RSC Advances

The facultative methylotroph model organism Methylorubrum extorquens AM1 is a known lanthanide user, which has shed light on the role of rare-earth metals in biochemistry. The characterization of a methanol dehydrogenase (MDH) protein which requires lanthanides as an enzymatic cofactor outlined the question of how these metals are acquired from the environment. It has been proposed that mesophilic organisms as M. extorquens AM1 can produce siderophore-like molecules, which chelate, transport and traffic rare-earth elements into the microbial cell. Therefore, we performed the bioinformatic and chemical investigation of M. extorquens AM1 by using genome mining, the CAS and arsenazo assay, molecular networking and chemical analytical techniques. Our results showed that indeed Methylorubrum extorquens AM1 harbored a gene cluster to produce metal chelators. The chemical analysis confirmed the production of the known hybrid hydroxamate-citrate siderophores schizokinen A and N-deoxyschizokinen A, which are very likely the side products of the transformation of schizokinen and N-deoxyschizokinen. The determination of the lanthanide chelation activity of the schizokinen siderophores series against three different lanthanides (La, Eu and Lu) showed no coordination activity, thus ruling out the involvement of schizokinen siderophores in rare-earth metal transport.

This paper presents the results of a structural analysis of chlorogenic acid isolated from a 70% ethanol extract of red clover (Trifolium pratense) callus culture. X-ray phase analysis showed that the sample was crystalline and single-phase and crystallized in an orthorhombic unit cell with the following parameters: a = 36.7548(5) [A], b = 11.0770(3) [A], c = 7.7947(2) [A], V = 3173.46(11) [A]3, R-Bragg = 0.347 %, Rexp = 4.75 %, Rwp = 5.83 %, Rp = 4.39 %, GOF = 1.23 %. NMR spectroscopy data (1H, 13C{1H}, 2D 1H1H-COSY, 1H13C-HSQC, 1H13C-HMBC) confirmed that the chemical structure and purity of the sample fully corresponded to chlorogenic acid, with no chemical impurities detected. Complete proton and carbon atom assignments are provided.

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen is well known for life-threatening acute infections among the human population. The bacterium can withstand most antibiotics by using their high levels of inherent and acquired resistance mechanisms such as Biofilm-EPS, Persistence, and Quorum sensing (QS). Owing to the importance of adaptive antibiotic multi-drug resistance of P. aeruginosa, the current investigation is aimed to explore the phytochemicals derived from mangrove plants as potential agents to control biofilm and drug resistance mechanisms through a multi-mechanistic computational approach. For identifying potential compounds and target, In-silico drug repurposing technique is implemented by docking/virtual screening of 49 phytochemical compounds against 18 proteins involved in the Persister Cell formation, QS, and EPS synthesis in P. aeruginosa which resulted the proteins RelA and SpoT (persistence), PqsA, and PqSR (QS), and PelA and PelB (EPS synthesis) and compounds Taraxerone and Taraxerol to be potential. The results of docking were well corroborated with MD simulations. These targets and compounds explored through in-silico approach, are found to target potential antimicrobial pathways involving EPS synthesis, persistence genes, and QS, aiming to enhance antibiotic efficacy. Further, this study could be reference for in-vivo and in-vitro investigations to evaluate the further effectiveness of the compounds and potentiality of the proteins for MDR therapeutics of P. aeruginosa.

Natural products are a rich source of bioactive molecules and undiscovered chemical scaffolds with significant potential for novel drug discovery. Among these, fungi are particularly promising, offering diverse metabolites and undiscovered structural motifs. Large, well-curated collections of crude extracts, or "libraries", are central to fungal natural product discovery, serving as starting material for bioassay-guided isolation of new compounds. However, the systematic influence of fungal selection strategies, culturing methods, and environmental factors on chemical diversity remains underexplored. In this study, we analyzed several large fungal libraries to assess how geographic origin, and phylogenetic classification shape fungal chemical profiles. We also evaluated whether culturing conditions that more closely mimic natural environments can enhance metabolite diversity. Our findings offer practical guidelines for optimizing fungal natural product library design, improving drug development efficiency and access to novel chemotypes for future drug discovery. Summary Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/709592v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@70a0e0org.highwire.dtl.DTLVardef@51f84eorg.highwire.dtl.DTLVardef@184dd90org.highwire.dtl.DTLVardef@1ee2813_HPS_FORMAT_FIGEXP M_FIG C_FIG

