR of (6). Figure S23: 13C-NMR of (6). Figure S24: HR-ESI-MS of (6). Figure S25: 1H-NMR of (7). Figure S26: 13C-NMR of (7). Figure S27: DEPT of (7). Figure S28: 1H-1H COSY of (7). Figure S29: HSQC of (7). Figure S30: HMBC of (7). Figure S31: NOSEY of (7). Figure S32: HR-ESI-MS of (7). Figure S33: 1H-NMR of (8). Figure S34: 13C-NMR of (8). Figure S35: HR-ESI-MS of (8). Figure S36: 1H-NMR of (9). Figure S37: 13C-NMR of (9). Figure S38: DEPT of (9). Figure S39: 1H-1H COSY of (9). Figure S40: HSQC of (9). Figure S41: HMBC of (9). Figure S42: NOSEY of (9). Figure S43: HR-ESI-MS of (9). Figure S44: 1H-NMR of (10). Figure S45: 13C-NMR of (10). Figure S46: HR-ESI-MS of (10). Figure S47: 1H-NMR of (11). Figure S48: 13C-NMR of (11). Figure S49: HR-ESI-MS of (11). Figure S50: 1H-NMR of (12). Figure S51: 13C-NMR of (12). Figure S52: HR-ESI-MS of (12). Figure S53: 1H-NMR of (13). Figure S54: 13C-NMR of (13). Figure S55: HR-ESI-MS of (13). Figure S56: 1H-NMR of (14). Figure S57: 13C-NMR of (14). Figure S58: HR-ESI-MS of (14). Table S1: Gibbs free energiesa and equilibrium populationsb of low-energy conformers of phomosterol C (7). Table S2: Cartesian coordinates for the low-energy reoptimized MMFF conformers of phomosterol C (7) at B3LYP/6-31G(d,p) level of theory in gas. Table S3: Gibbs free energiesa and equilibrium populationsb of low-energy conformers of phomopyrone E (9). Table S4: Cartesian coordinates for the low-energy reoptimized MMFF conformers of phomopyrone E (9) at B3LYP/6-31G(d,p) level of theory in gas.

Author Contributions

J.X. designed and supervised this research, structured the elucidation, and wrote the draft and final revision of the manuscript. J.W. performed the isolation. J.Y. assisted in the phylogenetic analysis. J.C. and Y.C. carried out the biological evaluation. The final revision of the manuscript was revised by all the authors. All authors have read and agreed to the published version of the manuscript.

