Eight UbiA-type prenyltransferases were mined in Laetiporus sulphureus by bioinformatic analysis, and phylogenetic analysis showed that they have unique functions. Through heterologous expression in Aspergillus oryzae and addition of exogenous hydroquinone (HYQ, 2) substrate, it was found that LaPT3 could transfer isoprenyl groups on 2. Substrate specificity studies revealed that LaPT3 was substrate specific and could only transfer dimethylallyl diphosphate (DMAPP) using 2 as substrate to produce the product 2-(3-methylbut-2-en-1-yl) benzene-1,4-diol (1). The key active sites of LaPT3 were analyzed and two key amino acid sites near the conserved motifs were targeted for mutation, and the product yields were reduced to 60% and 29% respectively by mutating N100 to S and G208 to A. Molecular docking and site-directed mutagenesis results indicate that these two amino acid sites play a crucial role in the catalytic generation of 2 by LaPT3 to produce 1.

Seven new polycyclic-fused cytochalasins (CYTs), harziachalasins A-G (1-7), together with three known analogues (8-10) were isolated from the solid culture of the endophytic fungus Trichoderma harzianum MLJ-4, which was originally isolated from the leaves of Asclepias curassavica. The planar and absolute structures of all new compounds were determined on the basis of extensive spectroscopic data (1D, 2D NMR and HR-ESI-MS), NMR calculations with DP4 + probability analysis, and theoretical simulations of ECD spectra. Compound 1 represents the first example of 5/6/6 tricyclic CYT featuring a 2-methyl-4-oxopentyl side chain at the C-14 position. This novel architecture originates from a 5/6/6/7 tetracyclic CYT precursor through sequential oxidative cleavage of the C-19-C-20 bond followed by decarboxylative elimination of C-19. Compound 2 features an unprecedented 5/6/6/6/7 pentacyclic scaffold incorporating a 1,3-dioxane moiety, may be constructed by the acetalization of the 7-OH and 13-OH on a 5/6/6/7 tetracyclic CYT with acetaldehyde. Compounds 1-10 were screened for HIV latency reversal activity using J-Lat A72 and J-Lat 10.6 cell models. Compound 4 showed strong activity, with half-maximal effective concentrations (EC₅₀) values of 2.68 μM (J-Lat A72 cells) and 2.99 μM (J-Lat 10.6 cells), demonstrating consistent potency. Mechanistic studies revealed 4 activated the NF-κB pathway to reverse HIV latency, offering insights for new therapeutic strategies targeting this pathway.

Sixteen previously undiscovered guaiane-type sesquiterpene dimers, xylanins A-P (1-16), along with six known analogues (17-22), were isolated from the branches and leaves of Xylopia vielana with chromatographic techniques. Their structures including absolute configurations were determined by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), nuclear magnetic resonance (NMR) data, electron circular dichroism (ECD) spectra, as well as X-ray crystallographic analysis. In cytotoxicity test, we found that five compounds (6, 7, 8, 9 and 12) had cytotoxic activities in vitro against the human pancreatic cancer (PANC-1) and human prostate cancer (PC-3) cell lines. Of these compounds, compound 8 showed a relatively greater cytotoxic effect against PANC-1 cell lines with half maximal inhibitory concentration (IC₅₀) value of 1.06 μM. Flow cytometry analysis showed that the apoptosis rate of PANC-1 cells increased with increasing concentrations of compound 8, and also demonstrated that the cell cycle of PANC-1 cells was arrested at S phase by the treatment of compound 8. By the invasion test, compound 8 was found to restrain the invasion of PANC-1 cells. In autophagy assay, we observed increased microtubule-associated protein 1 light chain 3 (LC3) by immunofluorescence in the compound 8-treated group.

Phytochemical study of the EtOAc extract of Ophiorrhiza brevidentata resulted in the discovery of 10 new monoterpene indole alkaloids (1-10), along with 13 known compounds (11 - 23). Compound 1 possess an unprecedented skeleton consisting of fused 6/5/6/7/6 polycyclic systems. The structural characterization of these compounds was achieved using Nuclear Magnetic Resonance, Mass Spectrometry and Quantum Chemical Calculations. Compounds 3-5 and 9 exhibited strong inhibition on lipopolysaccharide-induced B cell proliferation with IC₅₀ values ranging from 3.6-9.1 μM with excellent selectivity indices (SI > 10).

Antibiotic-induced depletion of the gut microbiota facilitated the colonization of vancomycin-resistant Enterococci (VRE) in the gastrointestinal tract, and then increased patients' susceptibility to secondary infections. Ellagic acid, a major constituent of fruits and nuts, showed various bioactivities except for antibacterial. Interestingly, it promoted the recovery of gut microbiota, enhanced microbial diversity and stimulated the proliferation of probiotic gut microbes, and then ameliorated the overgrowth of pathogens in vivo in our experiment. Moreover, ellagic acid activated Gpr41 and Gpr43 mainly by promoting the production of short chain fatty acids (SCFAs) such as acetic acid and propionic acid to inhibit the NF-κB signaling pathway. Then the dietary supplement with ellagic acid might treat infected gut to avoid antibiotic-associated intestinal diseases, and the finding also provided a novel strategy for exploring antibacterial agent besides screening in vitro.

The new species Cortinarius mapuveronicae from Andean-Patagonian Nothofagus-forests is described in a polythetic approach combining chemical analysis of the anthraquinonoid secondary metabolites, microscopical and morphological characteristics, as well as molecular phylogeny. C. mapuveronicae exhibits an intense red color reaction of the basidiomata by treatment with KOH, whereas the basidiospores are turning purplish brown. As responsible compound, the new anthraquinonoid pigment clavorubin-8-O-methylether (1), together with the known monomeric and dimeric anthraquinones (+)-7,7-emodinphyscion (2), emodin (3), emodin-6,8-di-O-methylether (4), questin (5), (+)-(S)-skyrin (6), (+)-(S)-aurantioskyrin (7), hypericin (8), dermolutein (9), and endocrocin (10) could be identified, showing also remarkable activity in a (photo)antimicrobial and (photo)cytotoxic assay. Phylogenetic analysis (ITS, LSU, rpb1) demonstrates a sister group relationship with the holotype of C. rubrobasalis.

Guided by the MS/MS-based molecular networking, eight previously undescribed clerodane diterpenoid glycosides, designated tinospinosides F-M (1-8), along with 12 known analogues (9-20), were isolated from the tuberous roots of Paratinospora sagittata. Structural elucidation of the undescribed compounds was achieved through comprehensive spectroscopic analyses (NMR, HRESIMS), with their absolute configurations confirmed via single-crystal X-ray diffraction, TD-DFT/ECD computational analyses, and chemical degradation. Immunomodulation evaluation on all the isolates revealed that compounds 6 and 7 exerted significant promoting effect toward NO production in RAW264.7 macrophages. Further study demonstrated that 6 could enhance the release of immune cytokines (e.g., TNF-α) and upregulate the protein expression of iNOS and COX-2, which was potentially mediated through the activation of NF-κB signaling pathway.

Thrombosis triggers various severe diseases, while antithrombotic drugs carry bleeding risks, making the development of novel natural anticoagulants a subject of widespread attention. Poecilobdella manillensis, a prevalent medicinal leech, exhibits remarkable anticoagulant and antithrombotic activities. However, the material basis underlying its anticoagulant effects remains insufficiently investigated. This study aims to mine anticoagulant peptides from P. manillensis by peptidomics analysis, elucidate the material basis of its anticoagulant activity, and provide candidate molecules for developing novel natural anticoagulant drugs. Proteins extracted from P. manillensis were enzymatically digested and fractionated using DEAE-52 and CN columns. The resulting peptide components were analyzed by UPLC-Q-Orbitrap HRMS, and peptide sequences were matched against proteomic databases using Proteome Discoverer. Anticoagulant peptides were predicted using the BIOPEP-UWM database and PeptideRanker server, followed by in vitro and in vivo activity validation. Results showed that the hydrolysate consisted predominantly of low-molecular-weight peptides. 1533 peptides with Mw < 3000 Da (length < 20 amino acids) were identified from the PM-A2 and PM-A3 fractions, accounting for 40.76% of the total. Four peptides selected through predictive screening demonstrated anticoagulant and antithrombotic activities in vitro. Among them, LE-11 significantly prolonged both APTT and TT (P < 0.0001). Furthermore, LE-11 effectively alleviated carrageenan-induced thrombosis in mice, outperforming the heparin control at the mid-concentration (20 mg/kg). In this study, the highly active anticoagulant peptide LE-11 was identified from P. manillensis through peptidomic analysis. These findings establish a solid foundation for developing anticoagulant drugs from this source and provide critical scientific support for its clinical application in treating thrombotic diseases.

A screen of ~ 4000 actinomycetes strains identified Streptomyces sp. W3009 as a producer of the antituberculosis agent pyridomycin. Using a mass spectrometry-based metabolomics approach coupled with molecular networking, we identified seven pyridomycin derivatives, six of which were novel. Three of these novel compounds were linear, featuring a unique 3-hydroxypicolinic acid-l-threonine-3-(3-pyridyl)-l-alanine (3HP-T-3PA) scaffold. Their structures were elucidated via detailed NMR studies. While two cyclic derivatives (4 and 5) showed modest antitubercular activity, the three linear derivatives, despite possessing the key 3HP-T-3PA moiety, exhibited no inhibitory activity. This intensive MS-based approach demonstrates the important role of such techniques in the discovery of novel biologically active core structures and their natural derivatives.

Thrombotic disorders remain a global health burden, necessitating novel antiplatelet agents with improved safety and efficacy. This study investigates the molecular mechanisms of two lignans, 6'-Hydroxyjusticidin B (6'-HJB) and Neojusticin A (Neo-A), isolated from Justicia procumbens L., through an innovative target-driven strategy integrating LC/MS, proteomics, network pharmacology, and biophysical validation. For the first time, integrin β₃ (ITGB3) was identified as their direct molecular target, with microscale thermophoresis (MST) confirming high-affinity binding, the dissociation constant (Kd) = 0.0642 ± 0.005 μM for 6'-HJB; 0.0097 ± 0.001 μM for Neo-A. This study not only elucidates the structural basis of their activity-C-6 hydroxylation in 6'-HJB enhances ITGB3 specificity, whereas Neo-A's fused furan ring optimizes COX-1 interaction, but also establishes a paradigm shift from phenotypic screening to target-validated natural product research. The findings position 6'-HJB and Neo-A as promising candidates for the development of safer, ITGB3-mediated antithrombotic therapies, with future efforts directed toward structural optimization and preclinical validation.

The Tiger Milk mushrooms (Lignosus spp.) have long been used as traditional folk medicines throughout Southeast Asia. However, the chemical constituents of these species remain largely unexplored. In this study, four type I sesquiterpene synthases (LrhTS1-LrhTS4) from Lignosus rhinocerus TM02^® were functionally characterized through genome mining, in vitro enzymatic assays, and heterologous expression in an Escherichia coli host engineered to overexpress an exogenous mevalonate pathway. The enzyme products were analyzed by GC-MS and further purified and structurally elucidated by NMR spectroscopy. A total of 11 sesquiterpenes (1-11) were identified, among which two were unstable and converted to stable derivatives (2 to 2a and 7 to 7a). Notably, compounds 1-10 are reported from the genus Lignosus for the first time. LrhTS1 and LrhTS3 exhibited high catalytic specificity, whereas LrhTS2 and LrhTS4 displayed product promiscuity. This work expands the knowledge of terpene synthase from mushroom, and advances the understanding of the chemical basis underlying the bioactivity of the Lignosus rhinocerus TM02^®.

In course of investigating the endophytic and nematode-associated fungus Polydomus karssenii for the production of secondary metabolites, seven previously undescribed natural products were isolated from liquid and solid-state fermentations. 1D and 2D NMR spectroscopy, together with HR-ESI-MS data, enabled the elucidation of the planar structures of 3-decalinoyltetramic acids polydosetins A-E (1-5) and cyclodepsipeptides pullularins G and H (6 and 7). The relative configurations of the decalin moiety of 1-5 were determined based on ROESY correlations and ¹H-¹H coupling constants. The configuration of the side chains was established through a detailed J-resolved analysis (Murata's method) in combination with chemical shift comparison to model compounds. Absolute stereochemistry of 1-5 was assigned based on ECD data, and confirmed by Mosher's method utilizing 3. Finally, the absolute configuration of amino acid residues in 6 and 7 was determined through advanced Marfey's method. Bioassays revealed that compounds 1, 3, 5, and 7 were active against Gram-positive bacteria, 3 and 5 exhibited antifungal activity, and 1 and 2 showed nematicidal effects. These results underscore the untapped chemical potential of P. karssenii and highlight the importance of exploring nematode-associated fungi as sources of new natural products with potential antimicrobial and nematicidal properties.

Five new α-pyrones, diaporpyrones G-K (1-5) and three new γ-butenolide derivatives, porbutenolides A-C (6-8), along with seven known compounds (9-15), were isolated from the culture extract of the endophytic fungus Diaporthe sp. CCY4. Their structures were elucidated by comprehensive spectroscopic analysis, including 1D/2D NMR and HRESIMS data. The absolute configurations of 7 and 8 were assigned using electronic circular dichroism (ECD) calculations. All compounds were evaluated for inhibitory activity against ubiquitin-specific peptidase 4 (USP4). Compounds 2, 5, 9, and 13 exhibited significant anti-ubiquitination effects at 40 μM, with 13 showing the most potent inhibition (IC₅₀ = 20.85 μM).

Hippophae rhamnoides L. (Elaeagnaceae), commonly known as sea buckthorn, is a medicinal plant valued for its diverse bioactive constituents and broad therapeutic potential. Phytochemical investigation of its roots led to the isolation of three new 14-noreudesmane-type sesquiterpenoids (1-3), together with sixteen known compounds (4-19). Their structures and absolute configurations were determined using comprehensive spectroscopic analyses and quantum chemical computations of electronic circular dichroism (ECD) spectra and ¹³C NMR shifts. Three new sesquiterpenoids (1-3) were tested for their antioxidant, anti-inflammatory, antibacterial, and α-glucosidase inhibitory activities. Unfortunately, none exhibited significant activity under the tested conditions. Among the isolated known compounds, all those displaying α-glucosidase inhibitory and antibacterial activities are pentacyclic triterpenoids. Hippophamide (17) showed significant DPPH and ABTS radical-scavenging activity. These results enriched the chemical profile of H. rhamnoides roots.