Lung cancer remains the leading cause of cancer-related deaths worldwide, with cisplatin as the primary chemotherapy despite its limitations. Organotin(IV) dithiocarbamates have emerged as promising anticancer agents due to their potent cytotoxicity and stability. This study reports the successful synthesis of four novel organotin(IV) dithiocarbamates: dimethyltin(IV) N-methyl-N-benzyldithiocarbamate (DioSn-1), diphenyltin(IV) N-methyl-N-benzyldithiocarbamate (DioSn-2), triphenyltin(IV) N-methyl-N-benzyldithiocarbamate (TriSn-3), and triphenyltin(IV) N-ethyl-N-benzyldithiocarbamate (TriSn-4). Their cytotoxicity against A549 lung carcinoma cells was evaluated via MTT assay, while Annexin V-FITC/PI staining determined the mode of cell death. DioSn-2, TriSn-3, and TriSn-4 exhibited potent cytotoxicity (IC: 0.52-1.86 M), whereas DioSn-1 was inactive (IC > 50 M). Apoptotic features such as cell shrinkage and membrane blebbing were observed, with apoptosis rates ranging from 58% to 91%. DioSn-2 was the most selective (SI = 6.45) and induced early DNA damage within 30 minutes, followed by mitochondrial depolarization and excessive ROS generation. Caspase-9 activation exceeded caspase-8, confirming intrinsic apoptosis. NAC treatment reduced apoptosis by 52%, highlighting oxidative stress as a key cytotoxic mechanism. These findings suggest DioSn-2 as a promising alternative to cisplatin for lung cancer therapy.

Belgian endive-derived biostimulant (BEE) was previously shown to enhance root and shoot growth of Arabidopsis thaliana and Plectranthus esculentus in in vitro culturing conditions. In this study, we evaluated the effect of BEE on A. thaliana subjected to abiotic stresses and assessed the translatability of its bioactivity on lettuce (Lactuca sativa) and sweet pepper (Capsicum annuum) cultured in substrate and soil. A first set of experiments tested the impact of BEE on protection during, and restoration after, osmotic or salt (NaCl) stress. BEE treatment had little to no rescuing effect when plants were exposed to osmotic stress. In contrast, BEE strongly promoted shoot development and leaf health both under standard and NaCl stress conditions. Under mild stress, BEE enhanced photosynthetic efficiency and chlorophyll content in Arabidopsis, whereas it did not significantly alleviate osmotic stress induced by sorbitol. To evaluate the effect under ex vitro conditions, BEE was applied via root drenching to substrate-grown A. thaliana, lettuce, and sweet pepper. BEE improved leaf greenness and photosynthesis enhancing Arabidopsis rosette development, but it did not increase lettuce head weight. In sweet pepper, BEE increased fruit yield and promoted fruit maturation. Under drought stress conditions, BEE application did not improve sweet pepper yield.

Catechinopyranocyanidins (Cpcs) which consist of diastereomers A and B are pigments derived from adzuki beans and are compounds in which the catechin and cyanidin skeletons are condensed to a pyrano ring. While catechins and anthocyanidins possess high antioxidant capacity, the physiological functions of Cpcs remains unclear. In this study, the antioxidant capacity of Cpcs was evaluated by in vitro antioxidant assays and by assessing their cytoprotective activity against oxidative stress in normal human dermal fibroblasts (NHDFs). Antioxidant capacity based on the hydrogen atom transfer (HAT) mechanism, as assessed by the ORAC assay revealed that Cpcs exhibit 14.1 mol TE/mol (Trolox equivalent antioxidant capacity: TEAC). Meanwhile, capacity based on the single electron transfer (SET) mechanism, as assessed by the DPPH, ABTS and CUPRAC assays revealed, they exhibit 2.1-3.6 mol TE/mol. Since TEAC value of Cpcs demonstrated by the HAT based mechanism higher than its SET based oxidative capacity suggesting that the antioxidant capacity of Cpcs is driven by the HAT mechanism. In cell culture experiments, Cpcs ameliorate cell toxicity in rotenone-induced injury model, suggesting to cytoprotective activity against mitochondrial dysfunction-dependent apoptosis. These results reveal novel physiological functions of Cpcs which may serve as a design guideline for elucidating in vivo dynamics based on antioxidant mechanisms.