Funding

This work was co-financed by the grants of the National Natural Science Foundation of China (No. 82160675/81973229), the Key Research Program of Hainan Province (ZDYF2021SHFZ108), the Collaborative Innovation Center Foundation of Hainan University (XTCX2022STB01), and the Guangdong Key Laboratory of Marine Materia Medica Open Fund (LMM2021-4). They are gratefully acknowledged.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original data presented in the study are included in the article/Supplementary Materials; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Lin, W.; Li, G.; Xu, J. Bio-Active Products from Mangrove Ecosystems. Mar. Drugs 2023, 21, 239. [Google Scholar] [CrossRef] [PubMed]
  2. Xu, J. Biomolecules Produced by Mangrove-Associated Microbes. Curr. Med. Chem. 2011, 18, 5224–5266. [Google Scholar] [CrossRef] [PubMed]
  3. Xu, J. Bioactive Natural Products Derived from Mangrove-Associated Microbes. RSC Adv. 2015, 5, 841–892. [Google Scholar] [CrossRef]
  4. Wu, J.; Xiao, Q.; Xu, J.; Li, M.-Y.; Pan, J.-Y.; Yang, M. Natural Products from True Mangrove Flora: Source, Chemistry and Bioactivities. Nat. Prod. Rep. 2008, 25, 955. [Google Scholar] [CrossRef] [PubMed]
  5. Thatoi, H.; Behera, B.C.; Mishra, R.R.; Dutta, S.K. Biodiversity and Biotechnological Potential of Microorganisms from Mangrove Ecosystems: A Review. Ann. Microbiol. 2013, 63, 1–19. [Google Scholar] [CrossRef]
  6. Chen, S.; Cai, R.; Liu, Z.; Cui, H.; She, Z. Secondary Metabolites from Mangrove-Associated Fungi: Source, Chemistry and Bioactivities. Nat. Prod. Rep. 2022, 39, 560–595. [Google Scholar] [CrossRef] [PubMed]
  7. Ancheeva, E.; Daletos, G.; Proksch, P. Lead Compounds from Mangrove-Associated Microorganisms. Mar. Drugs 2018, 16, 319. [Google Scholar] [CrossRef]
  8. Yin, S.; Fan, C.-Q.; Wang, X.-N.; Lin, L.-P.; Ding, J.; Yue, J.-M. Xylogranatins A−D: Novel Tetranortriterpenoids with an Unusual 9,10- s Eco Scaffold from Marine Mangrove Xylocarpus g Ranatum. Org. Lett. 2006, 8, 4935–4938. [Google Scholar] [CrossRef]
  9. Gong, K.-K.; Li, P.-L.; Qiao, D.; Zhang, X.-W.; Chu, M.-J.; Qin, G.-F.; Tang, X.-L.; Li, G.-Q. Cytotoxic and Antiviral Triterpenoids from the Mangrove Plant Sonneratia Paracaseolaris. Molecules 2017, 22, 1319. [Google Scholar] [CrossRef]
  10. Zhang, Q.; Satyanandamurty, T.; Shen, L.; Wu, J. Krishnolides A–D: New 2-Ketokhayanolides from the Krishna Mangrove, Xylocarpus Moluccensis. Mar. Drugs 2017, 15, 333. [Google Scholar] [CrossRef]
  11. Tiwari, P.; Bae, H. Endophytic Fungi: Key Insights, Emerging Prospects, and Challenges in Natural Product Drug Discovery. Microorganisms 2022, 10, 360. [Google Scholar] [CrossRef] [PubMed]
  12. Rutledge, P.J.; Challis, G.L. Discovery of Microbial Natural Products by Activation of Silent Biosynthetic Gene Clusters. Nat. Rev. Microbiol. 2015, 13, 509–523. [Google Scholar] [CrossRef] [PubMed]
  13. Knowles, S.L.; Raja, H.A.; Roberts, C.D.; Oberlies, N.H. Fungal–Fungal Co-Culture: A Primer for Generating Chemical Diversity. Nat. Prod. Rep. 2022, 39, 1557–1573. [Google Scholar] [CrossRef] [PubMed]
  14. Wu, J.; Chen, D.; Li, Q.; Feng, T.; Xu, J. Metabolomics-Guided Discovery of New Dimeric Xanthones from Co-Cultures of Mangrove Endophytic Fungi Phomopsis asparagi DHS-48 and Phomopsis Sp. DHS-11. Mar. Drugs 2024, 22, 102. [Google Scholar] [CrossRef] [PubMed]
