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Front Microbiol
2023 Jan 01;14:1144328. doi: 10.3389/fmicb.2023.1144328.
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The screening for marine fungal strains with high potential in alkaloids production by in situ colony assay and LC-MS/MS based secondary metabolic profiling.
Lu T
,
Liu Y
,
Zhou L
,
Liao Q
,
Nie Y
,
Wang X
,
Lei X
,
Hong P
,
Feng Y
,
Hu X
,
Zhang Y
.
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BACKGROUND: Alkaloids are the second primary class of secondary metabolites (SMs) from marine organisms, most of which have antioxidant, antitumor, antibacterial, anti-inflammatory, and other activities. However, the SMs obtained by traditional isolation strategies have drawbacks such as highly reduplication and weak bioactivity. Therefore, it is significantly important to establish an efficient strategy for screening strains and mining novel compounds.
METHODS: In this study, we utilized in situ colony assay combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the strain with high potential in alkaloids production. The strain was identified by genetic marker genes and morphological analysis. The secondary metabolites from the strain were isolated by the combine use of vacuum liquid chromatography (VLC), ODS column chromatography, and Sephadex LH-20. Their structures were elucidated by 1D/2D NMR, HR-ESI-MS, and other spectroscopic technologies. Finally, these compounds bioactivity were assay, including anti-inflammatory and anti-β aggregation.
RESULTS: Eighteen marine fungi were preliminarily screened for alkaloids production by in situ colony assay using Dragendorff reagent as dye, and nine of them turned orange, which indicated abundant alkaloids. By thin-layer chromatography (TLC), LC-MS/MS, and multiple approaches assisted Feature-Based Molecular Networking (FBMN) analysis of fermentation extracts, a strain ACD-5 (Penicillium mallochii with GenBank accession number OM368350) from sea cucumber gut was selected for its diverse alkaloids profiles especially azaphilones. In bioassays, the crude extracts of ACD-5 in Czapek-dox broth and brown rice medium showed moderate antioxidant, acetylcholinesterase inhibitory, anti-neuroinflammatory, and anti-β aggregation activities. Three chlorinated azaphilone alkaloids, compounds 1-3 (sclerotioramine, isochromophilone VI, and isochromophilone IX, respectively), were isolated from the fermentation products of ACD-5 in brown rice medium guided by bioactivities and mass spectrometry analysis. Compound 1 had shown remarkable anti-neuroinflammatory activity in liposaccharide induced BV-2 cells.
CONCLUSION: In summary, in situ colony screening together with LC-MS/MS, multi-approach assisted FBMN can act as an efficient screening method for strains with potential in alkaloids production.
FIGURE 1. The strains showed alkaloid positive reaction in the in situ colony screening by Dragendorff reagent. (A1–A6) Strains from seaweeds; (B1–B3): Strains from sea cucumber. (A1, S5-2-1; A2, S5-2-2; A3, S5-3; A4, S5-5-1; A5, S6-3 Y; A6, S7-3; B1, CD-1; B2, ACD-2; B3, ACD-5).
FIGURE 2. The TLC analysis of the positive strains. (A1–I1) UV pattern at 254 nm; (A2–I2) fluorescence pattern at 365 nm; (A3–I3) the results of Dragendorff reagent staining. The order of spotting on each plate from left to right is PSB, MEB, BRM, and CDB (representing different cultural medium used). BL-I was as a negative result and LJY-6 was as a positive result in TLC analysis.
FIGURE 3. The metabolic profile of the features in extracts ACD-5-CDB (A) and ACD-5-BRM (B) showing their retention times, precursor ion m/z values, and intensities (exported from MSDIAL).
FIGURE 4. The statics of the numbers of the total features (A), the annotated and unknown features (B), and the alkaloids (C) detected in the two crude extracts of strain ACD-5.
FIGURE 5. The FBMN molecular network based on positive ion MS/MS spectral similarity, showing a selection of amplified clusters. In the FBMN network, each node represents a feature marked with the mean m/z value of the parent ion, and the similarity of the secondary mass spectra between compounds was expressed by the cosine value, which was proportional to the similarity. The different colors of sections in the nodes represent different samples, i.e.,
: respectively, ACD-5-BRM and ACD-5-CDB. The thickness of the connecting lines between nodes was positively correlated with the cosine value. The node size reflects the feature abundance (ion intensity).
FIGURE 6. The DPPH scavenging and AChE inhibitory assays of the two crude extracts of strain ACD-5. (A) DPPH scavenging rate of ACD-5-BRM and ACD-5-CDB. Vitamin C was taken as a positive control. (B) The TLC-bioautography of the two extracts: (1) UV, (2) fluorescence, (3) Dragendorff reagent staining, (4) DPPH staining. (C) AChE inhibitory effect of ACD-5-BRM and ACD-5-CDB. The positive control was donepezil. (D) The TLC-bioautography of the two extracts: (1) UV, (2) fluorescence, (3) Dragendorff reagent staining, (5) AChE bioautography. The TLC developing system was dichloromethane: methanol = 5:1 (v/v). The results were expressed as the means ± SD (n = 3), ****: p < 0.0001 compared with positive control of the same concentration. A value of p < 0.05 was considered statistically significant.
FIGURE 7. The anti-neuroinflammatory, cytotoxic, and anti-Aβ aggregation effects of the extracts ACD-5-BRM and ACD-5-CDB. (A,B) Inhibition to liposaccharide induced nitric oxide (NO) production of BV-2 cells of ACD-5-BRM (A) and ACD-5-CDB (B), respectively. The results were expressed as the mean ± SD (n = 3). **p < 0.01, ****p < 0.0001, compared with the LPS group. A value of p < 0.05 was considered statistically significant. (C) The effect of ACD-5-BRM on the viability of BV-2 cells. Since the quantity of ACD-5-CDB was insufficient, no cytotoxicity test was conducted. (D) The Anti-Aβ aggregation effect of ACD-5-CDB and ACD-5-BRM in gold nanoparticle-mediated anti-Aggregation test. Albumin was taken as a positive control.
FIGURE 8. The colonial and microscopic morphology of ACD-5. (A) The 7-day old cultures of ACD-5 on CYA, CDA, MEA, YES, and PDA, showing variation in colony characters. The front view of colony on the left and the back view of colony on the right. (B) The microscopic morphology of strain ACD-5. 1–3 were morphologies observed under light microscope at 100 x magnification, scale bar = 2 μm; 4–6 were the morphologies observed under scanning electron microscope, the scale bar of 4 and 5 was 20 μm while the 6 was 5 μm. Co, Conidium; Cp, Conidiophore; Sh, Septa hypha.
FIGURE 9. Neighbor-jointing trees based on β-tubulin (A) and ITS rDNA (B) of ACD-5 and adjacent areas of the dominant fungal groups (Bootstrap values were calculated using 1,000 replications).
FIGURE 10. The anti-Aβ aggregation, anti-neuroinflammatory and cytotoxic effects of the primary fractions of ACD-5-BRM and the isolated compounds 1–3. (A) The anti-Aβ aggregation effects of the primary fractions of ACD-5-BRM and compounds. Since compounds 2 and 3 were not soluble in methanol at high concentrations, anti-Aβ aggregation assay was not performed on 500 μg/mL. Albumin was taken as a positive control. (B) The structure of compounds 1–3. (C) The inhibitory effect of compounds on LPS induced nitric oxide (NO) production of BV-2 cells. (D) The effect of compounds 1–3 on the viability of BV-2 cells. The results were expressed as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, ****p < 0.0001, compared with the control group. A value of p < 0.05 was considered statistically significant.
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