Volatilome Profiling and Apoptosis-sensitizing potential of secondary metabolites from Trichoderma lixii: Integrating GC–MS, Bioassays, and In Silico Docking Approaches

Volatilome Profiling and Apoptosis-sensitizing potential of secondary metabolites from Trichoderma lixii: Integrating GC–MS, Bioassays, and In Silico Docking Approaches | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Volatilome Profiling and Apoptosis-sensitizing potential of secondary metabolites from Trichoderma lixii: Integrating GC–MS, Bioassays, and In Silico Docking Approaches Nguyen Huy Thuan, Santhosh Sigamani, Saranyadevi Subburaj, Hue Thi Nguyen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9031969/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Purpose Trichoderma species are common soil-dwelling fungi that are well known for their extensive production of secondary metabolites, antagonistic qualities against phytopathogens, are remarkable ecological adaptability. Method By a thorough molecular and chemical analysis, a novel isolate known as Trichoderma sp. (TR5) was carefully characterized in this study. Pairwise ITS comparison identified TR5 as Trichoderma lixii and its phylogenetic analysis based on ITS rDNA sequences. The GC MS analysis primarily had contained fatty acids, alkenes, esters, and phthalates Results The most noticeable zones of inhibition was detected by T.lixii derived metabolites against pathogenic bacteria namely Pseudomonas aeruginosa (34.5 mm) and Bacillus cereus (27.4 mm). Additionally, they showed strong antioxidant properties and selective cytotoxicity in a variety of cancer cell lines, with HepG2 cells showing the strongest inhibitory effect (IC 50 =29.01 3.33 µg/ml). IC 50 values of 54.2 µg/ml for DPPH and 149. µg/ml for ABTS assays. Docking suggested favorable interactions of representative VOCs with apoptosis-relevant targets (IKKβ, topoisomerase IIα, β-tubulin, Bcl-2 family), and ADMET predictions indicated acceptable oral bioavailability with low toxicity liabilities. Integrating post hoc prioritization, cyclopentadecanol emerged as the more promising hit - predicted active across several cancer panels and blood - brain barrier permeable. In contrast, N -benzyloxy carbonyl-L-tyrosine exhibited drug-like ADME and strong protein contacts but was predicted inactive across cancer panels. Conclusion Overall TR5 is confirmed to be T. lixii and its volatilome provides a tractable source of apoptosis sensitizing leads. With multifaced biological applications the potential fungi based extracellular metabolites may serve as a potential candidate in pharmaceutical industries. Trichoderma ITS sequence cytotoxic activity docking ADMET7 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction In agricultural and forest ecosystems around the world, filamentous fungus belonging to genus Trichoderma (Ascomycota, order Hypocreales, family hypocreaceae) are common soil residents [ 1 ]. Fast growing septate hyphae, conidiphores with phialides, and green to whitish conidia that promote quick substrate colonization are their distinguishing features [ 2 ]. In terms of ecology, Trichoderma species are opportunistic mycoparasites and decomposers that actively oppose phytopathogens through competition for nutrients and space, secondary metabolites, and enzyme degradation [ 3 ]. Due to these characteristics, they are used extensively in agriculture as soil conditioners, plant growth promoters and biocontrol agents. They are also used as industrial manufacturers of cellulases, chitinases and proteases [ 4 ]. In addition to their agricultural and ecological functions, Trichoderma species are abundant chemical factories that can produce a wide range of natural products, such as terpenoids, peptaibols, polyketides and volatile organic compounds (VOCs) with antifungal, antibacterial and anticancer properties [ 5 , 6 ]. Their volatile organic compounds (VOCs), which diffuse via air or liquid headspace and have been linked to both inter-microbial interactions and mammalian cytotoxicity are particularly interesting. These compounds include low molecular weight fatty acids, alkanes, lactones and terpenes. By supplying polyunsaturated fatty acids and phthalate derivatives with documented pro-apoptotic effects, cyanobacteria further enhance this chemical repertory [ 7 , 8 ]. The term “cancer” is used to describe a wide range of illness, each of which has a unique set of treatment options. Drug resistance, ineffectiveness, relapses, and undesirable side effects continue to be problems despite significant advancements in therapy and a variety of available treatment options [ 9 ]. Despite being widely used, chemotherapy is often linked to a number of adverse effects, such as hematological changes and toxicity to organs such as kidneys, liver, heart, and nervous system [ 10 ]. Personalized cancer treatment through precision medicine seeks to overcome these issues by personalizing medicines to individual patients, yet it remains one of the most serious challenges in modern healthcare. The development of new therapeutic drugs, as well as the investigation of prospective drug combinations that could improve treatment efficacy, are examples of progress in this field. Fungal species are an excellent potential source for finding new anticancer compounds due to their high diversity and variety of environments [ 11 ]. Trichoderma species have drawn particular interest [ 12 ], and fungi have been acknowledged as a valuable natural source of bioactive compounds with medicinal potential [ 13 ]. Secondary metabolites exhibiting a range of bioactivities, such as antibacterial, immunomodulatory and anticancer properties have been generated from extracts of several Trichoderma species. Paracelsin, L-lysine α-oxidase and Trichodermin are a few examples of these compounds [ 14 ]. The potential of volatile chemicals produced by fungi, bacteria and plants as sources for the creation of new anticancer drugs is becoming increasingly apparent. A few essential oils including Terpenoid and phenylpyranoid-rich have considerable seasonal oscillations; Nepalese Ocimum species show compositional variations that greatly impact their bioactive qualities, especially cytotoxic effects [ 15 ]. The plant such as rose, eucalyptus, lemon, and clove have been confirmed for biological effects on neural cells apart from their well-established aromatic benefits, with wider therapeutic potential [ 16 ]. Beyond plants, volatile secondary metabolites of microbial origin, such as those produced by Trichoderma spp., include peptaibols, terpenoids, and polyketides with demonstrated antimicrobial and anticancer activities [ 5 ]. The pro-apoptotic properties of plant volatiles are further exemplified by Warburgia salutaris leaf extracts, which trigger apoptosis in MCF-7 breast cancer cells via caspase-dependent pathways [ 17 ]. At the microbial level, small volatile molecules function as chemical signals but also act as cytotoxins capable of perturbing mitochondrial integrity and redox balance in cancer cells [ 18 ]. Collectively, these findings highlight the chemical diversity of volatile compounds across biological kingdoms and their significant promise as anticancer agents. The present study interrogates the volatilome profiling of a newly isolated Trichoderma sp5 (TR5S2). GC - MS revealed twelve dominant compounds, including fatty acids, alkanes, esters, and phthalates. We evaluated the extracts for antibacterial, antioxidant, and cytotoxic activities against KB, HepG2, A549, and MCF-7 cells, complemented by in silico docking of each compound to apoptotic regulators (IKKβ, topoisomerase IIα, β-tubulin, and Bcl-2 family proteins) and ADMET predictions. By integrating chemical profiling, docking, and functional assays, this work seeks to identify Trichoderma -derived VOCs with apoptosis-sensitizing potential and pharmacokinetic properties suitable for further optimization as anticancer leads. 2. Material and methods 2.1. Morphological identification of the fungi The fungi sample was collected from Long Bien Ward, Hanoi city, Viet Nam and was morphologically identified as Trichoderma sp. It was cultured in potato dextrose agar in petri plates and after the growth appeared as a mat on petri plate a single hypha was picked and placed on microscopic slide. Morphological features were examined under an Olympus CX23 light microscope using 40 × and 100 ×. The characteristics of the fungi was recorded for the morphological identification. 2.2. Molecular Identification of isolated fungi The genomic DNA of Trichoderma sp5 was extracted by Vazyme™ Bacterial DNA Kit (Vazyme Biotech, Nanjing, China). Then its partial sequence was amplified with ITS primers forward and reverse and the PCR products were sent for gene sequencing by 1st Biobase (Malaysia). DNA quality was assessed on a 0.8% agarose gel and quantified with a NanoDrop 2000 (Thermo Scientific) [ 19 ]. The gene sequence was hit on NCBI database by nucleotide BLAST and it most identical sequences were chosen and it was submitted to NCBI for accession number. The phylogenetic tree was constructed from the most similar sequences to identify the family and genus that closely relates by using MEGA12 [ 20 ]. 2.3. Cultivation, chemical extraction and GC-MS Profiling The isolated fungal strain was then grown on potato dextrose agar broth for 10–12 days in shaking condition at 22℃. After incubation time the filtrate was collected by passing the culture media into the blotting paper leaving the mycelium on top. This filtrate was then exposed to ethyl acetate in the ratio of 1:2 of the solvent. This was then kept on shaker for 24 hrs for the extraction in room temperature. The ethyl acetate extracted Trichoderma sp5 filtrate (TEtOAc) was then poured into separating funnel and its organic layer was collected firmly into a beaker. This Trichoderma extract was then evaporated in rotary evaporator with vacuum and mild heat 35 ℃ to avoid leakage of metabolites during the concentration process. The fully concentrated TEtOAc sample was then distributed to glass vials and kept in 4℃ for further analysis [ 21 ]. Confrontation assays were incubated at 21 ± 1°C in darkness for 5 days. A PDMS/DVB solid-phase microextraction (SPME) fiber (Supelco, Bellefonte, PA, USA) was used to collect volatile organic compounds (VOCs), which were then desorbed at 180℃ for 30 seconds inside the injection port of an Agilent 7890 B gas chromatograph interfaced with a 5973-mass spectrometry detector. An HP-FFAP capillary column (30m × 0.25 mm, 0.25 µm film thickness) with helium as carrier gas at a flow rate of 1 ml per minute was used to accomplish chromatographic separation. The oven program was 40°C (5 min), ramped 3°C/min to 220°C (5 min), and followed by 300°C (3 min). Compounds were identified using the NIST/EPA/NIH Mass Spectral Database (version 11) and ChemStation (Agilent, Rev. D.04.00). Each treatment was analyzed in triplicate [ 22 ]. 2.4. Antibacterial action of fugal extracellular metabolites The antibacterial activity of the fungal extracellular metabolites was performed by well diffusion method [ 23 ]. Seven bacterial pathogens were spread on Muller Hinton agar plates using sterile swabs to create a bacterial lawn. Wells were made with 6 mm diameter using sterile cork borers. For the experimental tests, the TEtOAc stock solution (10 mg/ml) was made in 10% DMSO in amounts of 100, 150 and 200 µg/ well respectively. The positive control was streptomycin (10µl of 1 mg/ml stock solution) and the negative control was 10% DMSO. In order to assess antibacterial activity, the zones of inhibition surrounding each well were determined after the plates were incubated at 37℃ for whole day [ 24 ] 2.5. MIC of the fungal metabolites Nutrient broth was prepared and aliquoted into test tubes and closed the mouth with cotton plugs. These tubes were autoclaved at 121℃ for 20 mins at 15 lbs pressure and allowed to cool down gradually. After the media gets cooled down varying concentration of the positive control-streptomycin (syringe filtered) and Trichoderma lixii based extracellular metabolites viz., 7.8, 15.6, 31.2, 62.5, 125 and 250 mg/ml were added to all the tubes aseptically in biosafety cabinet. The tubes were then inoculated with 12 hrs grown bacterial pathogens and kept for 24 hrs incubation. After incubation the growth of the bacteria was observed visually and recorded for determining the MIC in UV spectrophotometer [ 25 , 26 ]. 2.6. Free radical scavenging Assay 2.6.1. DPPH assay 0.1 mM DPPH reagent was prepared and used it for the antioxidant assays. To 7.64 mg DPPH Reagent 100 ml of Ethanol analytical grade was added. Followed by these dilutions of the ascorbic acid were made and 2.0 ml of ethanol was added to it. Ascorbic acid stock at 1mg/ml concentration was prepared and used as standard. The stock was diluted to varying concentration of 5, 10, 15, 20 and 25 µg/ml. In the reaction tubes 1.0 ml of DPPH reagent was added and kept for incubation in dark room for 30 mins [ 27 ]. 2.6.2. ABTS assay ABTS reagent was prepared by mixing (1:1) 7 mM ABTS and 2.45 mM potassium persulphate in amber bottle. To 38.4 mg of ABTS, 10 ml of 6.6 mg Potassium per sulphate was added in 10 ml water and mixed both the reagents in the ratio of 1:1. The solution was kept for 16 hrs in room temperature and 1.0 ml of the reaction was mixed with 27 ml of distilled water. Its O.D was checked and it was diluted further till the O.D reached 0.7 that was read by spectrophotometer at 734 nm. Upon reaching the desired O.D the samples and control were taken for analysis. Dilutions were made with ascorbic acid (10, 20, 25 and 30 µg/ml) by adding water making the total volume to 2.0 ml. Later, 1.0 ml of ABTS reagent was added to each reaction tubes and keep for incubation 30 mins in dark room [ 28 ]. 