  15. Huang, Z.; Yang, R.; Guo, Z.; She, Z.; Lin, Y. A New Naphtho-γ-Pyrone from Mangrove Endophytic Fungus ZSU-H26. Chem. Nat. Compd. 2010, 46, 15–18. [Google Scholar] [CrossRef]
  16. Ouyang, M.-A. A New Adenosyl-Alkaloid from Ostrea rivularis. Nat. Prod. Res. 2006, 20, 79–83. [Google Scholar] [CrossRef] [PubMed]
  17. Ito, Y.; Kimura, A.; Osawa, T.; Hari, Y. Photoredox-Catalyzed Deformylative 1,4-Addition of 2′-Deoxy-5′- O -Phthalimidonucleosides for Synthesis of 5′-Carba Analogs of Nucleoside 5′-Phosphates. J. Org. Chem. 2018, 83, 10701–10708. [Google Scholar] [CrossRef] [PubMed]
  18. Duan, J.-L.; Li, X.-J.; Gao, J.-M.; Wang, D.-S.; Yan, Y.; Xue, Q.-H. Isolation and Identification of Endophytic Bacteria from Root Tissues of Salvia Miltiorrhiza Bge. and Determination of Their Bioactivities. Ann. Microbiol. 2013, 63, 1501–1512. [Google Scholar] [CrossRef]
  19. Venkateswarlu, V.; Aravinda Kumar, K.A.; Gupta, S.; Singh, D.; Vishwakarma, R.A.; Sawant, S.D. DMSO/I2 Mediated C–C Bond Cleavage of α-Ketoaldehydes Followed by C–O Bond Formation: A Metal-Free Approach for One-Pot Esterification. Org. Biomol. Chem. 2015, 13, 7973–7978. [Google Scholar] [CrossRef] [PubMed]
  20. Yu, F.-X.; Li, Z.; Chen, Y.; Yang, Y.-H.; Li, G.-H.; Zhao, P.-J. Four New Steroids from the Endophytic Fungus Chaetomium Sp. M453 Derived of Chinese Herbal Medicine Huperzia Serrata. Fitoterapia 2017, 117, 41–46. [Google Scholar] [CrossRef]
  21. Gao, L.; Xu, X.; Yang, J. Chemical Constituents of the Roots of Rheum Officinale. Chem. Nat. Compd. 2013, 49, 603–605. [Google Scholar] [CrossRef]
  22. Li, W.-S.; Hu, H.-B.; Huang, Z.-H.; Yan, R.-J.; Tian, L.-W.; Wu, J. Phomopsols A and B from the Mangrove Endophytic Fungus Phomopsis Sp. Xy21: Structures, Neuroprotective Effects, and Biogenetic Relationships. Org. Lett. 2019, 21, 7919–7922. [Google Scholar] [CrossRef] [PubMed]
  23. Watanadilok, R.; Sonchaeng, P.; Kijjoa, A.; Damas, A.M.; Gales, L.; Silva, A.M.S.; Herz, W. Tetillapyrone and Nortetillapyrone, Two Unusual Hydroxypyran-2-Ones from the Marine Sponge Tetilla japonica. J. Nat. Prod. 2001, 64, 1056–1058. [Google Scholar] [CrossRef] [PubMed]
  24. Ying, Y.-M.; Zhang, L.-W.; Shan, W.-G.; Zhan, Z.-J. Secondary Metabolites of Peyronellaea Sp. XW-12, an Endophytic Fungus of Huperzia Serrata. Chem. Nat. Compd. 2014, 50, 723–725. [Google Scholar] [CrossRef]
  25. Li, J.-T.; Yin, B.-L.; Liu, Y.; Wang, L.-Q.; Chen, Y.-G. Mono-Aromatic Constituents of Dendrobium longicornu. Chem. Nat. Compd. 2009, 45, 234–236. [Google Scholar] [CrossRef]
  26. Weber, D.; Gorzalczany, S.; Martino, V.; Acevedo, C.; Sterner, O.; Anke, T. Metabolites from Endophytes of the Medicinal Plant Erythrina crista-galli. Z. Für Naturforschung C 2005, 60, 467–477. [Google Scholar] [CrossRef] [PubMed]
  27. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [Google Scholar] [CrossRef] [PubMed]
  28. Kimura, M. A Simple Method for Estimating Evolutionary Rates of Base Substitutions through Comparative Studies of Nucleotide Sequences. J. Mol. Evol. 1980, 16, 111–120. [Google Scholar] [CrossRef] [PubMed]