Carbon monoxide (CO) poisoning is responsible for around 50,000 emergency department visits per year in the U.S. alone. With the present standard of care, persistent neurological sequelae occur in [~]30-40% of severe CO poisoning cases. Currently, there is no available targeted molecular antidote for CO poisoning. In previous work, we have developed an antidotal therapy for CO poisoning based on an engineered hemeprotein, human neuroglobin (Ngb-H64Q-CCC). Intravenous infusion of Ngb-H64Q-CCC removes CO from the circulating red blood cells and improves survival in a lethal CO-poisoning mouse model. However, the infusion of heme-containing proteins has inherent heme toxicity risks that may limit the dose that can be used safely without liver or kidney toxicity. In order to overcome these problems, we have investigated the development of immobilized Ngb in a solid matrix. This approach allows for the development of a CO removal system using an extracorporeal blood circulating system coupled with a stationary matrix with immobilized Ngb-H64Q-CCC. Such system avoids drug infusion and possible organ injury, allows for antidote recycling, and provides advantages for storage and handling of the antidote. By assessing the efficacy of Ngb-H64Q-CCC immobilized through different linkage strategies, we have identified N-hydroxysuccinimide agarose resin as a viable stationary phase. The immobilized protein shows preserved heme redox activity, can be chemically reduced/oxidized for activation/CO release purposes, and retains its CO removal capacity after successive regeneration cycles. We expect that this novel approach will advance the development of new scavenger-based therapies for CO poisoning.

This study presents the structural verification of baicalin isolated from a hydroethanolic extract of an in vitro Scutellaria baicalensis root culture using X-ray diffraction analysis and a set of NMR spectroscopy techniques. The crystalline molecular structure of the sample was found to correspond to baicalin. The 1H, 13C{1H}, 2D 1H1H-COSY, 1H13C-HSQC, 1H13C-HMBC spectra confirmed that the chemical shifts, signal multiplicities, integral intensities, and spin-spin coupling constants were fully consistent with the structure of the target compound. Minor impurity signals were detected in the aliphatic region of the spectra, with a total content not exceeding 5 mol%. These results confirm the high purity and structural individuality of baicalin, a biologically active flavonoid glycoside of considerable interest.

Globally, about 264 million individuals across all age groups are impacted by depression, a prevalent central nervous system (CNS) condition. Chronic and enduring depression might result in significant health consequences. Numerous pharmaceutical antidepressants exist for the management of mild to severe depression, largely functioning by modifying neurotransmitter levels in the brain. Nevertheless, these drugs frequently induce a variety of side effects, such as insomnia, constipation, exhaustion, drowsiness, and anxiety. Saffron (Crocus sativus L.) is widely acknowledged as a natural antidepressant with little adverse effects. This study investigated the potential antidepressant mechanisms of saffrons principal bioactive compounds safranal, crocin, and picrocrocin via molecular docking against critical target proteins associated with depression, namely the dopamine transporter (DAT), serotonin transporter (SERT), and monoamine oxidase B (MAO-B). Molecular docking was conducted with AutoDock 4.2 to assess the binding affinity and interaction energy of these drugs with the target proteins. Furthermore, Discovery Studio facilitated the viewing and study of both interacting and non-interacting residues at the docking sites, juxtaposing these interactions with those of established inhibitors in crystal structures. The permeability of the blood-brain barrier (BBB), pharmacokinetic characteristics, and toxicity profiles of saffron components were evaluated using SWISS ADME, DataWarrior, and Osiris Molecular Property Explorer. Among the evaluated elements, safranal had the greatest potential as a competitive inhibitor of the dopamine transporter, according to its notable blood-brain barrier permeability, robust binding affinity, and analogous interaction residues in comparison to nortriptyline, a recognized inhibitor. Our findings indicate that safranal may be a viable natural alternative to traditional antidepressants, with minimized adverse effects.

The M. tuberculosis ESX-1 secretion system EccA1 enzyme is involved in the secretion of virulence factors and is essential for virulence and bacterial survival within the phagosome. Development of the small molecular inhibitors abolishing EccA1 function can yield new antivirulence drugs. In this study, we modeled the full-length EccA1 (573 residues, Mw [~]62.4 kDa) structure, which contains N-terminal TPR domain and a C-terminal CbxX/CfqX type ATPase domain. We have identified five ZINC compounds having binding energy i. e. Z1 (ZINC000004513760, -43.45 kcal/mol), Z2 (ZINC000000001793, -49.56 kcal/mol), Z3 (ZINC000005390388, -55.83 kcal/mol), Z4 (ZINC000257294577, -52.33 kcal/mol), Z5 (ZINC000004824264, -44.44 kcal/mol) through virtual screening of the ZINC compounds targeting C-terminal ATPase pocket of EccA1. The Z1-Z5 compounds were compared with ADP substrate having binding energy (Adenosine diphosphate, -35.00 kcal/mol), p97 ATPase inhibitors i.e. NMS873 (3-[3-cyclopentylsulfanyl-5-[[3-methyl-4-(4 methylsulfonylphenyl)phenoxy]methyl]-1,2,4-triazol-4-yl]pyridine, -48.68 kcal/mol), and CB5083 (1-[4-(benzylamino)-5H,7H,8H-pyrano[4,3-d]pyrimidin-2-yl]-2-methyl-1H-indole-4-carboxamide, -50.88 kcal/mol) against EccA1. The Z1-Z5 compounds exhibited good Absorption, Distribution, Metabolism, and/or Excretion properties (ADMTE). Pharmacokinetic properties and Lipinskys rule of five for Z1-Z5 compounds showed drug-like properties. 100 ns dynamics simulation analysis on EccA1 complexed with (i) Z1-Z5 compounds (ii) ADP substrate and (iii) NMS873 and CB5083 inhibitors showed high stability and biologically relevant conformation during dynamics simulation. These data indicate that Z1-Z5 compounds may act as potential inhibitors against EccA1 and provide avenues for new antivirulence drug development after in vitro and in vivo clinical trials.

Prodigiosin is a red pigment produced by various bacteria, including Serratia marcescens. Despite its wide and promising range of biological activities, the large-scale production of prodigiosin is currently limited by its high cost and low yields. Here we propose and optimize an innovative, low-cost, peanut-based solid culture medium that enhances the yield of prodigiosin produced by Serratia marcescens. Colorimetric assays revealed that peanut significantly stimulates prodigiosin synthesis. Further HPLC-MS analysis allowed us to unambiguously identify prodigiosin and shows that our medium specifically improves the yield of prodigiosin. Overall, our innovative culture medium could help lower prodigiosin production costs and, ultimately, open new industrial applications.

Antimicrobial resistance (AMR) in plant pathogenic bacteria poses a serious threat to global agriculture, necessitating the development of novel antibacterial agents targeting virulence mechanisms. This study presents an integrated bioinformatics-driven framework for the rational design and computational validation of Solres, a newly designed small molecule targeting key virulence proteins in phytopathogenic bacteria. Approximately 10,000 active compounds from PubChem BioAssay (AID: 588726) were analyzed using structural clustering and scaffold mining to identify conserved molecular motifs associated with antibacterial activity. Guided by high-frequency substructures, Solres was designed de novo and screened for structural novelty against PubChem, ChEMBL, and WIPO databases. Drug-likeness evaluation using Lipinskis Rule of Five confirmed favorable physicochemical properties. Molecular docking was performed against essential virulence regulators, including PhcA, PhcR, HrpB, PehA, and Egl from Ralstonia solanacearum and Xanthomonas spp., with active sites predicted using CaspFold. Docking analyses revealed strong binding affinities and stable interactions with key catalytic and regulatory residues. Complex stability and conformational integrity were further validated through molecular dynamics simulations. Quantum chemical descriptors, including HOMO-LUMO energy gap and dipole moment, supported the electronic suitability and reactivity profile of Solres. Collectively, this study demonstrates the effective integration of cheminformatics, structural bioinformatics, molecular simulations, and quantum chemical analyses for plant-focused antibacterial discovery. The compound Solres represents a promising lead candidate for mitigating bacterial wilt disease and provides a computational framework for future experimental validation and sustainable crop protection strategies against AMR-driven phytopathogens.

Infectious diseases remain a persistent global health burden, with bacterial infections predominating. The growing global burden of drug-resistant infections has led to greater emphasis on the discovery and development of novel antibacterial compounds. In an attempt to discover new potent antibacterials, the antibacterial activity of novel 2-substituted benzimidazole derivatives (NR-1 to NR-9) was evaluated in this study against three bacteria, viz. M. smegmatis, B. subtilis and E. coli in vitro using Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). Among the nine derivatives assessed, two (NR-4 and NR-5) exhibited inhibitory activity against M. smegmatis, while two (NR-5 and NR-7) were active against B. subtilis, with MICs between 62.5 and 250 g/ml. Notably, NR-5 demonstrated antibacterial activity against both M. smegmatis and B. subtilis, with more efficacy against M. smegmatis (MIC: 62.5 g/ml), which was considerably closer to rifampicin (MIC: 31.25 g/ml). Cytotoxicity analysis of these derivatives in Vero cells indicated minimal toxicity for NR-4 and NR-5, and SwissADME evaluation suggested favourable physicochemical properties and drug-likeness, supporting good oral bioavailability. Moreover, the growth kinetics profiling of the NR-5 Benzimidazole derivative demonstrated that it inhibited the growth of M. smegmatis effectively, even after prolonged exposure. These findings highlighted the promise of the active benzimidazole derivative, NR-5, as a potential candidate for developing a more effective and less toxic antimycobacterial drug. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/710429v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@126a0fcorg.highwire.dtl.DTLVardef@1131372org.highwire.dtl.DTLVardef@161a70corg.highwire.dtl.DTLVardef@1e0dbe_HPS_FORMAT_FIGEXP M_FIG C_FIG

We have conducted all atom molecular dynamics simulations of POPC and DPPC lipid bilayers using AMBER Lipid21 force field with eight different water models, including SPC/E, TIP3P, TIP3P-FB, TIP4P-FB, TIP4P-Ew, TIP4P/2005, TIP4P-D, and OPC, to identify the most compatible one without any modification. A number of parameters have been computed in order to understand the structure of the lipid bilayer: Area per lipid, Isothermal compressibility modulus, average Volume per lipid, electron density profile, bilayer thickness, X-ray and neutron scattering form factors, deuterium order parameter, and radial distribution function. The estimated Area per lipid, Isothermal compressibility factor, volume per lipid and bilayer thickness are highly consistent with experimental results for the SPC/E water model, indicating its suitability with the AMBER Lipid21 force field, insted of any modification. The bilayer electron density profiles of both the lipid bilayers demonstrate a little augmentation of water penetration with respect to the membrane surface for TIP4P-D water model. However, the experimental X-ray and neutron scattering form factors are aligning well with the simulated results for all studied water models, and TIP4P-D shows better for X-ray data. The deuterium order parameter for lipid acyl chains value less than 0.25 for all observed water models, depicting their disorderness for both the lipid bilayers. The lateral diffusion and reorientation autocorrelation function of the lipid molecules in both the bilayers are computed to reveal their dynamics across all water models. In comparison to other water models, the simulated trajectories predict better structure and reasonably fair dynamic properties for the SPC/E water model. The TIP4P-Ew water model reproduces the lateral diffusion co-efficient in close agreement with experiment. Reorientational dynamics for both the lipids in the bilayers for eight different water models are observed; the presence of slow and slowest time components corresponds to the lipid axial motion (wobble motion) and Twist/Splay motions. So, in view of the overall performance of the different water models with the AMBER Lipid21 all atom force field in reproducing membrane physical properties, the SPC/E water model appears to be an optimal choice.

Forest tree seeds are mass produced for afforestation and forest restoration programs, but are mostly underutilized beyond propagation. Here, we aimed to evaluate the antioxidant, anti-inflammatory, anticancer, and tyrosinase-inhibitory activities of seed extracts of seven economically important forest tree species in the Republic of Korea to explore their potential as multifunctional natural bioresources. The seed extracts of Alnus japonica, Chamaecyparis obtusa, Cornus kousa, Phellodendron amurense, Pinus densiflora, Prunus sargentii, and Quercus glauca were comparatively assessed using multiple in vitro assays. The results revealed clear species-dependent functional profiles rather than uniform bioactivities across species. Quercus glauca exhibited strong antioxidant activity and significant anti-inflammatory and tyrosinase-inhibitory activities, suggesting multifunctional potential, while C. obtuse presented considerable anticancer activity against several cancer cell lines. Alnus japonica exhibited the highest tyrosinase-inhibitory activity, followed by Q. glauca and C. obtuse; A. japonica extract also showed a strong antioxidant capacity. Overall, the results demonstrated that forest tree seed extracts possess diverse and complementary bioactivities, supporting their potential as underexplored multifunctional natural materials. By focusing on seed resources generated within existing afforestation systems, we highlight a sustainable approach to valorize forest-derived by-products without additional pressure on natural ecosystems. Nevertheless, as bioactivities were evaluated using crude extracts, further studies are required to identify and elucidate the active compounds and their mechanisms of action.

Biological metal chelators are of great interest for investigation due to their capacity to retain or mobilize metals from the environment. While some biological and bioinspired chelators find use in medical applications, others are promising platforms for the mining or recycling of technologically important metal ions. In particular, the siderophores, which are primarily iron chelators, have been studied. Four siderophores of relevance are schizokinen and its derivatives, which have been isolated from bacterial and algae cultures, in addition to soil. These siderophores have shown metal chelating activity with different metals such as iron, copper, and aluminum. In the time of metabolomics, it is required to unambiguously determine the identity of the produced siderophores as quickly as possible. Thus, Liquid Chromatography coupled to High Resolution Mass Spectrometry and mass-tandem fragmentation (LC-HRMS-MS) provides a quick and applicable alternative for identification of schizokinen and its derivatives. Here, we report an analytical method for the identification and potential quantification of the schizokinen siderophore series. We developed a working method through LC-HRMS-MS, which provides the unequivocal identification of the four schizokinen derivatives, which has not been reported to date. Additionally, we constructed the molecular network for the four molecules to enable their identification using the Global Natural Products Social Molecular Networking (GNPS) platform. Most importantly, this contribution can help speed up the characterization of schizokinen producers and facilitate the dereplication process of siderophores.

Sea water desalination is a critical solution to global water scarcity, primarily relying on membrane-based technologies and reverse osmosis. Artificial water channels (AWCs) offer a promising solution for next-generation desalination membranes. Very promising channel building blocks for AWC is oligourea foldamers repeats. These biomimetic molecules, composed of unnatural amino acids, self-assemble into protein-like superhelical channels with water-filled pores, making them ideal candidates for selective water transport. In silico studies provide essential support to experimental efforts in designing novel oligoureas. However, such studies require a dedicated force field for now unavailable for these molecules. In this work, we developed a tailored force field for oligoureas by adapting parameters from two established protein force field families: CHARMM (CHARMM36m) and Amber (GAFF2), using their structural similarities to natural amino acids. Our objective was to identify a force field that reliably preserves the structural integrity of oligourea foldamers. We evaluated two distinct oligoureas using molecular dynamics (MD) simulations across increasing system sizes. By comparing simulation results with experimental data, we assessed key structural features, including folding patterns, stability, and pore shapes. Our findings demonstrate that the CHARMM-based force field consistently reproduces experimental observations for both foldamers, outperforming the Amber-based alternative. This newly developed CHARMM-based force field paves the way for further exploration of oligoureas, enabling deeper insights into their stability and efficiency in sea water desalination.

Endophytic fungi residing within medicinal plants are emerging as prolific sources of structurally diverse bioactive secondary metabolites with applications in drug discovery. Azadirachta indica (Neem) and Melia azedarach (Melia), members of the Meliaceae family, are renowned for their rich phytochemical composition; however, the contribution of their endophytic fungi communities to this chemical diversity remains largely unexplored. Herein, endophytic fungi were isolated from leaves and bark of Neem and Melia collected in Kenya and cultured under distinct physical conditions, solid (plates) and liquid (broth) media to assess how culture environment influences compound production. Compounds were extracted and analyzed using gas chromatography-mass spectrometry (GCMS) to profile the chemical diversity associated with each endophytic fungi, physical culturing state and host plant. GCMS analysis revealed that while the host plant identity influences the presence of specific compounds, the dominant determinant of chemical diversity was intrinsic biosynthetic capacity of the endophytic fungi themselves. Several compounds were unique to endophytic fungi cultures, highlighting their role as independent sources of bioactive compounds. Culture conditions moderately influence metabolite profiles, demonstrating the importance of optimizing growth environments in experimental design and natural product bioprospecting. From the Neem samples, we found 53 compounds uniquely present in the broth samples (consisting of Neem powder and endophytic fungi), 22 found exclusively with the endophytic fungi from the Neem, and 31 compounds shared between the broth and the endophytic fungi samples. In Melia samples, 109 compounds were uniquely present in broth samples from Melia plant (consisting of Melia powder and endophytic fungi), 22 compounds were found exclusively with the endophytic fungi from the Melia, and 55 were shared between the broth and the endophytic fungi samples. Our comparative analysis assessed the Neem and Melia endophytic fungi exclusive samples and reported 12 shared compounds. 10 compounds were unique to Neem and 10 unique to Melia; however, their identities varied between the two categories. While GCMS enabled the identification of volatile and semi-volatile metabolites, future studies employing complementary metabolomic approaches, such as liquid chromatography-mass spectrometry (LCMS), ultra-high-performance liquid chromatography MS/MS (UHPLC MS/MS), or nuclear magnetic resonance (NMR) spectroscopy, would expand coverage to non-volatile, polar, and high molecular weight compounds, providing a more comprehensive understanding of endophyte-derived chemical diversity. These findings provide insights into the interplay between medicinal plants and their endophytes and establish a foundation for leveraging endophytic fungi from Neem and Melia as scalable sources of structurally complex natural products for pharmaceutical and biotechnological applications while minimizing ecological impact.

Many agricultural soils are deficient in key macronutrients needed for healthy plant development. Relying on highly water-soluble commercial fertilizers for long durations can be costly and environmentally harmful. This study investigates a phosphorus-loaded Mg/Fe layered double hydroxide (LDH) dispersed on Douglas fir biochar (Mg/Fe-LDH biochar) as a controlled-release fertilizer and evaluates its impact on bush bean (Phaseolus vulgaris L.) growth. Emphasizing sustainability, the work integrates controlled-release fertilizers, biochar, and LDH modification to enhance nutrient use efficiency and mitigate environmental runoff. Mg/Fe-LDH was directly synthesized on biochar via a co-precipitation approach, loaded the composite with phosphate by anion exchange, and characterized the material using elemental analysis, N2 Brunauer-Emmett-Teller (BET) determinations surface area analysis, and x-ray photoelectron spectroscopy to confirm successful LDH modification on Douglas fir biochar, and high surface area with accessible active sites. The synthesis yielded a stable P-Mg/Fe-LDH biochar with enhanced dispersibility and phosphate-buffering capacity, enabling controlled-release fertilization. In greenhouse experiments, bush beans grown with the P-Mg/Fe-LDH biochar exhibited improved growth metrics, including increased yield (beans fresh weight of 31.7 g), biomass (plant dry weight of 6.3 g), plant height (32.8 cm), and improved nutrient uptakes (1.88 mg (P) g-1) at 100.88 kg (P2O5) ha-1 compared with unfertilized controls and conventional P fertilizers, indicating efficient, controlled-release phosphate delivery and sustained nutrient availability. The results demonstrate that integrating LDH-modified biochar can enhance P uptake and plant growth while reducing leaching losses. Overall, this study highlights the strategic significance of combining biochar, layered double hydroxides, and controlled-release formulations to advance sustainable nutrient management and improve crop performance in agroecosystems. The findings offer a promising pathway for environmentally conscious fertilizer design and soil amendment strategies that align with global goals for resource efficiency and food security. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/727001v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@316444org.highwire.dtl.DTLVardef@adcd48org.highwire.dtl.DTLVardef@8068aforg.highwire.dtl.DTLVardef@58d623_HPS_FORMAT_FIGEXP M_FIG C_FIG