2.7. Anticancer potential evaluation The MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] test, which was first developed by Mosmann [ 29 ] and then refined by research, which was used to evaluate th cytotoxic potential of the crude extract [ 30 ]. Briefly, complete culture media was used to seed human cancer cell lines (KB, HepG2, A549 and MCF-7) onto 96 well plates at densities ranging from 5×10 3 to 1×10 1 cells per well, and the cells were allowed to adhere overnight. The cells were then exposed to serial dilutions of the crude extract of Trichoderma sp. (0-400 µg/ml) for 24 to 48 hrs at standard incubation conditions (37℃, 5% CO 2 ). Following incubation, each well received 20 µl of MTT solution (5 mg/ml in PBS) and was incubated for three to four hours to allow metabolically active cells to decrease MTT dye into insoluble formazan crystals. After the medium was thoroughly aspirated, 100 µl of DMSO was used to dissolve the formazan crystals. A microplate reader was used to measure absorbance ar 570 nm, with 630 nm acting as reference wavelength. Cell viability percentages were computed in relation to untreated controls and non-linear regression analysis was used to create dose-response curves that yielded half-maximal inhibitory concentration (IC 50 ) values. A quantitative measure of cytotoxic efficacy is provided by the IC 50 values obtained for each cell line, which indicates the extract concentration required to block 50% of cell viability. 2.8. Virtual Screening and Docking Further, the docking was performed for the lead compounds towards Trichoderma with a UniProt ID of Q6A1B7 using the PyRx server. Initially, the ligands' PubChem IDs were imported into the PyRx (Table 1 ), and it was prepared by creating 3D structures, adding hydrogens using OpenBabel software to minimize the ligand energy. Subsequently, the protein was pre-processed by removing the water molecules, het atoms, and other undesirable components, by adding polar hydrogens and charges. The search space is then established by defining a grid box around the active site of a protein. AutoDock Vina, which is built into the PyRx tool, was then employed for the docking study. It predicts the best binding poses and ranks them according to binding affinity (kcal/mol). To determine which Trichoderma metabolites exhibit the strongest interaction with the target protein, the best docking conformations are examined for hydrogen bonding, hydrophobic interactions, and orientation within the pocket. The screened compound was further visualized in Discovery Studio [ 31 , 32 ]. Table 1 Antibacterial activity of the Trichoderma lixii extracellular metabolites S. No Name of the bacterial pathogen 100 µg 150 µg 200 µg Streptomycin 20 µg DMSO (10%) 1. Bacillus cereus 20.4 ± 0.9 22.9 ± 0.8 27.4 ± 0.8 23.4 ± 1.0 - 2. Bacillus Licheniformis 14.3 ± 0.7 16.5 ± 0.9 20.5 ± 0.9 35.4 ± 0.9 - 3. Staphylococcus aureus 9.1 ± 0.5 17.4 ± 0.9 19.6 ± 0.9 28.3 ± 1.0 - 4. Enterococcus faecalis 15.4 ± 0.9 19.5 ± 0.9 21.5 ± 0.9 27.4 ± 0.9 - 5. Vibrio parahaemolyticus 25.5 ± 0.8 30.9 ± 0.5 32.6 ± 0.9 11.6 ± 0.9 - 6. Klebsiella pneumoniae 18.4 ± 0.9 21.4 ± 0.4 23.4 ± 0.9 31.4 ± 0.9 - 7. Pseudomonas aeruginosa 24.9 ± 0.7 30.5 ± 0.9 34.5 ± 0.9 29.4 ± 0.9 - 2.9. Pharmacokinetic Study The SwissADME web-based server was used to assess the pharmacokinetic and drug-likeness characteristics of N-Benzyloxycarbonyl-L-tyrosine and Cyclopentadecanol. Individual compound’s canonical SMILES strings were obtained from the PubChem database and then uploaded to SWISSADME website. Pharmacokinetic profiles, aqueous solubility, lipophilicity, physiochemical features and drug likeliness criteria were among the important aspects that were carefully assessed. In order to provide a comprehensive in silico evaluation of the compounds, prediction analyses using the bioavailability radar, BOILED-Egg model, and structural warning systems (PAINS and Brenk) were carried out to evaluate adsorption, membrane permeability and potential structural liabilities. Subsequently, the pdCSM-cancer model was utilized to assess the anticancer potential of N -Benzyloxycarbonyl – L - tyrosine and cyclopentadecanol. The tool provided likelihood ratings and corresponding confidence levels after predicting their growth inhibition efficacy across several tumor cell lines and submitting the results to the server. The findings showed different patterns of action, which made it possible to compare the two drugs' efficacy and selectivity. In cancer research, this process offers a preliminary computer screening phase to rank these compounds for additional in vitro and in vivo confirmation [ 33 , 34 ]. 2.10. Statistical analysis All the experiments conducted in this research were performed in triplicate and its mean and standard values were calculated. Graph pad prism 8.0 was used for plotting the antioxidant and antibacterial assays. 3. Results 3.1. Morphological description Trichoderma sample Soil samples were serially diluted and plated onto agar PDA medium. After 5 days of incubation, discrete green to yellow-green colonies were selected and subcultured onto fresh PDA to obtain pure isolates. On PDA, colonies of the newly isolated Trichoderma strains were initially white, slightly floccose to finely velvety. With continued incubation, the colonies gradually turned light green and then deepened in colour, producing abundant branched conidiophores; hyphae rapidly covered the entire plate surface within 3–4 days. Conidia were ovoid with smooth walls. Colony morphology: initially white, velvety to cottony; after 4 days of incubation the colony turns green. The colony reverse is pale yellow. Hyphae branched. Conidia ovoid, smooth-walled. Conidiophores bear phialides arranged symmetrically (Fig. 1 ). 3.2. Phylogenetic of Trichoderma sp5 The 608 base pair ITS sequence of the fungus strain Trichodrma sp., is stored in Genebank with accession number PP754294 . With a 1000 bootstrap replicates and values more than 50% annotated at the nodes, the neighbor joining technique was used to create the phylogenetic tree. The query sequence was placed solidly within the Harzianum species complex by clustering with Trichoderma anaharzianum (NR174890.1), T.longicollom (NR198530.1), T. lixii (NR131264.1.), T.atrobrunneum (NR137298.1) and T.azevedoi (NR 173287.1). T.Simmonsi, T.achlamydosporum, T.longifilidicum, T.afarasin , and T.velutinum were other closely related taxa. T. stromaticum, T.solum, T.hainanense and T. cremeum showed distinct monophyletic clusters, while T. ghanense and T. pseudokoningi formed a well-supported subclade next to T. protrudens. The outgroup was Pseudocoleophoma polygonicola (NR 154274.1). The number of nucleotide changes per site is indicated by scale bar (Fig. 2 ). The evolutionary position of the query isolate PP54294.1 ( Trichoderma sp5) within genus Trichoderma is clarified by the phylogenetic analysis based on ITS sequences. T. anaharzianum (NR 174890.1), T. longicollum (NR 198520.1), T. lixii (NR13126.1), T. batrobrunneum (NR137298.1) and T. azevedoi (NR 173287.1) were among the members of Harzianum species complex with which the isolate showed good bootstrap support (82–87%). The isolate’s association with the Harzianum clade, a lineage known for its biocontrol powers, enzymatic activity, and ecological adaptability is supported by this close phylogenetic link. 3.3. GC-MS profiling of Trichoderma lixii based metabolites The ethyl acetate (TEtOAc) extract of the Trichoderma isolate designated T. lixxi was profiled for volatile and semi-volatile constituents by gas chromatography–mass spectrometry (GC–MS). The results of volatile compounds are summarized in the table below ( Fig S1 ). The GC–MS profile is strongly lipidic, dominated by long-chain fatty acids/derivatives and hydrocarbon–alcohol fractions. Major constituents include n-hexadecanoic (palmitic) acid (16.91%) and octadecanoic (stearic) acid (8.45%), alongside ricinoleic acid, several fatty alcohols (e.g., 1-octadecanol; n-heptadecanol-1; 9-/11-hexadecen-1-ol), n-alkanes (C12 – C20; octadecane and eicosane ≈ 5.3% each), and esters such as methyl stearate and isopropyl palmitate (Fig. 3 ) ( Table S2 ). 3.3. Antibacterial potential of TEtOAc Using the agar diffusion experiment, the antibacterial effectiveness of the TEtOAc extract was assessed against seven pathogenic bacterial strains at three different concentrations (100, 150 and 200 µg). The positive control streptomycin (20µg) whereas thew negative control was 10% DMSO. The mean and standard deviation of assays carried out in duplicate. With a strong zone of inhibition extending 34.5mm, the extract showed the most inhibitory action against Pseudomonas aeruginosa at the maximum dosage of 200µg. Vibrio parahaemolyticus (32.6mm), Bacillus cereus (27.4 mm), Klebsiella pneumoniae (23.4mm), Enterococcus faecalis (21.5mm), Staphylocoocus aureus (19.6 mm), and Bacillus licheniformis (20.5mm). Notably, the inhibitory zones at the maximum dose (200 µg) were similar to those generated by Streptomycin control for a number of pathogens including B. cereus, P.aeruginosa, and V.parahaemolyticus. The negative control showed no signs of inhibition (Fig. 4 ) (Table 1 ). As the extract efficacy increases from 100 to 200 µg, a dose dependent increase in activity was statistically significant (p < 0.05) for the tested bacterial pathogens. 3.4. MIC of TEtOAc for bacterial pathogens The extracellular metabolites of the fungi T. lixii showed minimum inhibitory concentration (MIC) against a range of bacterial pathogens were carefully assessed and compared to those of streptomycin. The antibacterial potency of tested strains varied as indicated by MIC values which were expressed in µg/ml. With MIC values of 15.6 µg/ml for few strains TEtOAc demonstrated strong bacteriostatic potential against V. parahaemolyticus and B. cereus . Accordingly, P. aeruginosa and K. pneumonia had MIC values of 15.6 and 62.5 respectively indicating that they were moderately susceptible. Alternatively, B. licheniformis, S. aureus and E. faecalis showed greater resistance to with MIC values above 62.5 µg/ml or below the quantification threshold more than 62.5 µg/ml, indicating a different spectrum of activity. Streptomycin in contrast showed better efficacy with MIC values that were generally lower specifically 7.8 µg/ml against majority of strains with the exception of E. faecalis and V. parahaemolyticus where MICs were slightly higher at 15.6 µg/ml. This demonstrates the strong properties of streptomycin as a standard positive control. This study involving antibacterial efficacy of TEtOAc are consistent with earlier research highlighting the antimicrobial potential of fungal secondary metabolites (Table 2 ). Table 2 MIC values of the bacterial pathogens tested for Trichoderma lixii extracts Samples Tested MIC values of the tested bacteria in µg/ml B. cereus B. licheniformis S. aureus E. faecalis V. parahaemolyticus K. pneumoniae P. aeruginosa TEtOAc 15.6 62.5 < 62.5 15.6 62.5 15.6 Streptomycin 7.8 7.8 7.8 15.6 15.6 7.8 7.8 3.5. Antioxidant profile of TEtOAc by free radicals The antioxidant potential of TEtOAc was evaluated by measuring its ability to scavenge free radicals using the DPPH assay. We tested various concentrations of TEtOAc to find their IC 50 values and percent inhibition. The results showed a significant concentration dependent increase in radical scavenging activity. Ascorbic acid was used as standard for comparison, it showed a calibration curve with an R 2 value of 0.9402 and achieved a 99% scavenging at 20–25 µg indicating exceptional potential. TEtOAc showed enhanced free radical inhibition. In the lowest concentration tested (100µg) we achieved 73.9% inhibition, while the highest concentration reached 99.8%. We calculated the IC 50 value for TEtOAc to be 54.2 µg/ml highlighting its prominent antioxidant capacity. In the ABTS radical scavenging test, we assessed the TEtOAc at various concentrations using ascorbic acid as a standard reference. Excellent linearity was demonstrated by the ascorbic acid calibration curve’s strong correlation coefficient (R2 = 0.98). The IC50 values of TEtOAc metabolite was 149.4 µg/ml. The extract showed a scavenging activity of 41.03% at the lowest tested concentration and an 82.16% suppression of ABTS free radical at highest concentration. The standard ascorbic acid reached > 90% scavenging at 30 µg whereas extract at 400 µg/ ml showed 82% (Fig. 5 a). The scavenging percentage was plotted against extract concentration (0–400 µg/mL) for DPPH (blue squares) and ABTS (olive circles). To determine the 50% inhibitory concentration a non-linear regression analysis was employed. When comparing ABTS radical with an IC 50 value of 149.4µg/ml a log IC 50 value of 2.17 and a R 2 value of 0.98 was achieved, the TEtOAc demonstrated superior efficacy against DPPH free radicals with an IC 50 value of 57µg/ml (log IC 50 -1.75; R 2 value of 0.99). This difference highlights the metabolites’ superior ability to scavenge DPPH radicals via electron or hydrogen atom donation. 