  29. Letunic, I.; Bork, P. Interactive Tree of Life (iTOL) v4: Recent Updates and New Developments. Nucleic Acids Res. 2019, 47, W256–W259. [Google Scholar] [CrossRef]
  30. Feng, T.; Wei, C.; Deng, X.; Chen, D.; Wen, Z.; Xu, J. Epigenetic Manipulation Induced Production of Immunosuppressive Chromones and Cytochalasins from the Mangrove Endophytic Fungus Phomopsis asparagi DHS-48. Mar. Drugs 2022, 20, 616. [Google Scholar] [CrossRef]
Marinedrugs 22 00332 g001
Figure 1. Structures of the isolated compounds 1–14.
Figure 1. Structures of the isolated compounds 1–14.
Marinedrugs 22 00332 g001
Marinedrugs 22 00332 g002
Figure 2. Unrooted neighbor-joining phylogenetic tree based on the ITS gene sequences showing the taxonomic positions of DHS-48, DHS-11, and type strains of closely related Phomopsis taxa. The values at each node represent the bootstrap values from 1000 replicates, and the scale bar represents 0.05 substitutions per nucleotide. Bacillus toyonensis BCT-7112T served as an outgroup.
Figure 2. Unrooted neighbor-joining phylogenetic tree based on the ITS gene sequences showing the taxonomic positions of DHS-48, DHS-11, and type strains of closely related Phomopsis taxa. The values at each node represent the bootstrap values from 1000 replicates, and the scale bar represents 0.05 substitutions per nucleotide. Bacillus toyonensis BCT-7112T served as an outgroup.
Marinedrugs 22 00332 g002
Marinedrugs 22 00332 g003
Figure 3. Key COSY and HMBC correlations of compounds 1, 7, and 9.
Figure 3. Key COSY and HMBC correlations of compounds 1, 7, and 9.
Marinedrugs 22 00332 g003
Marinedrugs 22 00332 g004
Figure 4. Key NOESY correlations of compounds 7 and 9.
Figure 4. Key NOESY correlations of compounds 7 and 9.
Marinedrugs 22 00332 g004
Marinedrugs 22 00332 g005
Figure 5. Experimental and calculated electronic circular dichroism (ECD) spectra of 7 and 9.
Figure 5. Experimental and calculated electronic circular dichroism (ECD) spectra of 7 and 9.
Marinedrugs 22 00332 g005
Table 1. 1H (400 MHz) and 13C (100 MHz) NMR data of 1 and 9 in CD3OD.
Table 1. 1H (400 MHz) and 13C (100 MHz) NMR data of 1 and 9 in CD3OD.
Position1 9
δC TypeδH Mult (J in Hz)HMBC (H to C)δC TypeδH Mult (J in Hz)HMBC (H to C)
1
2152.6, C C-5, 10, 11, 13168.3, C
3157.0, C C-5, 10, 13, 14 101.2, C
4 169.1, C
5141.5, CH8.23, sC-2, 3, 7, 10, 1395.8, CH6.43, sC-3, 4, 6, 7
6154.5, C C-5, 7, 8169.2, C
731.7, CH23.02, t, 7.5C-5, 6, 8, 936.6, CH2.86, mC-6, 8, 9, 12
836.2, CH22.64, t, 7.5C-6, 7, 938.2, CH21.90, m
1.73, m		C-6, 7, 9, 12
9179.0, C C-7, 8, 10, 1160.3, CH23.53, mC-7, 8
1030.4, CH23.08, t, 7.5C-2, 3, 11, 128.4, CH31.84, sC-2, 3, 4, 5
1135.3, CH22.66, t, 7.5C-2, 10, 1257.3, CH33.94, sC-4
12179.1, C 18.8, CH31.26, d, 7.0C-6, 7, 8
1328.2, CH22.87, q, 7.5C-2, 3, 14
1413.2, CH31.28, t, 7.5C-3, 13
Table 2. 1H (400 MHz) and 13C (100 MHz) NMR data of 7 and 8 in CD3OD.
Table 2. 1H (400 MHz) and 13C (100 MHz) NMR data of 7 and 8 in CD3OD.
Position78
δC TypeδH Mult (J in Hz)HMBCδC TypeδH Mult (J in Hz)
126.6, CH2Ha 2.29, dt, 14.5, 4.5C-2, 3, 5, 9, 10, 1926.6, CH2Ha 2.20, dt, 1.5, 13.4
Hb 1.50, m Hb 1.39, m
231.0, CH2Ha 1.87, mC-1, 3, 4, 1031.0, CH2Ha 1.78, m
Hb 1.46, m Hb 1.36, m
367.8, CH3.92, mC-1, 2, 4, 567.8, CH3.83, m
437.2, CH2Ha 2.02, m 37.1, CH2Ha 1.92, m
Hb 1.63, mC-2, 3, 5, 10Hb 1.52, m
580.2, C C-1, 3, 4, 6, 7, 1980.2, C