3.4. Cell cytotoxicity of TEtOAc against cancer cell lines Four cancer cell lines were treated with the TEtOAc at varying concentrations and their effect on inhibition was analyzed. The TEtOAc extract of Trichoderma lixii shows moderate, cell-line–dependent cytotoxicity: HepG2 is the most sensitive (IC₅₀ = 29.01 ± 3.33 µg mL⁻¹), whereas KB and A549 are intermediate (~ 51–55 µg mL⁻¹) and MCF-7 is weakly affected (91.2 ± 4.9 µg mL⁻¹) (Fig. 5 b) (Table 3 ). Table 3 IC 50 values of Trichoderma lixii ethyl acetate extract against different human cancer cell lines versus Tumor cell line Means of IC 50 values (µg/mL) ± SD Ellipticine Tricoderma lixii EtOA extract KB 0.46 ± 0.02 54.80 ± 2.15 HepG2 0.47 ± 0.02 29.01 ± 3.33 A549 0.45 ± 0.02 51.23 ± 5.13 MCF7 0.46 ± 0.02 91.17 ± 4.93 3.6. Binding Affinity and Interaction Analysis Table S3 summarizes the results of molecular docking analyses of bioactive chemicals identified in the GC-MS profile against Trichoderma protein (Q6A1B7), showing docking scores between − 4.7 and − 6.8 kcal/mol. With a docking score of − 6.8 kcal/mol, N-Benzyloxycarbonyl-L-tyrosine showed the highest binding affinity among the investigated ligands, closely followed by cyclopentadecanol (− 6.7 kcal/mol) and Bis (2-ethyl hexyl) phthalate (− 5.9 kcal/mol). Our docking results suggest that these compounds have the potential to be efficient binding molecules with the Trichoderma protein. Furthermore, derivatives such as octadecanoic acid, n-hexadecanoic acid, and 9-hexadecen-1-ol showed high binding affinities (− 5.4 to 55 kcal/mol), indicating intermediate stability and possible biological significance (Table 4 ). Table 4 Binding affinity scores of 13 compounds against the protein Q6A1B7. Q6A1B7 ( Trichoderma ) S. No. Ligand Details PubChem IDs Binding Affinity Scores 1 Tetradecane 12389 -5.1 2 N -Benzyloxycarbonyl-L-tyrosine 712438 -6.8 3 Octadecane 11635 -5.2 4 Eicosane 8222 -5.3 5 n-Hexadecanoic acid 985 -5.4 6 Octadecanoic acid, 2-(2-hydroxyethoxy) ethyl ester 7788 -5.5 7 n-Heptadecanol-1 15076 -5.2 8 9-Hexadecen-1-ol, (Z)- 5367661 -5.5 9 Cyclopentadecanol 107327 -6.7 10 Octadecanoic acid 5281 -5.5 11 Heneicosane 12403 -4.7 12 Pentadecane, 2-methyl- 15267 -5.2 13 Bis(2-ethylhexyl) phthalate (8343) 8343 -5.9 V-Benzylcarbonyl-L-tyrosine’s 2D interaction map showed several stabilizing interactions with the Q6A1B7 protein. Important hydrogen bonds were generated by key residues such as Asp248, Asn176, Ser111, Thr250, and Arg247 and additional interactions with Glu173, Gln174 and Lys241 improved binding stability (Fig. 6 ). Similarly, as shown in Fig. 6 , cyclopentadecanol mostly interacted with the Q6A1B7 protein via a hydrogen bond with Arg313. 3.7. Post Screening Analysis In initial screening the substances N-benzyloxycarbonyl-L-tyrosine and Cyclopentadecanol were pharmacokinetically purified using the SwissADME platform. N-Benzyloxycarbonyl-L-tyrosine emerges as a safer candidate, with moderate aqueous solubility, favorable drug-likeness, no violations of Lipinski’s rules, and strong gastrointestinal absorption, albeit with limited blood-brain barrier (BBB) permeability and no inhibition of key cytochrome p450 enzymes. The low log P value (~ 1.88) and higher polar surface area (95.86 Å 2 ) indicate limited transmembrane permeability but appropriate lipophilicity. Cyclopentadecanol had better BBB penetration, a smaller polar surface area (20.23 Å 2 ), and higher lipophilicity (Consensus Log P ~ 4.21), indicating a strong potential for CNS activation. Nonetheless, its decreased aqueous solubility and the existence of two lead likeliness violations- namely molecular weight and XLOGP surpassing 3.5 after formulation. 3.8. Anticancer Prediction The pdCSM cancer model indicated that the compound N -Benzyloxycarbonyl-L-tyrosine would be inactive. No discernible selective cytotoxicity against the tested cancer cell lines is shown by the fact that the anticipated probability values (~ 4.0–4.7) fall within a low, stable range. Specifically, all cell lines, including breast, CNS, colon, leukaemia, melanoma, lung, ovarian, prostate, renal, and small-cell lung cancer, have projected activity values that lie within a limited range (~ 4.0–4.7), suggesting no significant or specific cytotoxic effect. Notwithstanding its advantageous physicochemical characteristics (drug-like MW, LogP, and polar surface area), the molecule is ineffective against the cancer cell lines that were evaluated. Overall, the findings support the pdCSM cancer server’s "Inactive" and show limited anticancer potential. Moreover, the compound cyclopentadecanol was found to be active. With higher scores for breast (MDA-MB-468), melanoma (M14, SK-MEL-5), colon (HCT_15), and ovarian (OVCAR-5, OVCAR-8) cell lines, indicating increased activity in these malignancies, its projected values primarily fall between 4.0 and 5.5. ( Fig. 7 ) ( Table 7 ). 4. Discussion The soil fungus Trichoderma lixii generates VOCs that have significant antibacterial, antioxidant and cancer cell cytotoxic properties. Strong inhibition of infectious and cancer cell lines particularly HePG2 was demonstrated by its extracellular metabolites. Promising interactions between VOCs and cancer related targets were found by molecular docking, suggesting possible therapeutic advantages. Despite certain formulation issues, cyclopentadecanol emerged as a crucial compound with anticipated activity across several cancer cells. Colonial and micromorphological features were then examined and compared with the diagnostic descriptions of Sukmawaty et al., [ 35 ]. The query isolate PP754294.1 was firmly placed within the Trichoderma harzianum species complex, exhibiting close affiliation with T.anaharzianum (NR174890.1), T.longicollum (NR 198530.1), T.lixii (NR131264.1), T.atrobrunneum (NR137298.1), all supported by robust bootstrap values exceeding 80%. This phylogenetic grouping indicates a recent common ancestor among these taxa, which supports the isolate’s categorization in the Harzianum clade . The Trichoderma harzianum complex is well known for its ecological adaptability, biocontrol efficacy and bioactive chemical synthesis which highlights isolate PP754294.1’s potential functional importance [ 36 ]. Interestingly, although the genetic distance matrix revealed the smallest sequence difference between PP75429.1 and NR172576.1, phylogenetic reconstruction algorithms-by incorporating substitution patterns across all included taxa rather than relying simply on query sequence similarity-positioned the isolate in close proximity to T. anaharzianum [ 20 ]. Similar discrepancies have been noted in phylogenetic studies of Trichoderma , where distance-based and tree-based methods provide complementary insights into species delimitation [ 37 , 38 ]. This phylogenetic tree was rooted with Pseudomonas polygonicola (NR 154274.1) as the outgroup, which clearly separated from the Trichoderma cluster, establishing a phylogenetic baseline. The placement of isolate PP754294.1 within Harzianum species complex is consistent with prior research that has identified this clade as one of the genus most taxonomically varied and agriculturally relevant lineages. Among the beneficial chemicals discovered is the aromatic volatile 2-phenylethanol. The antibacterial and anti-inflammatory properties of saturated and unsaturated fatty acids, such as stearic, ricinoleic, and palmitic acids have been well investigated; these actions are mostly mediated by membrane disruption [ 39 ]. The reported antibacterial activities may be explained by the fact that long chain alcohols and their monoglyceride derivatives have also been demonstrated to inhibit Staphylococcus speices [ 40 ]. Antagonistic activity against molds and yeasts is probably facilitated by 2-phenylethanol, a well-known antifungal volatile released by a various microbe [ 41 ]. Additionally, fatty acid methyl esters, including methyl stearate, have been linked to nematicidal and larvicidal effects, indicating possible uses in vector-control bioassays if further research is done [ 42 ]. In sum, excluding the likely DEHP artifact, the profile is consistent with a lipid-rich extract whose FFAs, fatty alcohols and aromatic alcohols provide plausible mechanistic bases for the observed antimicrobial/antioxidant activities [ 39 , 43 ]. The GC–MS profile is dominated by free fatty acids (FFAs) and their derivatives (palmitic, stearic, ricinoleic acids; fatty alcohols/esters), a chemical class known to reduce cancer-cell viability by membrane perturbation and lipotoxic apoptosis - mechanisms involving mitochondrial dysfunction and ER stress [ 39 , 44 ]. Minor constituents may also contribute: retinoids (retinal) possess well-documented antiproliferative actions in epithelial cancers via nuclear receptor signalling [ 45 ]. Overall, the cytotoxic pattern - greatest toward hepatoma cells - fits a lipid-rich extract whose FFAs and related lipids plausibly underlie the observed effects; bioassay-guided fractionation and orthogonal confirmation (e.g., LC - MS, standards) are warranted to pinpoint the active principles [ 39 ]. In the present findings the extracellular metabolites from T. lixii demonstrated a strong dose dependent antibacterial activity mainly against gram positive bacillus cereus and gram-negative pathogens P. aeruginosa and V. parahaemolyticus . The TEtOAc outperformed streptomycin at 20 µg against P. aeruginosa (34.5 mm) and V. parahaemolyticus with 32.6 mm. These results are consistent with current research that demonstrated the potent antibacterial efficacy of Trichoderma metabolites against gram negative and positive bacteria. At lower concentrations of 40 µg/ml [ 46 ]. T. harzianum extracts showed high activity against P. aeruginosa and S. aureus whereas another study evidenced diterpenoid compounds from T. harzianum demonstrated a broad-spectrum activity against plant pathogens [ 47 ]. Similarly, Trichoderma virens from ampelopsis japonica roots exhibited significant biofilm inhibition and MIC values of 25 µg/ml against Methicillin resistant S. aureus . The inhibition of our extracellular metabolite was in line with these MIC levels [ 48 ]. Literature review on Trichoderma based metabolites confirms nearly 1000 compounds including gliotoxins, peptaibols, polyketides and volatile organic compounds that are shown to possess antifungal and antibacterial properties [ 12 , 49 ]. Notably TEtOAc was more active against V. parahaemolyticus than streptomycin suggesting that it could be useful against marine borne gram negative pathogens which are a focus that is not often studied in Trichoderma based research. In our patten of research the fungal metabolites showed more effectiveness against gram negative bacteria than the gram positive which is in line with findings from studies using silver nanoparticle mediated T. harzanium which showed gram negative were more vulnerable due to the cell wall structural variations [ 50 ]. According to recent research Trichoderma species have a wide variety of bioactive secondary metabolites possessing free radical scavenging potentials [ 12 , 51 ]. For instance, Trichoderma citriniviride is an endophytic fungus that produced sorbicillinoid derivatives with potent DPPH radical scavenging activity with an IC 50 value between (28 and 90 µM) both of them were potential as ascorbic acid [ 51 ]. Additionally, T. harzianum extracts showed IC 50 in the range of 10–100 µg/ml for DPPH radical [ 52 ]. In comparison to typical crude extracts our Trichoderma lixi i based extracts shows moderate activity with an IC 50 value of 54 µg/ml. Additionally T.harzianum crude metabolites have exhibited enzyme inhibitory and fre radical scavenging properties. The antioxidant potentials tested by ABTS and DPPH tests, its typical for extracts from Trichoderma to exhibit varying potencies. In a recent work performed by Kannan et al, [ 53 ], T. hazrianum outperformed many other taxa with an ABTS IC 50 value of 25 µg/ ml and that of DPPH to be 25 µg/ml [ 53 ]. In addition to directly scavenging radicals, Trichoderma sp. are known to increase the activity of antioxidant enzymes in host organisms. A meta-analysis found that Trichoderma inoculation increases the activity of plant enzymes such as glutathione reductase, ascorbate peroxidase, superoxide dismutase, and catalase, especially under stressful conditions like salinity and heavy metal exposure [ 54 ]. However, simple hydrocacrbons like derivatives of pentadecane, tetradecane and heneicosane showed weaker binding due to their non-polar geometries, suggesting limited interaction capacity. Overall, the results indicate that oxygenated fatty acids, alcohols and aromatic derivatives bind to proteins more effectively than long-chain alkanes [ 31 ]. In 2D interaction pattern other residues (Asn234, Lys241, Ser238, and Tyr314) also contribute through hydrophobic and van der Waals interactions. Because of its lengthy aliphatic chain, its binding is more dependent on the hydrophobic pocket than on the aromatic molecule. Cyclopentadecanol is better suited for CNS-targeted uses despite solubility limitations, whereas N-Benzyloxycarbonyl-L-tyrosine is more druggable and metabolically stable. Both compounds have similar bioavailability scores of ~ 0.55 [ 55 ]. From the anticancer evaluation the findings show that this compound has broad-spectrum anticancer potential, with considerable effects against cancer cell types, including ovarian, colon, breast, and melanoma [ 33 , 34 ]. This work authenticates Trichoderma sp5 (TR5S2) as T. lixii and establishes a soil-to-screen pipeline that links ITS - based phylogeny, GC–MS volatilome profiling, and functional assays. The extracts demonstrated antibacterial, antioxidant and cell line-specific cytotoxic activities are correlated with its lipid rich volatile organic compound (VOC) profile, which includes fatty acids, alkanes, esters, and phthalates. HepG2 cells showed the strongest inhibition (IC 50 value of 29µg/ml). Molecular docking studies confirm interactions with apoptosis-related targets, such as IKKβ, Topoisomerase IIα, β-tubulin, and members of the Bcl-2 protein family. Complementary in silico ADMET tests support the metabolite’s medicinal potential by revealing a generally favorable drug-likeliness profile. Following post-hoc prioritizing, cyclopentadecanol is identified as the leading candidate, owing to its expected multi-target action and blood barrier permeability, despite certain developability restrictions. In contrast, N-benzylcarbonyl-L-tyrosine, despite possessing druggable properties is expected to to inactive across oncological panels. In the future, we will pinpoint active volatiles by bioassay-guided fractionation and rigorous dereplication, then validate apoptosis mechanisms (caspase/PARP/Δψm, tubulin, IKKβ/Topo IIα) and optimize cyclopentadecanol via solubility/PK improvement and SAR. ADME/safety profiling, expanded cell panels and 3D models, plus in vivo tests will de-risk leads. Finally, headspace SPME-GC–MS and genome/BGC mining will map biosynthesis, and combination studies will assess therapeutic synergy. Abbreviations VOCs, volatile organic compounds; ITS, Internal transcribed spacer; ADMET, Absorption Distribution Metabolism Excretion Toxicity; GC-MS, Gas chromatography–mass spectrometry; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid; IC 50 , Half maximal inhibitory concentration. Declarations Acknowledgements This study was supported by the Vietnam Academy of Science and Technology (Project code: CSCL23.01/25-26). Author Contributions Santhosh Sigamani - Research Work, writing–original draft, Investigation, Saranyadevi Subbaraj- In Silico analysis, writing – original draft, Investigation, Pham The Thu - Project administration, writing – review & editing, Hue Thi Nguyen -Data curation, writing – review & editing Nguyen Van Giang -Visualization, Software, Nguyen Huy Thuan- Supervision, Resources and Project administration, Writing – review & editing Ethical Approval As no animals and humans were involved in this study hence ethical approval is not applicable for this research work. Consent to Participate Since this work does not involve studies on human trails and testing of drug on any individual was not performed, is not a case study either so the consent of participation is not required. Consent to Publish All the authors agree to evaluate and publish the study in this South African Journal of Botany. Availability of data and materials The datasets generated during and/or analyzed during the current study are available in the NCBI gene bank repository, [ Accession No: PP754294, https://www.ncbi.nlm.nih.gov/nuccore/PP754294 ] Conflicts of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Kubicek, C.P., Steindorff, A.S., Chenthamara, K., Manganiello, G., Henrissat, B., Zhang, J., Cai, F., Kopchinskiy, A.G., Kubicek, E.M., Kuo, A., Baroncelli, R., Sarrocco, S., Noronha, E.F., Vannacci, G., Shen, Q., Grigoriev, I.V., Druzhinina, I.S.: Evolution and comparative genomics of the most common Trichoderma species. BMC Genom. 20 , 485 (2019). https://doi.org/10.1186/s12864-019-5680-7 Harman, G.E., Howell, C.R., Viterbo, A., Chet, I., Lorito, M.: Trichoderma species — opportunistic, avirulent plant symbionts. Nat. Rev. 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Biomolecules. 13 , 194 (2023). https://doi.org/10.3390/biom13020194 Supplementary Files SupplementaryinformationWABVNew.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 05 Apr, 2026 Reviewers invited by journal 05 Apr, 2026 Editor invited by journal 29 Mar, 2026 Editor assigned by journal 04 Mar, 2026 First submitted to journal 04 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Thuan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIiWNgGAWjYPCCA0DMA2LYMDYwwNgEtbCBlaUx9pCq5TBhLeb8a8wkPu64k7jhfu8xiZ87zsvul0hgfPC2jSFxOw4tljPemEnOPPMsccMxvjTJ3jO3jXskEpgN5wK17GzArsXgxhkzad62w0AtPGYSvG23E4Fa2IAiDMYGB/Bo+QvVIvm37RxIC/tvvFrO95hJM0K1AA0/ALaFGahFDrctbMWWvW2HjWceyzG2lm1LNu4587BZcs45Cdxazh/eeONn22HZvsNnDG++bbOTbW9PPvjhTZkNDy4tDBIJLBIgWgGhAJwAJHCoBwL+A8wfQLR8A241o2AUjIJRMMIBALZ7Y4y87hpvAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-8891-7195","institution":"Duy Tan University: Dai Hoc Duy Tan","correspondingAuthor":true,"prefix":"","firstName":"Nguyen","middleName":"Huy","lastName":"Thuan","suffix":""},{"id":617740692,"identity":"6af58909-c500-4af2-a671-6021a6bb5c2f","order_by":1,"name":"Santhosh Sigamani","email":"","orcid":"","institution":"Duy Tan University: Dai Hoc Duy Tan","correspondingAuthor":false,"prefix":"","firstName":"Santhosh","middleName":"","lastName":"Sigamani","suffix":""},{"id":617740693,"identity":"0c8e5225-f356-488e-b67f-88bf7d5eb6f5","order_by":2,"name":"Saranyadevi Subburaj","email":"","orcid":"","institution":"Karpagam University: Karpagam Academy of Higher Education","correspondingAuthor":false,"prefix":"","firstName":"Saranyadevi","middleName":"","lastName":"Subburaj","suffix":""},{"id":617740694,"identity":"5eeeb9f2-8585-41f0-9df6-ca24a8e22547","order_by":3,"name":"Hue Thi Nguyen","email":"","orcid":"","institution":"Hanoi Open University","correspondingAuthor":false,"prefix":"","firstName":"Hue","middleName":"Thi","lastName":"Nguyen","suffix":""},{"id":617740695,"identity":"1f15424a-0580-4ada-927f-71b9c66afd0f","order_by":4,"name":"Nguyen Van Giang","email":"","orcid":"","institution":"Vietnam National University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Nguyen","middleName":"Van","lastName":"Giang","suffix":""}],"badges":[],"createdAt":"2026-03-04 15:34:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9031969/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9031969/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106519287,"identity":"bddb89f5-be91-47bb-a1ea-fe441a40f602","added_by":"auto","created_at":"2026-04-09 12:33:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":631246,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological characteristics and culture of\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eTrichoderma lixii\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e(TR5).\u003cstrong\u003e\u003cbr\u003e\n (A)\u003c/strong\u003ecolony morphology \u003cstrong\u003e(B)\u003c/strong\u003e Conidia and phialides, \u003cstrong\u003e(C)\u003c/strong\u003e Submerged culture of \u003cem\u003eT. lixii\u003c/em\u003e \u003cstrong\u003e(D)\u003c/strong\u003e Extracted metabolites of \u003cem\u003eT. lixii\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9031969/v1/4bdd139d741396259ec35c14.png"},{"id":106725024,"identity":"72505cab-5102-458e-a1d0-af2ff75cf160","added_by":"auto","created_at":"2026-04-12 18:31:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":116689,"visible":true,"origin":"","legend":"\u003cp\u003eNeighbor-joining phylogram showing the isolate PP754294.1\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9031969/v1/3c7d25893016a7ddae86438c.png"},{"id":106726014,"identity":"8ae640f4-1a6f-4d43-a053-e4ac005a13ee","added_by":"auto","created_at":"2026-04-12 18:34:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":162306,"visible":true,"origin":"","legend":"\u003cp\u003eGC–MS spectrum of the \u003cem\u003eTrichoderma lixii\u003c/em\u003e extracellular metabolites.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9031969/v1/b177f183ac37ef6cb82b40de.png"},{"id":106724571,"identity":"5515bba6-4691-482a-888b-e9e748a6771a","added_by":"auto","created_at":"2026-04-12 18:28:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":768165,"visible":true,"origin":"","legend":"\u003cp\u003eAntibacterial activity of TEtOAc against selected bacterial pathogens \u003cstrong\u003ea)\u003c/strong\u003e \u003cem\u003eBacillus cereus\u003c/em\u003e, \u003cstrong\u003eb)\u003c/strong\u003e \u003cem\u003eBacillus licheniformis\u003c/em\u003e \u003cstrong\u003ec)\u003c/strong\u003e \u003cem\u003eStaphylococcus aureus\u003c/em\u003e \u003cstrong\u003ed)\u003c/strong\u003e \u003cem\u003eEnterococcus faecalis\u003c/em\u003e \u003cstrong\u003ee)\u003c/strong\u003e \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e \u003cstrong\u003ef)\u003c/strong\u003e \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e \u003cstrong\u003eg)\u003c/strong\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9031969/v1/5dbd569302e4be45667d4c2d.png"},{"id":106519288,"identity":"f1a23176-1852-4156-acd3-81b8293fd2a0","added_by":"auto","created_at":"2026-04-09 12:33:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":97858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea) \u003c/strong\u003eFree radical scavenging profile of TEtOAc \u003cstrong\u003eb)\u003c/strong\u003e Cytotoxic potential evaluation of TEtOAcagainst cancer cell lines\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9031969/v1/3a16e8cf7b3edc104ed2885f.png"},{"id":106724799,"identity":"72c442de-4011-4ab3-8bcc-c25a03be25b6","added_by":"auto","created_at":"2026-04-12 18:29:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":335261,"visible":true,"origin":"","legend":"\u003cp\u003e2D interaction pattern of Q6A1B7 protein with \u003cstrong\u003ea)\u003c/strong\u003e N-Benzyloxycarbonyl-L-tyrosine \u003cstrong\u003eb)\u003c/strong\u003e Cyclopentadecanol\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9031969/v1/ab3335e40bbfb5f0b84c5c61.png"},{"id":106519290,"identity":"b140eb91-6de9-4b14-94c2-d1fd86edbc2f","added_by":"auto","created_at":"2026-04-09 12:33:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":142916,"visible":true,"origin":"","legend":"\u003cp\u003ePharmacokinetic study of \u003cstrong\u003ea)\u003c/strong\u003e \u003cem\u003eN\u003c/em\u003e-Benzyloxycarbonyl-L-tyrosine \u003cstrong\u003eb)\u003c/strong\u003e Cyclopentadecanol.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9031969/v1/0f095820c245fc3630d24589.png"},{"id":106727569,"identity":"bd0f6886-bf71-43b7-bc9c-44d0d4c649ec","added_by":"auto","created_at":"2026-04-12 18:39:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3982640,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9031969/v1/1653f142-c59c-4985-a631-b5c3a345cc6a.pdf"},{"id":106724806,"identity":"f1d5b8c5-1078-430d-80ad-5fa6f78b00ce","added_by":"auto","created_at":"2026-04-12 18:29:53","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":74532,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationWABVNew.docx","url":"https://assets-eu.researchsquare.com/files/rs-9031969/v1/285a2c92dcfcbc9eaf7b6235.docx"}],"financialInterests":"","formattedTitle":"Volatilome Profiling and Apoptosis-sensitizing potential of secondary metabolites from Trichoderma lixii: Integrating GC–MS, Bioassays, and In Silico Docking Approaches","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn agricultural and forest ecosystems around the world, filamentous fungus belonging to genus Trichoderma (Ascomycota, order Hypocreales, family hypocreaceae) are common soil residents [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Fast growing septate hyphae, conidiphores with phialides, and green to whitish conidia that promote quick substrate colonization are their distinguishing features [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In terms of ecology, Trichoderma species are opportunistic mycoparasites and decomposers that actively oppose phytopathogens through competition for nutrients and space, secondary metabolites, and enzyme degradation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Due to these characteristics, they are used extensively in agriculture as soil conditioners, plant growth promoters and biocontrol agents. They are also used as industrial manufacturers of cellulases, chitinases and proteases [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition to their agricultural and ecological functions, Trichoderma species are abundant chemical factories that can produce a wide range of natural products, such as terpenoids, peptaibols, polyketides and volatile organic compounds (VOCs) with antifungal, antibacterial and anticancer properties [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Their volatile organic compounds (VOCs), which diffuse via air or liquid headspace and have been linked to both inter-microbial interactions and mammalian cytotoxicity are particularly interesting. These compounds include low molecular weight fatty acids, alkanes, lactones and terpenes. By supplying polyunsaturated fatty acids and phthalate derivatives with documented pro-apoptotic effects, cyanobacteria further enhance this chemical repertory [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The term \u0026ldquo;cancer\u0026rdquo; is used to describe a wide range of illness, each of which has a unique set of treatment options. Drug resistance, ineffectiveness, relapses, and undesirable side effects continue to be problems despite significant advancements in therapy and a variety of available treatment options [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Despite being widely used, chemotherapy is often linked to a number of adverse effects, such as hematological changes and toxicity to organs such as kidneys, liver, heart, and nervous system [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Personalized cancer treatment through precision medicine seeks to overcome these issues by personalizing medicines to individual patients, yet it remains one of the most serious challenges in modern healthcare. The development of new therapeutic drugs, as well as the investigation of prospective drug combinations that could improve treatment efficacy, are examples of progress in this field. Fungal species are an excellent potential source for finding new anticancer compounds due to their high diversity and variety of environments [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Trichoderma species have drawn particular interest [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and fungi have been acknowledged as a valuable natural source of bioactive compounds with medicinal potential [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Secondary metabolites exhibiting a range of bioactivities, such as antibacterial, immunomodulatory and anticancer properties have been generated from extracts of several Trichoderma species. Paracelsin, L-lysine α-oxidase and Trichodermin are a few examples of these compounds [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The potential of volatile chemicals produced by fungi, bacteria and plants as sources for the creation of new anticancer drugs is becoming increasingly apparent. A few essential oils including Terpenoid and phenylpyranoid-rich have considerable seasonal oscillations; Nepalese Ocimum species show compositional variations that greatly impact their bioactive qualities, especially cytotoxic effects [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The plant such as rose, eucalyptus, lemon, and clove have been confirmed for biological effects on neural cells apart from their well-established aromatic benefits, with wider therapeutic potential [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBeyond plants, volatile secondary metabolites of microbial origin, such as those produced by \u003cem\u003eTrichoderma\u003c/em\u003e spp., include peptaibols, terpenoids, and polyketides with demonstrated antimicrobial and anticancer activities [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The pro-apoptotic properties of plant volatiles are further exemplified by \u003cem\u003eWarburgia salutaris\u003c/em\u003e leaf extracts, which trigger apoptosis in MCF-7 breast cancer cells via caspase-dependent pathways [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. At the microbial level, small volatile molecules function as chemical signals but also act as cytotoxins capable of perturbing mitochondrial integrity and redox balance in cancer cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Collectively, these findings highlight the chemical diversity of volatile compounds across biological kingdoms and their significant promise as anticancer agents. The present study interrogates the volatilome profiling of a newly isolated \u003cem\u003eTrichoderma\u003c/em\u003e sp5 (TR5S2). GC - MS revealed twelve dominant compounds, including fatty acids, alkanes, esters, and phthalates. We evaluated the extracts for antibacterial, antioxidant, and cytotoxic activities against KB, HepG2, A549, and MCF-7 cells, complemented by in silico docking of each compound to apoptotic regulators (IKKβ, topoisomerase IIα, β-tubulin, and Bcl-2 family proteins) and ADMET predictions. By integrating chemical profiling, docking, and functional assays, this work seeks to identify \u003cem\u003eTrichoderma\u003c/em\u003e-derived VOCs with apoptosis-sensitizing potential and pharmacokinetic properties suitable for further optimization as anticancer leads.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Morphological identification of the fungi\u003c/h2\u003e \u003cp\u003eThe fungi sample was collected from Long Bien Ward, Hanoi city, Viet Nam and was morphologically identified as \u003cem\u003eTrichoderma\u003c/em\u003e sp. It was cultured in potato dextrose agar in petri plates and after the growth appeared as a mat on petri plate a single hypha was picked and placed on microscopic slide. Morphological features were examined under an Olympus CX23 light microscope using 40 \u0026times; and 100 \u0026times;. The characteristics of the fungi was recorded for the morphological identification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Molecular Identification of isolated fungi\u003c/h2\u003e \u003cp\u003eThe genomic DNA of \u003cem\u003eTrichoderma\u003c/em\u003e sp5 was extracted by Vazyme\u0026trade; Bacterial DNA Kit (Vazyme Biotech, Nanjing, China). Then its partial sequence was amplified with ITS primers forward and reverse and the PCR products were sent for gene sequencing by 1st Biobase (Malaysia). DNA quality was assessed on a 0.8% agarose gel and quantified with a NanoDrop 2000 (Thermo Scientific) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe gene sequence was hit on NCBI database by nucleotide BLAST and it most identical sequences were chosen and it was submitted to NCBI for accession number. The phylogenetic tree was constructed from the most similar sequences to identify the family and genus that closely relates by using MEGA12 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Cultivation, chemical extraction and GC-MS Profiling\u003c/h2\u003e \u003cp\u003eThe isolated fungal strain was then grown on potato dextrose agar broth for 10\u0026ndash;12 days in shaking condition at 22℃. After incubation time the filtrate was collected by passing the culture media into the blotting paper leaving the mycelium on top. This filtrate was then exposed to ethyl acetate in the ratio of 1:2 of the solvent. This was then kept on shaker for 24 hrs for the extraction in room temperature. The ethyl acetate extracted \u003cem\u003eTrichoderma\u003c/em\u003e sp5 filtrate (TEtOAc) was then poured into separating funnel and its organic layer was collected firmly into a beaker. This \u003cem\u003eTrichoderma\u003c/em\u003e extract was then evaporated in rotary evaporator with vacuum and mild heat 35 ℃ to avoid leakage of metabolites during the concentration process. The fully concentrated TEtOAc sample was then distributed to glass vials and kept in 4℃ for further analysis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConfrontation assays were incubated at 21\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C in darkness for 5 days. A PDMS/DVB solid-phase microextraction (SPME) fiber (Supelco, Bellefonte, PA, USA) was used to collect volatile organic compounds (VOCs), which were then desorbed at 180℃ for 30 seconds inside the injection port of an Agilent 7890 B gas chromatograph interfaced with a 5973-mass spectrometry detector. An HP-FFAP capillary column (30m \u0026times; 0.25 mm, 0.25 \u0026micro;m film thickness) with helium as carrier gas at a flow rate of 1 ml per minute was used to accomplish chromatographic separation. The oven program was 40\u0026deg;C (5 min), ramped 3\u0026deg;C/min to 220\u0026deg;C (5 min), and followed by 300\u0026deg;C (3 min). Compounds were identified using the NIST/EPA/NIH Mass Spectral Database (version 11) and ChemStation (Agilent, Rev. D.04.00). Each treatment was analyzed in triplicate [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Antibacterial action of fugal extracellular metabolites\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of the fungal extracellular metabolites was performed by well diffusion method [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Seven bacterial pathogens were spread on Muller Hinton agar plates using sterile swabs to create a bacterial lawn. Wells were made with 6 mm diameter using sterile cork borers. For the experimental tests, the TEtOAc stock solution (10 mg/ml) was made in 10% DMSO in amounts of 100, 150 and 200 \u0026micro;g/ well respectively. The positive control was streptomycin (10\u0026micro;l of 1 mg/ml stock solution) and the negative control was 10% DMSO. In order to assess antibacterial activity, the zones of inhibition surrounding each well were determined after the plates were incubated at 37℃ for whole day [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. MIC of the fungal metabolites\u003c/h2\u003e \u003cp\u003eNutrient broth was prepared and aliquoted into test tubes and closed the mouth with cotton plugs. These tubes were autoclaved at 121℃ for 20 mins at 15 lbs pressure and allowed to cool down gradually. After the media gets cooled down varying concentration of the positive control-streptomycin (syringe filtered) and \u003cem\u003eTrichoderma lixii\u003c/em\u003e based extracellular metabolites viz., 7.8, 15.6, 31.2, 62.5, 125 and 250 mg/ml were added to all the tubes aseptically in biosafety cabinet. The tubes were then inoculated with 12 hrs grown bacterial pathogens and kept for 24 hrs incubation. After incubation the growth of the bacteria was observed visually and recorded for determining the MIC in UV spectrophotometer [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Free radical scavenging Assay\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1. DPPH assay\u003c/h2\u003e \u003cp\u003e0.1 mM DPPH reagent was prepared and used it for the antioxidant assays. To 7.64 mg DPPH Reagent 100 ml of Ethanol analytical grade was added. Followed by these dilutions of the ascorbic acid were made and 2.0 ml of ethanol was added to it. Ascorbic acid stock at 1mg/ml concentration was prepared and used as standard. The stock was diluted to varying concentration of 5, 10, 15, 20 and 25 \u0026micro;g/ml. In the reaction tubes 1.0 ml of DPPH reagent was added and kept for incubation in dark room for 30 mins [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2. ABTS assay\u003c/h2\u003e \u003cp\u003eABTS reagent was prepared by mixing (1:1) 7 mM ABTS and 2.45 mM potassium persulphate in amber bottle. To 38.4 mg of ABTS, 10 ml of 6.6 mg Potassium per sulphate was added in 10 ml water and mixed both the reagents in the ratio of 1:1. The solution was kept for 16 hrs in room temperature and 1.0 ml of the reaction was mixed with 27 ml of distilled water. Its O.D was checked and it was diluted further till the O.D reached 0.7 that was read by spectrophotometer at 734 nm. Upon reaching the desired O.D the samples and control were taken for analysis. Dilutions were made with ascorbic acid (10, 20, 25 and 30 \u0026micro;g/ml) by adding water making the total volume to 2.0 ml. Later, 1.0 ml of ABTS reagent was added to each reaction tubes and keep for incubation 30 mins in dark room [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Anticancer potential evaluation\u003c/h2\u003e \u003cp\u003eThe MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] test, which was first developed by Mosmann [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and then refined by research, which was used to evaluate th cytotoxic potential of the crude extract [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Briefly, complete culture media was used to seed human cancer cell lines (KB, HepG2, A549 and MCF-7) onto 96 well plates at densities ranging from 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e to 1\u0026times;10\u003csup\u003e1\u003c/sup\u003e cells per well, and the cells were allowed to adhere overnight. The cells were then exposed to serial dilutions of the crude extract of \u003cem\u003eTrichoderma\u003c/em\u003e sp. (0-400 \u0026micro;g/ml) for 24 to 48 hrs at standard incubation conditions (37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eFollowing incubation, each well received 20 \u0026micro;l of MTT solution (5 mg/ml in PBS) and was incubated for three to four hours to allow metabolically active cells to decrease MTT dye into insoluble formazan crystals. After the medium was thoroughly aspirated, 100 \u0026micro;l of DMSO was used to dissolve the formazan crystals. A microplate reader was used to measure absorbance ar 570 nm, with 630 nm acting as reference wavelength. Cell viability percentages were computed in relation to untreated controls and non-linear regression analysis was used to create dose-response curves that yielded half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values. A quantitative measure of cytotoxic efficacy is provided by the IC\u003csub\u003e50\u003c/sub\u003e values obtained for each cell line, which indicates the extract concentration required to block 50% of cell viability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Virtual Screening and Docking\u003c/h2\u003e \u003cp\u003eFurther, the docking was performed for the lead compounds towards \u003cem\u003eTrichoderma\u003c/em\u003e with a UniProt ID of Q6A1B7 using the PyRx server. Initially, the ligands' PubChem IDs were imported into the PyRx (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and it was prepared by creating 3D structures, adding hydrogens using OpenBabel software to minimize the ligand energy. Subsequently, the protein was pre-processed by removing the water molecules, het atoms, and other undesirable components, by adding polar hydrogens and charges. The search space is then established by defining a grid box around the active site of a protein. AutoDock Vina, which is built into the PyRx tool, was then employed for the docking study. It predicts the best binding poses and ranks them according to binding affinity (kcal/mol). To determine which \u003cem\u003eTrichoderma\u003c/em\u003e metabolites exhibit the strongest interaction with the target protein, the best docking conformations are examined for hydrogen bonding, hydrophobic interactions, and orientation within the pocket. The screened compound was further visualized in Discovery Studio [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibacterial activity of the \u003cem\u003eTrichoderma lixii\u003c/em\u003e extracellular metabolites\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS. No\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eName of the bacterial pathogen\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100 \u0026micro;g\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e150 \u0026micro;g\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e200 \u0026micro;g\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eStreptomycin 20 \u0026micro;g\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eDMSO (10%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBacillus cereus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e20.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e22.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e27.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e23.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBacillus Licheniformis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e16.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e20.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e35.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e17.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e19.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e28.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eEnterococcus faecalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e15.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e19.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e21.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e27.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e25.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e30.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e32.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e11.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e18.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e23.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e31.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e24.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e30.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e34.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e29.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Pharmacokinetic Study\u003c/h2\u003e \u003cp\u003eThe SwissADME web-based server was used to assess the pharmacokinetic and drug-likeness characteristics of N-Benzyloxycarbonyl-L-tyrosine and Cyclopentadecanol. Individual compound\u0026rsquo;s canonical SMILES strings were obtained from the PubChem database and then uploaded to SWISSADME website. Pharmacokinetic profiles, aqueous solubility, lipophilicity, physiochemical features and drug likeliness criteria were among the important aspects that were carefully assessed. In order to provide a comprehensive in silico evaluation of the compounds, prediction analyses using the bioavailability radar, BOILED-Egg model, and structural warning systems (PAINS and Brenk) were carried out to evaluate adsorption, membrane permeability and potential structural liabilities. Subsequently, the pdCSM-cancer model was utilized to assess the anticancer potential of \u003cem\u003eN\u003c/em\u003e-Benzyloxycarbonyl \u0026ndash; L - tyrosine and cyclopentadecanol. The tool provided likelihood ratings and corresponding confidence levels after predicting their growth inhibition efficacy across several tumor cell lines and submitting the results to the server. The findings showed different patterns of action, which made it possible to compare the two drugs' efficacy and selectivity. In cancer research, this process offers a preliminary computer screening phase to rank these compounds for additional in vitro and in vivo confirmation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll the experiments conducted in this research were performed in triplicate and its mean and standard values were calculated. Graph pad prism 8.0 was used for plotting the antioxidant and antibacterial assays.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Morphological description \u003cem\u003eTrichoderma\u003c/em\u003e sample\u003c/h2\u003e \u003cp\u003eSoil samples were serially diluted and plated onto agar PDA medium. After 5 days of incubation, discrete green to yellow-green colonies were selected and subcultured onto fresh PDA to obtain pure isolates. On PDA, colonies of the newly isolated \u003cem\u003eTrichoderma\u003c/em\u003e strains were initially white, slightly floccose to finely velvety. With continued incubation, the colonies gradually turned light green and then deepened in colour, producing abundant branched conidiophores; hyphae rapidly covered the entire plate surface within 3\u0026ndash;4 days. Conidia were ovoid with smooth walls. Colony morphology: initially white, velvety to cottony; after 4 days of incubation the colony turns green. The colony reverse is pale yellow. Hyphae branched. Conidia ovoid, smooth-walled. Conidiophores bear phialides arranged symmetrically (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Phylogenetic of \u003cem\u003eTrichoderma\u003c/em\u003e sp5\u003c/h2\u003e \u003cp\u003eThe 608 base pair ITS sequence of the fungus strain \u003cem\u003eTrichodrma\u003c/em\u003e sp., is stored in Genebank with accession number \u003cb\u003ePP754294\u003c/b\u003e. With a 1000 bootstrap replicates and values more than 50% annotated at the nodes, the neighbor joining technique was used to create the phylogenetic tree. The query sequence was placed solidly within the Harzianum species complex by clustering with Trichoderma anaharzianum (NR174890.1), T.longicollom (NR198530.1), T. lixii (NR131264.1.), T.atrobrunneum (NR137298.1) and T.azevedoi (NR 173287.1). \u003cem\u003eT.Simmonsi, T.achlamydosporum, T.longifilidicum, T.afarasin\u003c/em\u003e, and \u003cem\u003eT.velutinum\u003c/em\u003e were other closely related taxa. \u003cem\u003eT. stromaticum, T.solum, T.hainanense\u003c/em\u003e and \u003cem\u003eT. cremeum\u003c/em\u003e showed distinct monophyletic clusters, while \u003cem\u003eT. ghanense\u003c/em\u003e and \u003cem\u003eT. pseudokoningi\u003c/em\u003e formed a well-supported subclade next to T. protrudens. The outgroup was \u003cem\u003ePseudocoleophoma polygonicola\u003c/em\u003e (NR 154274.1). The number of nucleotide changes per site is indicated by scale bar (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The evolutionary position of the query isolate \u003cb\u003ePP54294.1 (\u003c/b\u003e\u003cb\u003eTrichoderma\u003c/b\u003e \u003cb\u003esp5)\u003c/b\u003e within genus Trichoderma is clarified by the phylogenetic analysis based on ITS sequences. \u003cem\u003eT. anaharzianum\u003c/em\u003e (NR 174890.1), \u003cem\u003eT. longicollum\u003c/em\u003e (NR 198520.1), \u003cem\u003eT. lixii\u003c/em\u003e (NR13126.1), \u003cem\u003eT. batrobrunneum\u003c/em\u003e (NR137298.1) and \u003cem\u003eT. azevedoi\u003c/em\u003e (NR 173287.1) were among the members of Harzianum species complex with which the isolate showed good bootstrap support (82\u0026ndash;87%). The isolate\u0026rsquo;s association with the Harzianum clade, a lineage known for its biocontrol powers, enzymatic activity, and ecological adaptability is supported by this close phylogenetic link.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3. GC-MS profiling of \u003cem\u003eTrichoderma lixii\u003c/em\u003e based metabolites\u003c/h2\u003e \u003cp\u003eThe ethyl acetate (TEtOAc) extract of the \u003cem\u003eTrichoderma\u003c/em\u003e isolate designated \u003cem\u003eT. lixxi\u003c/em\u003e was profiled for volatile and semi-volatile constituents by gas chromatography\u0026ndash;mass spectrometry (GC\u0026ndash;MS). The results of volatile compounds are summarized in the table below (\u003cb\u003eFig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe GC\u0026ndash;MS profile is strongly lipidic, dominated by long-chain fatty acids/derivatives and hydrocarbon\u0026ndash;alcohol fractions. Major constituents include n-hexadecanoic (palmitic) acid (16.91%) and octadecanoic (stearic) acid (8.45%), alongside ricinoleic acid, several fatty alcohols (e.g., 1-octadecanol; n-heptadecanol-1; 9-/11-hexadecen-1-ol), n-alkanes (C12 \u0026ndash; C20; octadecane and eicosane\u0026thinsp;\u0026asymp;\u0026thinsp;5.3% each), and esters such as methyl stearate and isopropyl palmitate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e) (\u003cb\u003eTable S2\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.3. Antibacterial potential of TEtOAc\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eUsing the agar diffusion experiment, the antibacterial effectiveness of the TEtOAc extract was assessed against seven pathogenic bacterial strains at three different concentrations (100, 150 and 200 \u0026micro;g). The positive control streptomycin (20\u0026micro;g) whereas thew negative control was 10% DMSO. The mean and standard deviation of assays carried out in duplicate. With a strong zone of inhibition extending 34.5mm, the extract showed the most inhibitory action against \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e at the maximum dosage of 200\u0026micro;g. \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e (32.6mm), \u003cem\u003eBacillus cereus\u003c/em\u003e (27.4 mm), \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e (23.4mm), \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (21.5mm), \u003cem\u003eStaphylocoocus aureus\u003c/em\u003e (19.6 mm), and Bacillus licheniformis (20.5mm). Notably, the inhibitory zones at the maximum dose (200 \u0026micro;g) were similar to those generated by Streptomycin control for a number of pathogens including \u003cem\u003eB. cereus, P.aeruginosa, and V.parahaemolyticus.\u003c/em\u003e The negative control showed no signs of inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As the extract efficacy increases from 100 to 200 \u0026micro;g, a dose dependent increase in activity was statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for the tested bacterial pathogens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4. MIC of TEtOAc for bacterial pathogens\u003c/h2\u003e \u003cp\u003eThe extracellular metabolites of the fungi \u003cem\u003eT. lixii\u003c/em\u003e showed minimum inhibitory concentration (MIC) against a range of bacterial pathogens were carefully assessed and compared to those of streptomycin. The antibacterial potency of tested strains varied as indicated by MIC values which were expressed in \u0026micro;g/ml. With MIC values of 15.6 \u0026micro;g/ml for few strains TEtOAc demonstrated strong bacteriostatic potential against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e and \u003cem\u003eB. cereus\u003c/em\u003e. Accordingly, \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eK. pneumonia\u003c/em\u003e had MIC values of 15.6 and 62.5 respectively indicating that they were moderately susceptible. Alternatively, \u003cem\u003eB. licheniformis, S. aureus\u003c/em\u003e and \u003cem\u003eE. faecalis\u003c/em\u003e showed greater resistance to with MIC values above 62.5 \u0026micro;g/ml or below the quantification threshold more than 62.5 \u0026micro;g/ml, indicating a different spectrum of activity. Streptomycin in contrast showed better efficacy with MIC values that were generally lower specifically 7.8 \u0026micro;g/ml against majority of strains with the exception of \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eV. parahaemolyticus\u003c/em\u003e where MICs were slightly higher at 15.6 \u0026micro;g/ml. This demonstrates the strong properties of streptomycin as a standard positive control. This study involving antibacterial efficacy of TEtOAc are consistent with earlier research highlighting the antimicrobial potential of fungal secondary metabolites (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMIC values of the bacterial pathogens tested for \u003cem\u003eTrichoderma lixii\u003c/em\u003e extracts\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSamples Tested\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c8\" namest=\"c2\"\u003e \u003cp\u003eMIC values of the tested bacteria in \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eB. cereus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eB. licheniformis\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eE. faecalis\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eV. parahaemolyticus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP. aeruginosa\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTEtOAc\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;62.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;62.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;62.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e15.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e62.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e15.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStreptomycin\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e15.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Antioxidant profile of TEtOAc by free radicals\u003c/h2\u003e \u003cp\u003eThe antioxidant potential of TEtOAc was evaluated by measuring its ability to scavenge free radicals using the DPPH assay. We tested various concentrations of TEtOAc to find their IC\u003csub\u003e50\u003c/sub\u003e values and percent inhibition. The results showed a significant concentration dependent increase in radical scavenging activity. Ascorbic acid was used as standard for comparison, it showed a calibration curve with an R\u003csup\u003e2\u003c/sup\u003e value of 0.9402 and achieved a 99% scavenging at 20\u0026ndash;25 \u0026micro;g indicating exceptional potential. TEtOAc showed enhanced free radical inhibition. In the lowest concentration tested (100\u0026micro;g) we achieved 73.9% inhibition, while the highest concentration reached 99.8%. We calculated the IC\u003csub\u003e50\u003c/sub\u003e value for TEtOAc to be 54.2 \u0026micro;g/ml highlighting its prominent antioxidant capacity.\u003c/p\u003e \u003cp\u003eIn the ABTS radical scavenging test, we assessed the TEtOAc at various concentrations using ascorbic acid as a standard reference. Excellent linearity was demonstrated by the ascorbic acid calibration curve\u0026rsquo;s strong correlation coefficient (R2\u0026thinsp;=\u0026thinsp;0.98). The IC50 values of TEtOAc metabolite was 149.4 \u0026micro;g/ml. The extract showed a scavenging activity of 41.03% at the lowest tested concentration and an 82.16% suppression of ABTS free radical at highest concentration. The standard ascorbic acid reached\u0026thinsp;\u0026gt;\u0026thinsp;90% scavenging at 30 \u0026micro;g whereas extract at 400 \u0026micro;g/ ml showed 82% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The scavenging percentage was plotted against extract concentration (0\u0026ndash;400 \u0026micro;g/mL) for DPPH (blue squares) and ABTS (olive circles). To determine the 50% inhibitory concentration a non-linear regression analysis was employed. When comparing ABTS radical with an IC\u003csub\u003e50\u003c/sub\u003e value of 149.4\u0026micro;g/ml a log IC\u003csub\u003e50\u003c/sub\u003e value of 2.17 and a R\u003csup\u003e2\u003c/sup\u003e value of 0.98 was achieved, the TEtOAc demonstrated superior efficacy against DPPH free radicals with an IC\u003csub\u003e50\u003c/sub\u003e value of 57\u0026micro;g/ml (log IC\u003csub\u003e50\u003c/sub\u003e-1.75; R\u003csup\u003e2\u003c/sup\u003e value of 0.99). This difference highlights the metabolites\u0026rsquo; superior ability to scavenge DPPH radicals via electron or hydrogen atom donation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Cell cytotoxicity of TEtOAc against cancer cell lines\u003c/h2\u003e \u003cp\u003eFour cancer cell lines were treated with the TEtOAc at varying concentrations and their effect on inhibition was analyzed. The TEtOAc extract of \u003cem\u003eTrichoderma lixii\u003c/em\u003e shows moderate, cell-line\u0026ndash;dependent cytotoxicity: HepG2 is the most sensitive (IC₅₀ = 29.01\u0026thinsp;\u0026plusmn;\u0026thinsp;3.33 \u0026micro;g mL⁻\u0026sup1;), whereas KB and A549 are intermediate (~\u0026thinsp;51\u0026ndash;55 \u0026micro;g mL⁻\u0026sup1;) and MCF-7 is weakly affected (91.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9 \u0026micro;g mL⁻\u0026sup1;) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e values of \u003cem\u003eTrichoderma lixii\u003c/em\u003e ethyl acetate extract against different human cancer cell lines versus\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTumor cell line\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMeans of IC\u003csub\u003e50\u003c/sub\u003e values (\u0026micro;g/mL)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEllipticine\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eTricoderma lixii\u003c/em\u003e EtOA extract\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e54.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHepG2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e29.01\u0026thinsp;\u0026plusmn;\u0026thinsp;3.33\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA549\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e51.23\u0026thinsp;\u0026plusmn;\u0026thinsp;5.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCF7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e91.17\u0026thinsp;\u0026plusmn;\u0026thinsp;4.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Binding Affinity and Interaction Analysis\u003c/h2\u003e \u003cp\u003e \u003cb\u003eTable S3\u003c/b\u003e summarizes the results of molecular docking analyses of bioactive chemicals identified in the GC-MS profile against Trichoderma protein (Q6A1B7), showing docking scores between \u0026minus;\u0026thinsp;4.7 and \u0026minus;\u0026thinsp;6.8 kcal/mol. With a docking score of \u0026minus;\u0026thinsp;6.8 kcal/mol, N-Benzyloxycarbonyl-L-tyrosine showed the highest binding affinity among the investigated ligands, closely followed by cyclopentadecanol (\u0026minus;\u0026thinsp;6.7 kcal/mol) and Bis (2-ethyl hexyl) phthalate (\u0026minus;\u0026thinsp;5.9 kcal/mol). Our docking results suggest that these compounds have the potential to be efficient binding molecules with the \u003cem\u003eTrichoderma\u003c/em\u003e protein. Furthermore, derivatives such as octadecanoic acid, n-hexadecanoic acid, and 9-hexadecen-1-ol showed high binding affinities (\u0026minus;\u0026thinsp;5.4 to 55 kcal/mol), indicating intermediate stability and possible biological significance (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBinding affinity scores of 13 compounds against the protein Q6A1B7.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eQ6A1B7 (\u003cem\u003eTrichoderma\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS. No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLigand Details\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePubChem IDs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBinding Affinity Scores\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTetradecane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-5.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eN\u003c/em\u003e-Benzyloxycarbonyl-L-tyrosine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e712438\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-6.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOctadecane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11635\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-5.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEicosane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-5.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en-Hexadecanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e985\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-5.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOctadecanoic acid, 2-(2-hydroxyethoxy) ethyl ester\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7788\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-5.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en-Heptadecanol-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15076\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-5.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9-Hexadecen-1-ol, (Z)-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5367661\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-5.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCyclopentadecanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e107327\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-6.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOctadecanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5281\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-5.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHeneicosane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12403\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-4.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePentadecane, 2-methyl-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15267\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-5.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBis(2-ethylhexyl) phthalate (8343)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8343\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-5.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eV-Benzylcarbonyl-L-tyrosine\u0026rsquo;s 2D interaction map showed several stabilizing interactions with the Q6A1B7 protein. Important hydrogen bonds were generated by key residues such as Asp248, Asn176, Ser111, Thr250, and Arg247 and additional interactions with Glu173, Gln174 and Lys241 improved binding stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Similarly, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e, cyclopentadecanol mostly interacted with the Q6A1B7 protein via a hydrogen bond with Arg313.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Post Screening Analysis\u003c/h2\u003e \u003cp\u003eIn initial screening the substances N-benzyloxycarbonyl-L-tyrosine and Cyclopentadecanol were pharmacokinetically purified using the SwissADME platform. N-Benzyloxycarbonyl-L-tyrosine emerges as a safer candidate, with moderate aqueous solubility, favorable drug-likeness, no violations of Lipinski\u0026rsquo;s rules, and strong gastrointestinal absorption, albeit with limited blood-brain barrier (BBB) permeability and no inhibition of key cytochrome p450 enzymes. The low log P value (~\u0026thinsp;1.88) and higher polar surface area (95.86 \u0026Aring;\u003csup\u003e2\u003c/sup\u003e) indicate limited transmembrane permeability but appropriate lipophilicity. Cyclopentadecanol had better BBB penetration, a smaller polar surface area (20.23 \u0026Aring;\u003csup\u003e2\u003c/sup\u003e), and higher lipophilicity (Consensus Log P\u0026thinsp;~\u0026thinsp;4.21), indicating a strong potential for CNS activation. Nonetheless, its decreased aqueous solubility and the existence of two lead likeliness violations- namely molecular weight and XLOGP surpassing 3.5 after formulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Anticancer Prediction\u003c/h2\u003e \u003cp\u003eThe pdCSM cancer model indicated that the compound \u003cem\u003eN\u003c/em\u003e-Benzyloxycarbonyl-L-tyrosine would be inactive. No discernible selective cytotoxicity against the tested cancer cell lines is shown by the fact that the anticipated probability values (~\u0026thinsp;4.0\u0026ndash;4.7) fall within a low, stable range. Specifically, all cell lines, including breast, CNS, colon, leukaemia, melanoma, lung, ovarian, prostate, renal, and small-cell lung cancer, have projected activity values that lie within a limited range (~\u0026thinsp;4.0\u0026ndash;4.7), suggesting no significant or specific cytotoxic effect. Notwithstanding its advantageous physicochemical characteristics (drug-like MW, LogP, and polar surface area), the molecule is ineffective against the cancer cell lines that were evaluated. Overall, the findings support the pdCSM cancer server\u0026rsquo;s \"Inactive\" and show limited anticancer potential. Moreover, the compound cyclopentadecanol was found to be active.\u003c/p\u003e \u003cp\u003eWith higher scores for breast (MDA-MB-468), melanoma (M14, SK-MEL-5), colon (HCT_15), and ovarian (OVCAR-5, OVCAR-8) cell lines, indicating increased activity in these malignancies, its projected values primarily fall between 4.0 and 5.5. \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e (\u003cb\u003eTable\u0026nbsp;7\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe soil fungus \u003cem\u003eTrichoderma lixii\u003c/em\u003e generates VOCs that have significant antibacterial, antioxidant and cancer cell cytotoxic properties. Strong inhibition of infectious and cancer cell lines particularly HePG2 was demonstrated by its extracellular metabolites. Promising interactions between VOCs and cancer related targets were found by molecular docking, suggesting possible therapeutic advantages. Despite certain formulation issues, cyclopentadecanol emerged as a crucial compound with anticipated activity across several cancer cells. Colonial and micromorphological features were then examined and compared with the diagnostic descriptions of Sukmawaty et al., [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The query isolate PP754294.1 was firmly placed within the \u003cem\u003eTrichoderma harzianum\u003c/em\u003e species complex, exhibiting close affiliation with \u003cem\u003eT.anaharzianum\u003c/em\u003e (NR174890.1), \u003cem\u003eT.longicollum\u003c/em\u003e (NR 198530.1), \u003cem\u003eT.lixii\u003c/em\u003e (NR131264.1), \u003cem\u003eT.atrobrunneum\u003c/em\u003e (NR137298.1), all supported by robust bootstrap values exceeding 80%. This phylogenetic grouping indicates a recent common ancestor among these taxa, which supports the isolate\u0026rsquo;s categorization in the \u003cem\u003eHarzianum clade\u003c/em\u003e. The \u003cem\u003eTrichoderma harzianum\u003c/em\u003e complex is well known for its ecological adaptability, biocontrol efficacy and bioactive chemical synthesis which highlights isolate PP754294.1\u0026rsquo;s potential functional importance [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Interestingly, although the genetic distance matrix revealed the smallest sequence difference between PP75429.1 and NR172576.1, phylogenetic reconstruction algorithms-by incorporating substitution patterns across all included taxa rather than relying simply on query sequence similarity-positioned the isolate in close proximity to \u003cem\u003eT. anaharzianum\u003c/em\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Similar discrepancies have been noted in phylogenetic studies of \u003cem\u003eTrichoderma\u003c/em\u003e, where distance-based and tree-based methods provide complementary insights into species delimitation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This phylogenetic tree was rooted with \u003cem\u003ePseudomonas polygonicola\u003c/em\u003e (NR 154274.1) as the outgroup, which clearly separated from the Trichoderma cluster, establishing a phylogenetic baseline. The placement of isolate PP754294.1 within Harzianum species complex is consistent with prior research that has identified this clade as one of the genus most taxonomically varied and agriculturally relevant lineages. Among the beneficial chemicals discovered is the aromatic volatile 2-phenylethanol. The antibacterial and anti-inflammatory properties of saturated and unsaturated fatty acids, such as stearic, ricinoleic, and palmitic acids have been well investigated; these actions are mostly mediated by membrane disruption [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The reported antibacterial activities may be explained by the fact that long chain alcohols and their monoglyceride derivatives have also been demonstrated to inhibit Staphylococcus speices [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Antagonistic activity against molds and yeasts is probably facilitated by 2-phenylethanol, a well-known antifungal volatile released by a various microbe [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Additionally, fatty acid methyl esters, including methyl stearate, have been linked to nematicidal and larvicidal effects, indicating possible uses in vector-control bioassays if further research is done [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In sum, excluding the likely DEHP artifact, the profile is consistent with a lipid-rich extract whose FFAs, fatty alcohols and aromatic alcohols provide plausible mechanistic bases for the observed antimicrobial/antioxidant activities [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The GC\u0026ndash;MS profile is dominated by free fatty acids (FFAs) and their derivatives (palmitic, stearic, ricinoleic acids; fatty alcohols/esters), a chemical class known to reduce cancer-cell viability by membrane perturbation and lipotoxic apoptosis - mechanisms involving mitochondrial dysfunction and ER stress [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Minor constituents may also contribute: retinoids (retinal) possess well-documented antiproliferative actions in epithelial cancers via nuclear receptor signalling [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Overall, the cytotoxic pattern - greatest toward hepatoma cells - fits a lipid-rich extract whose FFAs and related lipids plausibly underlie the observed effects; bioassay-guided fractionation and orthogonal confirmation (e.g., LC - MS, standards) are warranted to pinpoint the active principles [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn the present findings the extracellular metabolites from \u003cem\u003eT. lixii\u003c/em\u003e demonstrated a strong dose dependent antibacterial activity mainly against gram positive bacillus cereus and gram-negative pathogens \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eV. parahaemolyticus\u003c/em\u003e. The TEtOAc outperformed streptomycin at 20 \u0026micro;g against \u003cem\u003eP. aeruginosa\u003c/em\u003e (34.5 mm) and \u003cem\u003eV. parahaemolyticus\u003c/em\u003e with 32.6 mm. These results are consistent with current research that demonstrated the potent antibacterial efficacy of \u003cem\u003eTrichoderma\u003c/em\u003e metabolites against gram negative and positive bacteria. At lower concentrations of 40 \u0026micro;g/ml [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. \u003cem\u003eT. harzianum\u003c/em\u003e extracts showed high activity against \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e whereas another study evidenced diterpenoid compounds from \u003cem\u003eT. harzianum\u003c/em\u003e demonstrated a broad-spectrum activity against plant pathogens [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eSimilarly, \u003cem\u003eTrichoderma virens\u003c/em\u003e from ampelopsis japonica roots exhibited significant biofilm inhibition and MIC values of 25 \u0026micro;g/ml against Methicillin resistant \u003cem\u003eS. aureus\u003c/em\u003e. The inhibition of our extracellular metabolite was in line with these MIC levels [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Literature review on \u003cem\u003eTrichoderma\u003c/em\u003e based metabolites confirms nearly 1000 compounds including gliotoxins, peptaibols, polyketides and volatile organic compounds that are shown to possess antifungal and antibacterial properties [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Notably TEtOAc was more active against \u003cem\u003eV. parahaemolyticus\u003c/em\u003e than streptomycin suggesting that it could be useful against marine borne gram negative pathogens which are a focus that is not often studied in \u003cem\u003eTrichoderma\u003c/em\u003e based research. In our patten of research the fungal metabolites showed more effectiveness against gram negative bacteria than the gram positive which is in line with findings from studies using silver nanoparticle mediated \u003cem\u003eT. harzanium\u003c/em\u003e which showed gram negative were more vulnerable due to the cell wall structural variations [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. According to recent research \u003cem\u003eTrichoderma\u003c/em\u003e species have a wide variety of bioactive secondary metabolites possessing free radical scavenging potentials [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. For instance, \u003cem\u003eTrichoderma citriniviride\u003c/em\u003e is an endophytic fungus that produced sorbicillinoid derivatives with potent DPPH radical scavenging activity with an IC\u003csub\u003e50\u003c/sub\u003e value between (28 and 90 \u0026micro;M) both of them were potential as ascorbic acid [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Additionally, \u003cem\u003eT. harzianum\u003c/em\u003e extracts showed IC\u003csub\u003e50\u003c/sub\u003e in the range of 10\u0026ndash;100 \u0026micro;g/ml for DPPH radical [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. In comparison to typical crude extracts our \u003cem\u003eTrichoderma lixi\u003c/em\u003ei based extracts shows moderate activity with an IC\u003csub\u003e50\u003c/sub\u003e value of 54 \u0026micro;g/ml. Additionally T.harzianum crude metabolites have exhibited enzyme inhibitory and fre radical scavenging properties. The antioxidant potentials tested by ABTS and DPPH tests, its typical for extracts from \u003cem\u003eTrichoderma\u003c/em\u003e to exhibit varying potencies. In a recent work performed by Kannan et al, [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], \u003cem\u003eT. hazrianum\u003c/em\u003e outperformed many other taxa with an ABTS IC\u003csub\u003e50\u003c/sub\u003e value of 25 \u0026micro;g/ ml and that of DPPH to be 25 \u0026micro;g/ml [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In addition to directly scavenging radicals, Trichoderma sp. are known to increase the activity of antioxidant enzymes in host organisms. A meta-analysis found that Trichoderma inoculation increases the activity of plant enzymes such as glutathione reductase, ascorbate peroxidase, superoxide dismutase, and catalase, especially under stressful conditions like salinity and heavy metal exposure [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. However, simple hydrocacrbons like derivatives of pentadecane, tetradecane and heneicosane showed weaker binding due to their non-polar geometries, suggesting limited interaction capacity. Overall, the results indicate that oxygenated fatty acids, alcohols and aromatic derivatives bind to proteins more effectively than long-chain alkanes [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In 2D interaction pattern other residues (Asn234, Lys241, Ser238, and Tyr314) also contribute through hydrophobic and van der Waals interactions. Because of its lengthy aliphatic chain, its binding is more dependent on the hydrophobic pocket than on the aromatic molecule. Cyclopentadecanol is better suited for CNS-targeted uses despite solubility limitations, whereas N-Benzyloxycarbonyl-L-tyrosine is more druggable and metabolically stable. Both compounds have similar bioavailability scores of ~\u0026thinsp;0.55 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. From the anticancer evaluation the findings show that this compound has broad-spectrum anticancer potential, with considerable effects against cancer cell types, including ovarian, colon, breast, and melanoma [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This work authenticates \u003cem\u003eTrichoderma\u003c/em\u003e sp5 (TR5S2) as \u003cem\u003eT. lixii\u003c/em\u003e and establishes a soil-to-screen pipeline that links ITS - based phylogeny, GC\u0026ndash;MS volatilome profiling, and functional assays. The extracts demonstrated antibacterial, antioxidant and cell line-specific cytotoxic activities are correlated with its lipid rich volatile organic compound (VOC) profile, which includes fatty acids, alkanes, esters, and phthalates. HepG2 cells showed the strongest inhibition (IC\u003csub\u003e50\u003c/sub\u003e value of 29\u0026micro;g/ml). Molecular docking studies confirm interactions with apoptosis-related targets, such as IKKβ, Topoisomerase IIα, β-tubulin, and members of the Bcl-2 protein family. Complementary in silico ADMET tests support the metabolite\u0026rsquo;s medicinal potential by revealing a generally favorable drug-likeliness profile. Following post-hoc prioritizing, cyclopentadecanol is identified as the leading candidate, owing to its expected multi-target action and blood barrier permeability, despite certain developability restrictions. In contrast, N-benzylcarbonyl-L-tyrosine, despite possessing druggable properties is expected to to inactive across oncological panels.\u003c/p\u003e \u003cp\u003eIn the future, we will pinpoint active volatiles by bioassay-guided fractionation and rigorous dereplication, then validate apoptosis mechanisms (caspase/PARP/Δψm, tubulin, IKKβ/Topo IIα) and optimize cyclopentadecanol via solubility/PK improvement and SAR. ADME/safety profiling, expanded cell panels and 3D models, plus in vivo tests will de-risk leads. Finally, headspace SPME-GC\u0026ndash;MS and genome/BGC mining will map biosynthesis, and combination studies will assess therapeutic synergy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eVOCs, volatile organic compounds; ITS,\u0026nbsp;Internal transcribed spacer; ADMET, Absorption Distribution Metabolism Excretion Toxicity; GC-MS,\u0026nbsp;Gas chromatography\u0026ndash;mass spectrometry; DPPH,\u0026nbsp;2,2-diphenyl-1-picrylhydrazyl; ABTS,\u0026nbsp;2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid; IC\u003csub\u003e50\u003c/sub\u003e, Half maximal inhibitory concentration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Vietnam Academy of Science and Technology (Project code: CSCL23.01/25-26).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSanthosh Sigamani\u003c/strong\u003e- Research Work, writing\u0026ndash;original draft, Investigation, \u003cstrong\u003eSaranyadevi Subbaraj-\u003c/strong\u003e \u003cem\u003eIn Silico\u003c/em\u003e analysis, writing \u0026ndash; original draft, Investigation, \u003cstrong\u003ePham The Thu\u003c/strong\u003e- Project administration, writing \u0026ndash; review \u0026amp; editing, \u003cstrong\u003eHue Thi Nguyen\u003c/strong\u003e-Data curation, writing \u0026ndash; review \u0026amp; editing \u003cstrong\u003eNguyen Van Giang\u003c/strong\u003e-Visualization, Software, \u003cstrong\u003eNguyen Huy Thuan-\u0026nbsp;\u003c/strong\u003eSupervision, Resources and Project administration, Writing \u0026ndash; review \u0026amp; editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs no animals and humans were involved in this study hence ethical approval is not applicable for this research work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince this work does not involve studies on human trails and testing of drug on any individual was not performed, is not a case study either so the consent of participation is not required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors agree to evaluate and publish the study in this South African Journal of Botany.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available in the NCBI gene bank repository, [\u003cstrong\u003eAccession No:\u003c/strong\u003e PP754294, https://www.ncbi.nlm.nih.gov/nuccore/PP754294 \u0026nbsp;]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKubicek, C.P., Steindorff, A.S., Chenthamara, K., Manganiello, G., Henrissat, B., Zhang, J., Cai, F., Kopchinskiy, A.G., Kubicek, E.M., Kuo, A., Baroncelli, R., Sarrocco, S., Noronha, E.F., Vannacci, G., Shen, Q., Grigoriev, I.V., Druzhinina, I.S.: Evolution and comparative genomics of the most common Trichoderma species. 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Biomolecules. \u003cb\u003e13\u003c/b\u003e, 194 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/biom13020194\u003c/span\u003e\u003cspan address=\"10.3390/biom13020194\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Trichoderma, ITS sequence, cytotoxic activity, docking, ADMET7","lastPublishedDoi":"10.21203/rs.3.rs-9031969/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9031969/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eTrichoderma species are common soil-dwelling fungi that are well known for their extensive production of secondary metabolites, antagonistic qualities against phytopathogens, are remarkable ecological adaptability.\u003c/p\u003e\u003ch2\u003eMethod\u003c/h2\u003e \u003cp\u003eBy a thorough molecular and chemical analysis, a novel isolate known as \u003cem\u003eTrichoderma\u003c/em\u003e sp. (TR5) was carefully characterized in this study. Pairwise ITS comparison identified TR5 as \u003cem\u003eTrichoderma lixii\u003c/em\u003e and its phylogenetic analysis based on ITS rDNA sequences. The GC MS analysis primarily had contained fatty acids, alkenes, esters, and phthalates\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe most noticeable zones of inhibition was detected by \u003cem\u003eT.lixii\u003c/em\u003e derived metabolites against pathogenic bacteria namely \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (34.5 mm) and \u003cem\u003eBacillus cereus\u003c/em\u003e (27.4 mm). Additionally, they showed strong antioxidant properties and selective cytotoxicity in a variety of cancer cell lines, with HepG2 cells showing the strongest inhibitory effect (IC\u003csub\u003e50\u003c/sub\u003e=29.01 3.33 \u0026micro;g/ml). IC\u003csub\u003e50\u003c/sub\u003e values of 54.2 \u0026micro;g/ml for DPPH and 149. \u0026micro;g/ml for ABTS assays. Docking suggested favorable interactions of representative VOCs with apoptosis-relevant targets (IKKβ, topoisomerase IIα, β-tubulin, Bcl-2 family), and ADMET predictions indicated acceptable oral bioavailability with low toxicity liabilities. Integrating post hoc prioritization, cyclopentadecanol emerged as the more promising hit - predicted active across several cancer panels and blood - brain barrier permeable. In contrast, \u003cem\u003eN\u003c/em\u003e-benzyloxy carbonyl-L-tyrosine exhibited drug-like ADME and strong protein contacts but was predicted inactive across cancer panels.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOverall TR5 is confirmed to be \u003cem\u003eT. lixii\u003c/em\u003e and its volatilome provides a tractable source of apoptosis sensitizing leads. With multifaced biological applications the potential fungi based extracellular metabolites may serve as a potential candidate in pharmaceutical industries.\u003c/p\u003e","manuscriptTitle":"Volatilome Profiling and Apoptosis-sensitizing potential of secondary metabolites from Trichoderma lixii: Integrating GC–MS, Bioassays, and In Silico Docking Approaches","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-09 12:33:55","doi":"10.21203/rs.3.rs-9031969/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-04-05T09:57:11+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-05T08:43:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Waste and Biomass Valorization","date":"2026-03-29T07:56:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-05T00:59:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Waste and Biomass Valorization","date":"2026-03-04T10:34:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"71381a91-22bf-47d3-82de-f8e1ffa604fc","owner":[],"postedDate":"April 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-09T12:33:55+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-09 12:33:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9031969","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9031969","identity":"rs-9031969","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}