6200.2, C 200.1, C
7120.9, CH5.58, d, 2.0C-5, 6, 8, 9, 14120.9, CH5,49, s
8165.1, C 165.0, C
976.2, C 76.1, C
1042.8, C 42.8, C
1129.3, CH2Ha 1.97, m
Hb 1.76, m		C-8, 9, 10, 12, 1329.1, CH2Ha 1.70, m
Hb 1.64, m
1236.2, CH2Ha 1.89, m
Hb 1.72, m		C-9, 11, 13, 14, 1836.2, CH2Ha 1.80, m
Hb 1.62, m
1346.2, C 46.2, C
1452.8, CH2.75, ddd, 11.6, 9.8, 2.6C-7, 8, 9, 12, 13, 15, 1852.8, CH2.66, dd, 11.5, 7.6
1523.4, CH2Ha 1.60, m
Hb 1.52, m		C-19, 25, 2423.4, CH2Ha 1.52, m
Hb 1.42, m
1628.5, CH2Ha 1.87, m
Hb 1.30, m		C-13, 14, 15, 17, 2029.3, CH2Ha 1.89, m
Hb 1.36, m
1758.2, CH1.46, mC-13, 14, 15, 16, 20, 21, 2257.4, CH1.34, m
1812.7, CH30.68, sC-12, 13, 14, 1720.6, CH30.90, s
1920.6, CH30.99, sC-1, 5, 9, 1012.6, CH30.57, s
2035.9, CH2.41, mC-13, 16, 17, 21, 22, 2341.7, CH1.97, m
2121.2, CH30.98, d, 6.8C-17, 20, 2221.6, CH30.96, d, 6.6
22132.5, CH4.94, d, 9.6C-17, 20, 21, 23, 24, 29133.6, CH5.13, dd, 15.2, 8.2
23137.3, C C-20, 22, 24, 25, 28136.7, CH5.19, dd, 15.2, 7.6
2451.8, CH1.68, mC-22, 23, 25, 26, 27, 28, 2944.4, CH1.76, m
2532.0, CH1.55, mC-23, 24, 26, 27, 2834.4, CH1.39, m
2622.2, CH30.8, d, 6.6C-24, 25, 2720.5, CH30.77, d, 6.8
2720.6, CH30.87, d, 6.6C-24, 25, 2620.1, CH30.75, d, 6.8
2817.5, CH30.97, d, 6.8C-23, 24, 2518.2, CH30.85, d, 6.8
2913.4, CH31.53, sC-22, 23, 24
Table 3. Cytotoxicity of compounds 1–14.
Table 3. Cytotoxicity of compounds 1–14.
CompoundIC50 (µM) a
HepG2Hela
765.97 ± 2.5672.34 ± 2.03
877.41 ± 4.1272.02 ± 2.89
1473.37 ± 2.2587.30 ± 0.74
1–6, 9–13--
Adriamycin b\0.88 ± 0.71
Fluorouracil c179.03 ± 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.
Table 4. Immunosuppressive activity of compounds 1–14.
Table 4. Immunosuppressive activity of compounds 1–14.
CompoundIC50 (µM) a
ConA-Induced T-Cell ProliferationLPS-Induced B-Cell Proliferation
1125.1 ± 1.12133.87 ± 3.43
2–7--
835.75 ± 1.0947.65 ± 1.21
9108.21 ± 1.32112.76 ± 2.11
10111.01 ± 1.02123.84 ± 1.25
11–14--
cyclosporin A b4.39 ± 0.0225.11 ± 0.43
a data are presented as mean ± SD from three separate experiments. b positive control. ‘-’ stands for no inhibitory effect at 200 µM.
Table 5. Acetylcholinesterase inhibitory activity of compounds 1–14.
Table 5. Acetylcholinesterase inhibitory activity of compounds 1–14.
CompoundIC50 (µM) a
1186.11 ± 1.56
1–10, 12–14-
Donepezil b0.25 ± 0.76
a data are presented as mean ± SD from three separate experiments. b positive control. ‘-’ stands for no inhibitory effect at 200 µM.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Wu, J.; Ye, J.; Cen, J.; Chen, Y.; Xu, J. 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. Mar. Drugs 2024, 22, 332. https://doi.org/10.3390/md22080332

AMA Style

Wu J, Ye J, Cen J, Chen Y, Xu J. 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. Marine Drugs. 2024; 22(8):332. https://doi.org/10.3390/md22080332

Chicago/Turabian Style

Wu, Jingwan, Jingjing Ye, Juren Cen, Yuanjie Chen, and Jing Xu. 2024. "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" Marine Drugs 22, no. 8: 332. https://doi.org/10.3390/md22080332

APA Style

Wu, J., Ye, J., Cen, J., Chen, Y., & Xu, J. (2024). 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. Marine Drugs, 22(8), 332. https://doi.org/10.3390/md22080332

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop