Microbial Biotransformation of the Sesquiterpene Carotol: Generation of Hydroxylated Metabolites with Potential Cytotoxic and Target-Specific Binding Activities

Microbial Biotransformation of the Sesquiterpene Carotol: Generation of Hydroxylated Metabolites with Potential Cytotoxic and Target-Specific Binding Activities | 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 Microbial Biotransformation of the Sesquiterpene Carotol: Generation of Hydroxylated Metabolites with Potential Cytotoxic and Target-Specific Binding Activities Hanan G. Sary, Mohammed A. Khedr, Mohamed M. Radwan, Mickey Vinodh, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6893326/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Carotol, a major sesquiterpene alcohol found in carrot essential oil, exhibits promising biological activities including cytotoxic effects against various cancer cell lines. Despite its bioactivity, the metabolic fate and biotransformation pathways of carotol remain largely unexplored, particularly through microbial systems that can offer novel insights into its structural modifications and potential pharmacological applications. Results In this study, seventeen microbial strains were screened for their ability to biotransform carotol, with Absidia coerulea ATCC 6647 identified as the most effective strain. Preparative-scale fermentation using this strain led to the isolation and purification of three metabolites (CM1, CM2, and CM3). Spectroscopic analysis, including 1D and 2D NMR, HRMS, and single crystal X-ray diffraction, elucidated the structures of these metabolites as 9α-hydroxydaucol (CM1), 9α,13-dihydroxydaucol (CM2), and a diol derivative of daucol (CM3). Cytotoxicity evaluation against human liver (HepG-2), colon (HCT-116), breast (MCF-7), and lung (A-549) carcinoma cell lines, alongside normal lung fibroblasts (MRC-5), revealed that carotol exhibited the highest anticancer activity followed by CM2, CM3, and CM1. Molecular docking studies against human NADPH oxidase demonstrated that carotol and CM2 have stronger binding affinities and more stable interactions compared to the other metabolites, suggesting NADPH oxidase inhibition as a possible mechanism for their anticancer effects. Conclusion This study provides the first comprehensive microbial biotransformation pathway for carotol, leading to the identification of novel hydroxylated metabolites with varying cytotoxic activities. The findings highlight the potential of Absidia coerulea as a biocatalyst for producing bioactive carotol derivatives and underscore the relevance of NADPH oxidase inhibition in their anticancer mechanism. These results lay a foundation for future pharmacokinetic and drug development research involving carotol and its metabolites. Carotol Absidia coerulea Microbial biotransformation Sesquiterpene alcohol Hydroxydaucol Carrot seed essential oil Molecular docking Anticancer activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background The carrot ( Daucus carota L. subsp. carota , Family Apiaceae) is an herbaceous, biennial flowering plant native to Europe, Asia, and Africa. It ranks among the ten most economically significant vegetable crops worldwide ( 1 ). Carrots provide substantial nutritional and health benefits. Historically, they were first used medicinally before being incorporated into diets as food ( 2 ). Phytochemical-rich diets help prevent cancer, heart disease, neurological disorders, and birth defects ( 3 , 4 ). Carrots are a unique vegetable due to their abundant carotenoids, phenolics, vitamin C, and polyacetylenes, which contribute to reduced risks of cardiovascular disease and cancer ( 5 ). Ancient Egyptians employed D. carota as a stimulant, carminative, diuretic, anthelmintic, and a treatment for infantile diarrhea ( 6 ). Additionally, D. carota extracts are used for hepatic and renal insufficiency and skin disorders, including burns and furunculosis ( 7 ). Wild D. carota extracts exhibit iron-chelating, antioxidant ( 8 ), and antibacterial activities ( 9 ). Carrot seed essential oil is widely utilized in food flavoring, perfumes, cosmetics, and soaps ( 8 – 10 )and has demonstrated antibacterial, antioxidant, anti-aging, cytotoxic, and mosquito-repellent properties ( 11 – 18 ). Major components of carrot seed oil include carotol, α -pinene, geranyl acetate, β -pinene, daucol, limonene, β -bisabolene, geraniol, β -elemene, cis - β -bergamotene, γ -decalactone, β -farnesene, caryophyllene, methyl eugenol, caryophyllene oxide, nerolidol, eugenol, trans -asarone, vanillin, α -terpineol, terpinen-4-ol, coumarin, γ -decanolactone, β -selinene, palmitic acid, and butyric acid ( 19 ). Carotol, a sesquiterpene alcohol, is the predominant compound in carrot seed oil ( 10 , 20 , 21 ), displaying strong antifungal ( 22 ), cytotoxic ( 10 ), and mosquito-deterrent activities ( 16 ). Drug metabolism studies traditionally rely on in vivo animal models or in vitro enzyme-based systems, such as microsomal preparations and tissue cultures. Microbial biotransformation provides an alternative, overcoming certain limitations of traditional methods. The concept of "microbial models of mammalian metabolism" involves using bacteria, yeasts, and fungi to simulate human drug metabolism. Microbial systems serve as efficient, selective, and scalable tools for generating metabolites that closely resemble those produced in mammalian systems ( 23 ). Their advantages include operational efficiency under mild conditions and the ability to catalyze diverse reactions beyond their natural substrates while exhibiting chemo-, regio-, and stereoselectivity ( 24 ). These models facilitate metabolite production in quantities unattainable from animal-based systems or chemical synthesis, making them predictive tools for human metabolism. Fungi, as eukaryotes, share enzymatic similarities with mammals, reinforcing their role as models for mammalian metabolism. Previous studies have reported carotol biotransformation by Rhizopus oryzae , yielding two lactate derivatives: 9 α -hydroxydaucol-9-lactate and 9 α -hydroxydaucol-3,9-dilactate ( 25 ). These metabolites exhibited in vitro inhibition of cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), and butyrylcholinesterase (BChE) ( 25 ). Furthermore, carotol chemical modifications have yielded 3-bromcarotol ether, carotol aldehyde, 4-phenoxy carotol, and daucene ( 26 ), ketone mixtures ( 27 ), and carota-1,4-dienaldehyde ( 28 ). To date, no carotol metabolites of human or mammalian origin have been reported. This study investigates the microbial biotransformation of carotol to generate novel derivatives in quantities sufficient for cytotoxicity assessment, with the aim of supporting their potential therapeutic applications. Furthermore, molecular docking analyses were performed to compare the binding interactions of the newly formed metabolites and carotol with NADPH oxidase, an enzyme implicated as a therapeutic target in cancer treatment strategies. Materials and methods Materials Substrate Preparation Pure carotol ( 1 ) was obtained from carrot essential oil as previously described ( 16 ). Briefly, therapeutic-grade carrot essential oil was purchased from Edens Garden (1322 Calle Avanzado, San Clemente, CA). Successive chromatographic purification yielded a carotol-rich fraction, which was further purified using preparative high-performance liquid chromatography (HPLC) to obtain pure carotol. Microorganisms The microbial cultures were sourced from the American Type Culture Collection (ATCC, Rockville, MD, USA), the Northern Regional Research Laboratories (NRRL), and Assiut University Mycology Center (AUMC). The following microorganisms were screened for their ability to catalyze the biotransformation of carotol: Absidia coerulea ATCC 6647, Fusarium sp. ATCC 11599, Phanerochaete chrysosporium ATCC 24725, Rhizopus oryzae (syn. R. nigricans ) ATCC 34121, Rhizopus stolonifer ATCC 6227a, Aspergillus flavus AUMC 4787, Aspergillus niger NRRL 599, Cunninghamella blacksleeana AUMC, Cunninghamella echinulata NRRL 132, Mucor sp. AUMC, Rhizopus sp. ATCC 36060, Saccharomyces cerevisiae (Baker’s yeast), Cladosporium sp., Cordyceps sinensis AUMC, Botrytis allii ATCC 9435, Streptomyces griseus ATCC 13968, Streptomyces spheroids ATCC 23965. Stock cultures were maintained on agar slants using media recommended by ATCC and stored at 4°C. Methods General Experimental Procedure IR spectra were recorded as a chloroform film using Jasco FT/IR-4100 type A Spectrophotometer. The 1 H and 13 C NMR spectra were obtained on a Bruker Avance II-600 spectrometer operating at 600 and 150 MHz, respectively. Both 1 H and 13 C NMR spectra were recorded in CDCl 3 , and the chemical shift values were expressed in d (ppm) relative to the internal standard TMS. For the 13 C NMR spectra, spectral editing was determined by DEPT. 2D NMR data were obtained using the standard pulse sequence of the Bruker Avance II-600 for COSY, HSQC and HMBC. EIHRMS analysis were carried out on a Thermo Scientific High-Resolution GC/MS-DFS (Double Focusing Sector) mass spectrometer. Column chromatography was carried out on Silica gel 60 (230–400 mesh ASTM, Merck). TLC analysis was carried out on silica gel 60 F254 (Merck) plates. Compounds were detected by UV and p -anisaldehyde/ H 2 SO 4 spraying reagent followed by heating at 105°C for 1–2 min. Preparative HPLC A Waters preparative HPLC system, equipped with a 2998 Photodiode Array detector, a 2545 quaternary gradient module, a FlexInject manual dual injector, and a Fraction Collector III, was used to isolate pure carotol. The separation was performed on a Waters XBridge™ Prep C18 (10 × 150 mm, 5 µm) column using isocratic elution with 30% methanol in acetonitrile at a flow rate of 1.7 mL/min. The detection wavelength was set at 218 nm. The sample was prepared by dissolving 100 µL of carotol-rich fraction in 1.5 mL of 30% methanol in acetonitrile, filtered through a 0.1 µm PTFE membrane, and injected into the system. Screening Procedure The microbial screening was conducted following a standard two-stage protocol ( 23 , 29 ). Fermentation medium alpha was prepared using 2% glucose, 0.5% yeast extract, 0.5% peptone, 0.5% NaCl, and 0.5% K₂HPO₄ in distilled water, sterilized at 121°C for 15 minutes. In Stage I, 250-mL culture flasks containing 50 mL of sterile medium alpha were inoculated with the selected microbial strains and incubated at 150 rpm for 72 hr at room temperature. Stage II involved transferring 5 mL of the Stage I culture to fresh medium, followed by 24 hr of incubation before substrate addition. Carotol ( 1 ) was prepared as a 15% solution in N,N -dimethylformamide (DMF) and added to Stage II cultures at a final concentration of 0.5 mg/mL, then the cultures were incubated at 5 ºC for two weeks. Control groups included substrate control where sterile medium with carotol (5 mg/100 µL DMF) incubated without microorganisms at 5 ºC. However, in culture controls microorganisms were grown under identical conditions without carotol. After two weeks, cultures were harvested and analyzed via thin-layer chromatography (TLC). Preparative-Scale Fermentation Since Absidia coerulea ATCC 6647 exhibited clear capability to metabolize carotol, it was cultured in five 250-mL flasks containing 50 mL of medium alpha (Stage I). A total of 304 mg of carotol, dissolved in 450 µL DMF, was distributed among nine 1-L flasks containing 24-hr-old Stage II cultures. After 14 days, the cultures were analyzed using TLC with an n -hexane–acetone (8.5:1.5) solvent system. Visualization with p -anisaldehyde/H₂SO₄ revealed the transformation of carotol ( 1 ) into three polar metabolites. Each culture flask was filtered, and the filtrates were extracted with chloroform (1.5 L × 3). The combined extracts were dried over anhydrous Na₂SO₄ and evaporated to dryness under reduced pressure at 38°C, yielding a brownish residue (525 mg). Purification was performed using flash silica gel column chromatography (68 g, 2.7 × 33 cm, 230–400 mesh) with CHCl₃–acetone (9.3:0.7) as the eluent. Fractions were collected (25 mL each) and pooled together based on TLC analysis. Single Crystal X-Ray Diffraction Analysis Single crystals of CM1 used in this study were obtained via slow solvent evaporation. X-ray diffraction data were collected at 150 K using a Bruker X8 Prospector diffractometer equipped with Cu-Kα radiation. Reflection frames were integrated using the Bruker SAINT software package with a narrow-frame algorithm. The crystal structure was solved with the SHELXTL software suite and refined using SHELXL-2019/3. All non-hydrogen atoms were refined anisotropically, while hydrogen atoms were placed in calculated positions and refined using a riding model. Cytotoxicity Evaluation The cytotoxic activities of pure carotol ( 1 ) and its metabolites were evaluated against human liver (HepG-2), colon (HCT-116), breast (MCF-7), and lung (A-549) carcinoma cell lines, as well as normal human lung fibroblasts (MRC-5), using a standard cell viability (MTT) assay ( 30 ). The assay was conducted at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt. Cancer and normal cells were seeded into 96-well plates at a density of 5 × 10⁴ cells per well and incubated for 24 hours prior to treatment. Test compounds were added in triplicate at twelve different concentrations. Vehicle controls (media only or media containing 0.5% DMSO) were included. After a 24-hour incubation period, cell viability was assessed via the MTT assay. Briefly, the media were replaced with 100 µL of fresh RPMI-1640 medium, followed by the addition of 10 µL of a 12 mM MTT stock solution (5 mg/mL in PBS) to each well, including untreated controls. Plates were incubated at 37°C in a 5% CO₂ atmosphere for 4 hours. Subsequently, 85 µL of medium was removed from each well, and 50 µL of DMSO was added to dissolve the resulting formazan crystals. The plates were thoroughly mixed and incubated for an additional 10 minutes at 37°C. Absorbance was measured at 590 nm using a microplate reader (SunRise, TECAN Inc., USA). Cell viability percentages were calculated, and IC₅₀ values (µM) were determined from dose-response curves using GraphPad Prism software (San Diego, CA, USA) ( 31 ). Docking Study The crystal structure of human NADPH oxidase was downloaded from protein data bank (pdb code = 8gz3) ( 32 ). The structure was published in 2022, with resolution = 3.3 Å. Molecular docking was done using Molecular Operating Environment (MOE 2022.02) package license purchased from Chemical Computing Group Inc., Sherbooke St, Montreal, QC, Canada. Triangle matcher was used as a placement method. London ΔG scoring method was applied to estimate the free energy of binding (Kcal/mol). Root mean square deviation RMSD of the docked ligands was computed to measure the deviation from the co-crystalized ligand. The root mean square fluctuations RMSF was computed to compare between carotol and its metabolites. Results and Discussion Preparative HPLC fractionation resulted in the isolation of two major compounds (Fig. 1 ), designated as Car-1 (t R = 5.9 min) and Car-2 (t R = 7.5 min). Spectral analysis confirmed that Car-2 corresponds to pure carotol. With the exception of a single reference ( 25 ), a comprehensive literature search revealed no prior documentation or investigation detailing either microbial or mammalian metabolic pathways of carotol. Although carotol has been previously investigated for its biological activities, including cytotoxic and antimicrobial properties, its metabolic fate, particularly via microbial biotransformation, remains largely unexplored. The current findings thus provide novel insights into the metabolism of carotol, laying the foundation for future pharmacokinetic and bioactivity studies, and underscoring the value of microbial systems in elucidating the biotransformation pathways of bioactive sesquiterpenes. The metabolic capabilities of seventeen microbial cultures were assessed for carotol biotransformation, and Absidia coerulea ATCC 6647 exhibited significant metabolic activity. Consequently, this strain was selected for preparative-scale fermentation, yielding three metabolites: CM1, CM2, and CM3 (Fig. 2 ). The metabolites were purified using column chromatography, with multiple fractions collected and analyzed by TLC. Fractions 19, 21, and 25 were identified as promising based on their TLC profiles compared to the culture control. Fraction 19 (123 mg) was subjected to further purification over a silica gel 60 column (17 g, 30 × 1.5 cm) with toluene-acetone (9:1) as the mobile phase. Fractions (5 mL each) were collected and analyzed by TLC using the same solvent system. Similar fractions were pooled, affording a pure, crystalline, non-UV-active compound, designated CM1 ( 2 ) (104.3 mg, 30% yield), with R f =026 (toluene-acetone; 8.5:1.5). Likewise, fraction 25 (36.8 mg), after further purification yielded 24.7 mg (6.64% yield) of pure, non-UV-active amorphous powder, designated CM2 ( 3 ) with an R f value = 0.27 (toluene-acetone; 8.5:1.5). Moreover, fraction 21 (30.5 mg) was purified using a silica gel column (15 × 1.4 cm, 6.4 g) and eluted with toluene-acetone (8.7:1.3). Fractions (2 mL each) were collected and monitored by TLC using the same mobile phase. Similar fractions were pooled, yielding CM3 ( 4 ) (11.5 mg, 3.28% yield) as a pure, non-UV-active amorphous powder. The R f value was 0.27 in toluene-acetone (9:1). CM1 ( 2 ) structure was characterized based on detailed spectroscopic analysis. Its molecular formula was established as C₁₅H₂₆O₃, supported by the molecular ion peak at m/z 254.1873, along with its 1 H and 13 C NMR data (Tables 1 and 2 ). The 13 C NMR spectrum exhibited 15 resonances, categorized as three singlets, four doublets, four triplets, and four quartets. Compared to carotol, CM1 lacked the characteristic olefinic signals observed at δ C 138.8 (singlet) and δ C 122.3 (doublet), indicating the absence of the double bond present in the parent compound. Conversely, CM1 displayed four oxygenated aliphatic carbon signals at δ C 71.5 (doublet, C-3), δ C 85.6 (singlet, C-4), δ C 92.6 (singlet, C-7), and δ C 73.3 (doublet, C-9). Signal assignments were confirmed through 2D NMR experiments (HSQC and HMBC), which enabled unambiguous correlation of proton and carbon resonances. Notably, several of these chemical shifts closely matched those reported for daucol ( 5 ), a major sesquiterpene constituent of carrot seed oil ( 12 ). The identity and absolute configuration of CM1 were further confirmed by single crystal X-ray diffraction. Table 1 1 H NMR spectroscopic data (600 MHz, CDCl 3 ) of carotol and its metabolites. # ẟ H, multiplicity ( J in Hz) Carotol CM1 CM2 CM3 2 1.73, dd (12.1, 8.4) 2.29, d (16.2) 1.22, dd (12.0, 12.0) 1.75, dd (12.9, 6.0) 1.79, dd (12.2, 5.6) 2.27, m 1.39, m 1.55, dd (12.0, 10.2) 3 5.35, d 3.76, m 3.85, m 3.62, d (10.2) 5 2.10, dd (5.4, 5.4) - 1.34, dd (5.4, 1.2) 1.85, m 1.35, m 1.92, m 1.46, m 1.88, m 6 1.65, m 1.97, m 1.54, ddd (13.0, 13.0, 4.8) 2.14, m 1.25, m 1.78, m 1.68, m 1.74, m 8 1.82, m 1.85, m 2.45, d (8.0) 1.47, m 9 1.52, m 1.67, m 4.47, ddd (7.2, 7.2, 0.6) 4.56, m 1.45, m 1.63, m 10 1.32, m 1.58, m 1.45, dd (13.2, 0.6) 1.80, ddd (13.8, 7.2, 0.6) 1.60, m 1.65, m 1.47, m 1.93, m 11 0.96, s 1.36, s 1.38, s 1.01, s 12 1.68, s 1.30, s 1.37, s 1.24, s 13 1.83, m 2.10, m - 1.64, m 14 1.02, d (6.6) 1.04, d (6.0) 1.47, s 0.98, d (6.6) 15 0.97, d (6.0) 1.17, d (6.6) 1.48, s 0.90, d (6.6) Table 2 13 C NMR spectroscopic data (150 MHz, CDCl 3 ) of carotol and its metabolites. # ẟc, multiplicity a Carotol CM1 CM2 CM3 1 49.3, s 44.8, s 45.5, s 47.5, s 2 38.8, t 40.8, t 43.4, t 38.7, t 3 122.3, d 71.5, d 71.1, d 75.4, d 4 138.8, s 85.6, s 87.4, s 75.5, s 5 29.6, t 29.2, t 29.1, t 35.5, t 6 34.6, t 41.7, t 40.8, t 41.3, t 7 84.8, s 92.6, s 91.9, s 83.6, s 8 52.7, d 58.8, d 60.1, d 56.6, d 9 24.6, t 73.3, d 74.3, d 26.5, t 10 39.6, t 45.4, t 43.7, t 34.1, t 11 21.7, q 23.1, q 23.0, q 25.9, q 12 25.2, q 24.1, q 24.3, q 24.4, q 13 27.8, d 29.4, d 74.1, s 28.9, d 14 24.3, q 26.0, q 31.1, q 23.5, q 15 21.6, q 22.7, q 30.2, q 21.8, q a Carbon multiplicities were determined by DEPT 135°. The crystal structure of CM1 obtained from single crystal diffraction analysis is depicted in Fig. 3 and important crystallographic parameters of this crystal are provided in Table 3 . Table 3 Summary on the various crystallographic parameters of the compound CM1 Parameter Value Chemical formula C 16 H 30 O 4 M r 286.40 Crystal system, space group Orthorhombic, P 2 1 2 1 2 1 Temperature (K) 150 a , b , c (Å) 6.4762 ( 3 ), 15.7204 ( 9 ), 15.9679 ( 9 ) α, β, γ (°) 90, 90, 90 V (Å 3 ) 1625.67 ( 15 ) Z 4 Radiation type Cu K α µ (mm − 1 ) 0.66 Crystal size (mm) 0.21 × 0.19 × 0.15 Diffractometer Bruker APEX -II CCD Absorption correction Multi-scan SADABS2016 /2 - Bruker AXS area detector scaling and absorption correction T min , T max 0.86, 0.91 No. of measured, independent & observed [ I > 2σ( I )] reflections 10554, 2864, 2713 R int 0.037 (sin θ/λ) max (Å −1 ) 0.596 R [F 2 > 2σ(F 2 )], wR(F 2 ), S 0.037, 0.099, 1.08 No. of reflections 2864 No. of parameters 189 H-atom treatment Constrained Δρ max , Δρ min (e Å −3 ) 0.13, − 0.20 CM1 crystallizes in the orthorhombic crystal system, space group P 2 1 2 1 2 1 . Methanol molecules are incorporated into the crystal lattice as space-filling solvents, stabilizing the crystal structure through non-bonding interactions with adjacent CM1 molecules. The crystal structure of CM1 reveals the presence of six chiral carbon atoms in the molecule. These chiral centers include C-1 ( R ), C-3 ( S ), C-4 ( S ), C-7 ( S ), C-8 ( S ), and C-9 ( S ), which are color-labeled in Fig. 4 . Thus, CM1 identity was established as 9α-hydroxydaucol ( 2 ) (Fig. 2 ). On the other hand, CM2 ( 3 ) was determined to have the molecular formula C₁₅H₂₆O₄, as established by its molecular ion peak at m/z 270.1826, supported by its 1 H and 13 C NMR data (Tables 1 and 2 ). The 13 C NMR spectrum displayed 15 carbon resonances, comprising four singlets, three doublets, four triplets, and four quartets. Five of these resonances were clearly in the oxygenated aliphatic region. Comparison with the NMR data of CM1 revealed the presence of an additional oxygenated carbon in CM2, resonating at δ C 74.1 as a singlet, and the absence of the doublet observed in CM1 at δ C 29.4 (assigned to C-13). This suggested that the additional oxygen functionality in CM2 was located at C-13. This assignment was further supported by downfield chemical shifts of the adjacent methyl carbons, C-14 (δ C 31.1, Δ = 4.9 ppm) and C-15 (δ C 30.2, Δ = 7.5 ppm), indicating deshielding due to proximity to the oxygenated center. The remaining spectral data were consistent with those observed for CM1 (Tables 1 and 2 ). Based on these findings, the structure of CM2 was established as 9α,13-dihydroxydaucol (Fig. 2 ). Likewise, CM3 ( 4 ) was shown to have a molecular formula of C 15 H 28 O 3 based on its HRMS that shows the molecular ion peak at m / z 256.2032 and its 1 H and 13 C NMR data. Again, there were 15 carbon resonances distributed as three singlets, three doublets, five triplets, and four quarters. Of these resonances, three appear in the oxygenated aliphatic region at δ C 75.4 (doublet, C-3), δ C 75.5 (singlet, C-4), and δ C 83.6 (singlet, C-7). Comparison of the 13 C NMR data of CM3 with those published for daucol indicates that CM3 is a diol derivative resulting from the cleavage of the daucol ether linkage (Fig. 2 ). Notably, CM3 is confirmed as a true microbial metabolite, as pure carotol was used as the sole substrate in the microbial cultures. The unambiguous assignment of all 1 H and 13 C resonances was aided through DEPT 135°, HSQC, and HMBC experiments. CM1 ( 2 ): colorless prims (MeOH): mp 122–124°C; IR (neat) υ max 3434 (OH), 2935 (saturated C-H) cm − 1 ; 1 H NMR (CDCl 3 , 600 MHz) see Table 1; 13 C NMR (CDCl 3 , 150 MHz) see Table 2 ; EIHRMS m / z 254.1873 [M] + (calcd for C 15 H 26 O 3 254.1882). CM2 ( 3 ): colorless amorphous powder; IR (neat) υ max 3386 (OH), 2935 (saturated C-H) cm − 1 ; 1 H NMR (CDCl 3 , 600 MHz) see Table 1; 13 C NMR (CDCl 3 , 150 MHz) see Table 2 ; HRMS m / z 270.1826 [M] + (calcd for C 15 H 26 O 4 270.1831). CM3 ( 4 ): gummy residue; IR (neat) υ max 3380 (OH), 2910 (saturated C-H) cm − 1 ; 1 H NMR (CDCl 3 , 600 MHz) see Table 1; 13 C NMR (CDCl 3 , 150 MHz) see Table 2 ; HRMS m / z 256.2032 [M] + (calcd for C 15 H 28 O 3 256.2038). The cytotoxic effects of carotol and its metabolites were evaluated against several cancer cell lines (HepG2, HCT-116, MCF-7, and A549) as well as the normal human lung fibroblast cell line (MRC-5). Among the tested compounds, the parent molecule, carotol, exhibited the most potent cytotoxic activity, followed by its metabolites CM2, CM3, and CM1, in descending order of efficacy, as shown in Table 4 . Notably, carotol and its metabolites demonstrated selectivity toward cancer cells over normal cells. Previous studies have reported the cytotoxicity of carrot seed essential oils from various geographical sources, along with pure carotol, using green monkey kidney epithelial cells (VERO) and human hypopharyngeal squamous cell carcinoma cells (FaDu). Moroccan and French essential oils exhibited comparable cytotoxic effects, while the Polish essential oil displayed lower activity. In contrast, pure carotol demonstrated moderate cytotoxicity on both VERO and FaDu cell lines, with no apparent selectivity (IC₅₀ = 39.7 µg/mL and 38.3 µg/mL, respectively) ( 10 ). Table 4 IC 50 values (mean ± SD) of carotol along with its metabolites. Cell line a IC 50 (µM) ± SD Carotol CM1 CM2 CM3 HepG-2 52.34 ± 4.23 220.74 ± 12.26 154.45 ± 9.39 195.53 ± 10.57 HCT-116 25.68 ± 0.53 339.38 ± 15.56 180.64 ± 9.95 226.38 ± 11.04 MCF-7 68.38 ± 6.04 400.75 ± 20.28 212.85 ± 13.72 242.14 ± 15.68 A-549 28.65 ± 2.75 225.38 ± 13.40 138.21 ± 9.20 205.24 ± 10.61 MRC-5 175.61 ± 10.93 466.17 ± 20.32 247.62 ± 11.24 316.36 ± 15.17 a HepG-2: hepatocellular carcinoma, HCT-116: colon carcinoma, MCF-7: breast carcinoma, A-549: lung carcinoma, MRC-5: normal human lung fibroblasts. It has been previously documented that sesquiterpene alcohols can act as inhibitors of NADPH oxidase, an enzyme implicated in oxidative stress and cancer progression. The inhibition of NADPH oxidase is regarded as a key mechanism contributing to the anticancer potential of this class of compounds ( 33 ). In the present study, molecular docking simulations were performed to assess the binding interactions, free binding energy (ΔG), and binding stability of carotol and its metabolites with NADPH oxidase. As shown in Table 5 , carotol exhibited the most favorable binding profile, with the highest binding affinity (ΔG = − 5.65 kcal/mol) and the lowest RMSD value (1.35 Å), indicating a stable and specific interaction. CM2 followed with a ΔG of − 5.41 kcal/mol and an RMSD of 1.50 Å. In contrast, CM1 and CM3 showed lower binding affinities (ΔG = − 5.11 and − 5.16 kcal/mol, respectively), suggesting weaker interactions with the enzyme. These computational findings are in good agreement with the experimental cytotoxicity data presented in Table 4 and further support the hypothesis that carotol and CM2 exert anticancer effects, at least in part, through NADPH oxidase inhibition. Table 5 Docking results against NADPH oxidase (PBD = 8gz3) Compound Free energy of binding (Kcal/mol) RMSD (Å) Interacted residues Carotol -5.65 1.35 His338, Trp361 CM1 -5.11 1.91 Trp361 CM2 -5.41 1.50 Gly359, His338 CM3 -5.16 1.85 Thr362 Further analysis of the molecular docking interactions revealed distinct binding modes for carotol and its metabolites with NADPH oxidase (Fig. 5 ). Carotol formed a hydrogen bond with His338 and exhibited potential hydrophobic interactions with Trp361 (Fig. 5 A), suggesting a stable and favorable binding conformation. CM1 established a single hydrogen bond with the –NH group of Trp361 (Fig. 5 B), indicating a moderately stable interaction. Notably, CM2 was the only metabolite to exhibit an intramolecular hydrogen bond, which may contribute to enhanced binding stability. Additionally, CM2 formed two hydrogen bonds with Gly359 and His338 (Fig. 5 C), reinforcing its strong binding affinity. In contrast, CM3 interacted via a single hydrogen bond with Thr362 (Fig. 5 D), suggesting a comparatively weaker interaction. These distinct binding interactions further support the differential binding affinities observed in the docking scores and correlate well with the experimental cytotoxicity data. To further investigate the stability of binding interactions with NADPH oxidase, molecular dynamics (MD) simulations were performed over a 50 ns timescale for the best-docked complexes of carotol and its metabolites (CM1, CM2, and CM3). The resulting trajectories were analyzed to compute the root mean square fluctuations (RMSF) of NADPH oxidase residues in each complex, providing insight into local flexibility and binding stability (Fig. 6 ). Carotol exhibited the lowest RMSF values (blue trace), indicating a highly stable interaction with the enzyme throughout the simulation. CM2 displayed RMSF values comparable to those of carotol (orange trace), supporting its strong and stable binding. In contrast, CM1 (purple trace) showed higher fluctuations relative to carotol and CM2, suggesting a less stable interaction. The metabolite CM3 (green trace) demonstrated the highest residue fluctuations, with RMSF values exceeding 3.5 Å in certain regions, indicating the least stable binding among the tested compounds. These findings align well with the cytotoxicity results and further support the superior stability and potential biological activity of CM2 relative to the other metabolites. Conclusions This study successfully isolated pure carotol from carrot essential oil and evaluated its microbial biotransformation by diverse microorganisms. Among the strains tested, Absidia coerulea ATCC 6647 demonstrated significant metabolic activity, producing three distinct metabolites (CM1, CM2, and CM3). Structural elucidation of these metabolites was achieved through comprehensive spectroscopic analyses and single-crystal X-ray diffraction, confirming their identities as oxygenated sesquiterpene derivatives. Cytotoxicity assays revealed that carotol exhibited the strongest anticancer activity against multiple human carcinoma cell lines, with its metabolites showing moderate but selective cytotoxicity toward cancer versus normal cells. Molecular docking and dynamics simulations supported these findings, indicating that carotol and CM2 bind NADPH oxidase with higher affinity and stability, potentially underpinning their anticancer mechanisms through enzyme inhibition. This work provides novel insights into the microbial metabolism of carotol, a bioactive sesquiterpene with promising anticancer properties, and highlights the utility of microbial biotransformation as a tool for generating and characterizing novel derivatives. Future studies are warranted to further explore the pharmacokinetics, in vivo efficacy, and safety profiles of carotol metabolites, ultimately contributing to the development of new therapeutic agents derived from natural products. Abbreviations API: Active Pharmaceutical Ingredient ATCC: American Type Culture Collection AUMC: Assiut University Mycology Center BChE: Butyrylcholinesterase CM1, CM2, CM3: Carotol metabolites 1, 2, and 3 COX-2: Cyclooxygenase-2 COSY: Correlation Spectroscopy DEPT: Distortionless Enhancement by Polarization Transfer DMF: N,N -Dimethylformamide EIHRMS: Electron Impact High-Resolution Mass Spectrometry HPLC: High-Performance Liquid Chromatography HSQC: Heteronuclear Single Quantum Coherence HMBC: Heteronuclear Multiple Bond Correlation IC₅₀: Half Maximal Inhibitory Concentration IR: Infrared Spectroscopy MD: Molecular Dynamic MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide NRRL: Northern Regional Research Laboratories PBS: Phosphate-Buffered Saline PTFE: Polytetrafluoroethylene RMSD: Root Mean Square Deviation RMSF: Root Mean Square Fluctuations TLC: Thin-Layer Chromatography TMS: Tetramethylsilane UV: Ultraviolet XRD: X-Ray Diffraction Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information files. Competing interests The authors declare that they have no competing interests. Funding Spectroscopic and single crystal X-ray analyses were carried out at the Research Sector Projects Unit (RSPU), College of Science, Kuwait University, with support from grant numbers GS01/01 and GS01/03 (for spectral analyses), and GS03/08 (for X-ray analyses). Authors’ Contributions KYO and MMR conceived and designed the experiments; MMR, KYO and AA isolated carotol; HGS purified carotol; HGS and KYO performed the biotransformations; KYO and HGS analyzed the spectral data; MV performed single crystal X-ray analysis; MAK performed docking study; KYO interpreted the results; KYO and HGS wrote the paper; KYO administered and supervised the project. All authors read and approved the final manuscript. Acknowledgments Spectral analyses were done at RSPU facilities, College of Science, Kuwait University. Cytotoxicity evaluations were carried out at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt. Authors’ information KYO: Department of Pharmaceutical Chemistry, College of Pharmacy, Kuwait University, Safat 13110, Kuwait. HGS: Department of Pharmaceutical Chemistry, College of Pharmacy, Kuwait University, Safat 13110, Kuwait, and Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, Cairo, Egypt. MAK: Department of Pharmaceutical Chemistry, College of Pharmacy, Kuwait University, Safat 13110, Kuwait. Current address: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Ain Helwan, Cairo 11795, Egypt. MMR: Department of Biomolecular Sciences and National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS 38677, USA. AA: National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS 38677, USA. MV: Research Sector Project Units, College of Science, Kuwait University, Safat 13060, Kuwait. Supplementary information All 1D and 2D NMR spectra of carotol and its metabolites accompany this paper at https://doi.org/xxxxxxxxxxxxx. References Simon PW. Domestication, historical development, and modern breeding of carrot. Plant Breed Rev. 2000;19:157-90. Da Silva Dias J. Nutritional and health benefits of carrots and their seed extracts. Food and Nutrition Sciences. 2014;5:2147-56. Blancquaert D, Storozhenko S, Loizeau K, De Steur H, De Brouwer V, Viaene J, et al. Folates and folic acid: From fundamental research toward sustainable health. Crit Rev Plant Sci. 2010;29:14–35. Liu RH. Dietary bioactive compounds and their health implications. J Food Sci. 2013;78(A):18–A25. Ahmad T, Cawood M, Iqbal Q, Ariño A, Batool A, Tariq R, et al. Phytochemicals in Daucus carota and their health benefits-Review article. Foods 2019;8(9):424. Van Wyk B, Wink M. Medicinal plants of the world: an illustrated scientific guide to important medicinal plants and their uses. Portland, Oregon, USA and London, UK: Timber Press; 2004. Glisic S, Misic D, Stamenic M, Zizovic I, Asanin R, Skala D. Supercritical carbon dioxide extraction of carrot fruit essential oil: Chemical composition and antimicrobial activity. Food Chemistry 2007;105:346–52. Marzouki H, Khaldi A, Falconieri D, Piras A, Marongiu B, Molicotti P, Zanetti S. Essential oils of Daucus carota subsp. carota of Tunisia obtained by supercritical carbon dioxide extraction. Natural Product Communications. 2010;5(12):1955-8. Kumarasamy Y, Nahar L, Byres M, Delazar A, Sarker SD. The assessment of biological activities associated with the major constituents of the methanol extract of wild carrot (Daucus carota L.) seeds. Journal of Herbal Pharmacotherapy. 2005;5:61-72. Sieniawska E, Świątek L, Rajtar B, Kozioł E, Polz-Dacewicz M, Skalicka-Woźniak K. Carrot seed essential oil-Source of carotol and cytotoxicity study. Industrial Crops and Products. 2016;92:109-15. Alves-Silva J, Zuzarte M, Gonc¸ alves M, Cavaleiro C, Cruz M, Cardoso S, Salgueiro L. New claims for wild carrot (Daucus carota ssp carota) essential oil. Evid Based Complement Altern Med. 2016. Jasicka-Misiak I, Lipok J, Nowakowska EM, Wieczorek PP, Młynarz P, Kafarski P. Antifungal activity of the carrot seed oil and its major sesquiterpene compounds. Z Naturforsch C J Biosci. 2004;59(11-12):791-6. Khan I, Abourashed E. Leung’s Encyclopedia of common natural ingredients used in food, drugs, and cosmetics, 3rd ed John Wiley & Sons, Inc., Hoboken, US, NJ. 2010. p. 147–9. Radulovic N, Ðordevic N, Stojanovic-Radic Z. Volatiles of Balkan Endemic Daucus guttatus ssp: zahariadii and cultivated and wild-growing D. carota- a comparison study. Food Chem 2011;125:35-43. Mahboubi. M, Kazempour N, Mahboubi A. The efficacy of essential oils as natural preservatives in vegetable oil. J Diet Suppl 2014;11:334-46. Ali A, Radwan MM, Wanas AS, Khan IA. Repellent activity of carrot seed essential oil and its pure compound, carotol, against mosquitoes. Journal of the American Mosquito Control Association. 2018;34(4):272-80. Musnaini M, Fransisca S, Leslie W. Effectiveness of cream formulation of carrot seed oil as anti-aging. International Journal of Health and Pharmaceutical (IJHP). 2022;3(2):331–40. Manjunath C, Malpani A, Mahurkar N. In vitro cytotoxicity of cardamom oil and carrot seed oil on mouse fibroblast and urinary bladder cell line RGUHS Journal of Pharmaceutical Sciences. 2017;7(1):7-12. Leung AY, Foster S. Encyclopedia of Common Natural Ingredients used in food, drugs, and cosmetics. 2nd ed: John Wiley & Sons, Inc., New York, USA; 1996. p. 131. Ozcan MM, Chalchat JC. Chemical composition of carrot seeds (Daucus carota L.) cultivated in Turkey: characterization of the seed oil and essential oil. Grasas y Aceites. 2007;58:359–65. Chahal K, Kaur P, Kataria D, Kaur R. Carotol: A sesquiterpenoid isolated from carrot seed oil. Asian Journal of Chemistry. 2016;28(5):1004-6. Sharma MK, Chahal KK, Kaur R, Singh R, Kataria D. Antifungal potential and structure activity relationship of carrot seed constituents. Journal of Food Biochemistry. 2019;43(9). Orabi KY. Microbial models of mammalian metabolism. Sampangines. Atta-Ur-Rahman, editor. New York: Elsevier; 2000. 3-49 p. Loughlin WA. Biotransformation in organic synthesis. Biores Technol. 2000;74:49-62. Soliman AF, Abdel Bar FM, Sallam A, Galala AA. New neuroprotective sesquiterpene lactate esters from carotol biotransformation. South African Journal of Botany. 2023;153:163-71. Kataria D, Chahal K, Kumar A. Chemical transformations of carotol isolated from carrot seed oil. Asian Journal of Chemistry. 2016;28:1790–2. Kula J, Bonikowski R, Staniszewska M, Krakowiak A, Wieczorek M, Majzner W, Bujacz G. Transformation of Carotol into the Hydroindane-Derived Musk Odorant. Eur J Org Chem. 2002;11:1826-9. Hashidoko Y, Tahara S, Mizutani J. Conversion of (+)-Carotol into (−)-Carota-1,4-dienaldehyde. Bioscience, Biotechnology, and Biochemistry. 1992;56(11):1892-3. Smith RV, Rosazza JP. Microbial models of mammalian metabolism. J Pharm Sci. 1975;64(11):1737-59. Özlem Sultan A. In vitro cytotoxicity and cell viability assays: Principles, advantages, and disadvantages. In Genotoxicity. Marcelo LL, Sonia, S., editor: 10.5772/intechopen.71923: Rijeka, Croatia, 2017.Ch. 1. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1-2):55-63. Rui L, Kangcheng S, Jing-Xiang W, Xiao-Peng G, Zheng L, Xiaoyin G, et al. Structure of human phagocyte NADPH oxidase in the resting state. eLife 11:e83743 [Internet]. 2022. Available from: https://doi.org/10.7554/eLife.83743. Mishra SK, Bae YS, Lee Y-M, Kim J-S, Oh SH, Kim HM. Sesquiterpene Alcohol Cedrol Chemosensitizes Human Cancer Cells and Suppresses Cell Proliferation by Destabilizing Plasma Membrane Lipid Rafts. Frontiers in Cell and Developmental Biology. 2021;Volume 8 - 2020. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInofrmationOrabielal.pdf Cite Share Download PDF Status: Posted Version 1 posted 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. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6893326","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472676330,"identity":"addf01ea-0d76-45cc-97c0-baf8288ae142","order_by":0,"name":"Hanan G. 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Radwan","email":"","orcid":"","institution":"The University of Mississippi, University","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"M.","lastName":"Radwan","suffix":""},{"id":472676333,"identity":"fb8a957c-946e-4617-b672-3f6c06a481f1","order_by":3,"name":"Mickey Vinodh","email":"","orcid":"","institution":"Kuwait University","correspondingAuthor":false,"prefix":"","firstName":"Mickey","middleName":"","lastName":"Vinodh","suffix":""},{"id":472676334,"identity":"d11842e7-0b39-45c1-a609-d98e924236ce","order_by":4,"name":"Abbas Ali","email":"","orcid":"","institution":"The University of Mississippi, University","correspondingAuthor":false,"prefix":"","firstName":"Abbas","middleName":"","lastName":"Ali","suffix":""},{"id":472676335,"identity":"9d8de757-2aa6-42ea-bcb5-8725c6348852","order_by":5,"name":"Khaled Y. Orabi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACCQh1gIefB0SzwUWI0CLZQ6oWBoMzxGqRnJHA+Lii5o6M8ZkzBgwfyg6DRPBrkZZIYDY8c+wZj9nZHgPGGecOg0Twa5GTSGCTbGA7zGN2nseAmbftMIOcNFFa/h3mMe4HavlLjBZpkJbGtsM8Brw9BsyMQC3ShLRI9jxsNmzse8YjceZYwcGec+k8kvMf4NcicTz54MOGb3fs+XuSNz74UWYtJ3HmAH4tDAKJDXA2SC0PAfVAwE/IzFEwCkbBKBgFAHkrP40VobXTAAAAAElFTkSuQmCC","orcid":"","institution":"Kuwait University","correspondingAuthor":true,"prefix":"","firstName":"Khaled","middleName":"Y.","lastName":"Orabi","suffix":""}],"badges":[],"createdAt":"2025-06-14 10:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6893326/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6893326/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84984442,"identity":"17d808bd-3f6c-4d57-b35f-d57ed0306caa","added_by":"auto","created_at":"2025-06-19 14:07:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":43261,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram depicting the separation of two major compounds. Car-2 (t\u003csub\u003eR\u003c/sub\u003e = 7.5 min) was identified as pure carotol based on spectral analysis\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6893326/v1/47acd7173ccb582585ad15eb.png"},{"id":84985225,"identity":"72268b5e-9894-49e8-bc97-14fed7baade1","added_by":"auto","created_at":"2025-06-19 14:15:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":134523,"visible":true,"origin":"","legend":"\u003cp\u003eStructures of carotol, its metabolites and daucol.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6893326/v1/7c40a3252c4dc124559b14a1.png"},{"id":84984447,"identity":"fec29fa7-93a0-4fbb-966c-e52b1517f91d","added_by":"auto","created_at":"2025-06-19 14:07:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":189051,"visible":true,"origin":"","legend":"\u003cp\u003eCrystal structure (thermal ellipsoid representation; 30% probability) of CM1 \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eobtained from single crystal diffraction. Color code: gray-carbon; red-oxygen and black-hydrogen.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6893326/v1/c13f6495f698e3c59493539c.png"},{"id":84986757,"identity":"71bd76e4-1103-4904-afe5-cfe6e5e88719","added_by":"auto","created_at":"2025-06-19 14:31:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":155036,"visible":true,"origin":"","legend":"\u003cp\u003eCrystal structure of CM1 in which the chiral atoms are color-labeled.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6893326/v1/ec8db2e1c81c7878a066c7fe.png"},{"id":84986222,"identity":"8e11d0c4-3983-4379-a78b-c33db30133f5","added_by":"auto","created_at":"2025-06-19 14:23:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":533100,"visible":true,"origin":"","legend":"\u003cp\u003eDocking interactions of NADPH oxidase with: (A) Carotol, (B) CM1, (C) CM2, and (D) CM3. Hydrogen bonds and key interacting residues are highlighted to illustrate binding modes of each compound.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6893326/v1/55f22a1903a8285f988e7947.png"},{"id":84984449,"identity":"b5929fe4-522d-4b5a-8ce3-12a8bfa3eb0c","added_by":"auto","created_at":"2025-06-19 14:07:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":500190,"visible":true,"origin":"","legend":"\u003cp\u003eRoot mean square fluctuations (RMSF) of NADPH oxidase residues in complex with carotol, CM1, CM2, and CM3 over a 50 ns molecular dynamics simulation. The RMSF profiles illustrate the residue-wise flexibility upon binding of each compound, providing insight into the stability of the protein–ligand complexes.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6893326/v1/ae84594c8c9e56c3d358a584.png"},{"id":91016858,"identity":"b32fd3e3-5569-4264-bd6a-b7837987af28","added_by":"auto","created_at":"2025-09-10 17:08:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2573359,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6893326/v1/913f4ce7-04ef-402f-9929-5b7b18741686.pdf"},{"id":84984444,"identity":"49a52bf2-39c3-44c0-b57c-0571cf4f3bb7","added_by":"auto","created_at":"2025-06-19 14:07:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1494942,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInofrmationOrabielal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6893326/v1/4f8158c823aa2903ffa7285c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Microbial Biotransformation of the Sesquiterpene Carotol: Generation of Hydroxylated Metabolites with Potential Cytotoxic and Target-Specific Binding Activities","fulltext":[{"header":"Background","content":"\u003cp\u003eThe carrot (\u003cem\u003eDaucus carota\u003c/em\u003e L. subsp. \u003cem\u003ecarota\u003c/em\u003e, Family Apiaceae) is an herbaceous, biennial flowering plant native to Europe, Asia, and Africa. It ranks among the ten most economically significant vegetable crops worldwide (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Carrots provide substantial nutritional and health benefits. Historically, they were first used medicinally before being incorporated into diets as food (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Phytochemical-rich diets help prevent cancer, heart disease, neurological disorders, and birth defects (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Carrots are a unique vegetable due to their abundant carotenoids, phenolics, vitamin C, and polyacetylenes, which contribute to reduced risks of cardiovascular disease and cancer (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAncient Egyptians employed \u003cem\u003eD. carota\u003c/em\u003e as a stimulant, carminative, diuretic, anthelmintic, and a treatment for infantile diarrhea (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Additionally, \u003cem\u003eD. carota\u003c/em\u003e extracts are used for hepatic and renal insufficiency and skin disorders, including burns and furunculosis (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Wild \u003cem\u003eD. carota\u003c/em\u003e extracts exhibit iron-chelating, antioxidant (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), and antibacterial activities (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Carrot seed essential oil is widely utilized in food flavoring, perfumes, cosmetics, and soaps (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)and has demonstrated antibacterial, antioxidant, anti-aging, cytotoxic, and mosquito-repellent properties (\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16 CR17\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Major components of carrot seed oil include carotol, \u003cem\u003eα\u003c/em\u003e-pinene, geranyl acetate, \u003cem\u003eβ\u003c/em\u003e-pinene, daucol, limonene, \u003cem\u003eβ\u003c/em\u003e-bisabolene, geraniol, \u003cem\u003eβ\u003c/em\u003e-elemene, \u003cem\u003ecis\u003c/em\u003e-\u003cem\u003eβ\u003c/em\u003e-bergamotene, \u003cem\u003eγ\u003c/em\u003e-decalactone, \u003cem\u003eβ\u003c/em\u003e-farnesene, caryophyllene, methyl eugenol, caryophyllene oxide, nerolidol, eugenol, \u003cem\u003etrans\u003c/em\u003e-asarone, vanillin, \u003cem\u003eα\u003c/em\u003e-terpineol, terpinen-4-ol, coumarin, \u003cem\u003eγ\u003c/em\u003e-decanolactone, \u003cem\u003eβ\u003c/em\u003e-selinene, palmitic acid, and butyric acid (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCarotol, a sesquiterpene alcohol, is the predominant compound in carrot seed oil (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), displaying strong antifungal (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), cytotoxic (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), and mosquito-deterrent activities (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDrug metabolism studies traditionally rely on \u003cem\u003ein vivo\u003c/em\u003e animal models or \u003cem\u003ein vitro\u003c/em\u003e enzyme-based systems, such as microsomal preparations and tissue cultures. Microbial biotransformation provides an alternative, overcoming certain limitations of traditional methods. The concept of \"microbial models of mammalian metabolism\" involves using bacteria, yeasts, and fungi to simulate human drug metabolism. Microbial systems serve as efficient, selective, and scalable tools for generating metabolites that closely resemble those produced in mammalian systems (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Their advantages include operational efficiency under mild conditions and the ability to catalyze diverse reactions beyond their natural substrates while exhibiting chemo-, regio-, and stereoselectivity (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). These models facilitate metabolite production in quantities unattainable from animal-based systems or chemical synthesis, making them predictive tools for human metabolism.\u003c/p\u003e \u003cp\u003eFungi, as eukaryotes, share enzymatic similarities with mammals, reinforcing their role as models for mammalian metabolism. Previous studies have reported carotol biotransformation by \u003cem\u003eRhizopus oryzae\u003c/em\u003e, yielding two lactate derivatives: 9\u003cem\u003eα\u003c/em\u003e-hydroxydaucol-9-lactate and 9\u003cem\u003eα\u003c/em\u003e-hydroxydaucol-3,9-dilactate (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). These metabolites exhibited \u003cem\u003ein vitro\u003c/em\u003e inhibition of cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), and butyrylcholinesterase (BChE) (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Furthermore, carotol chemical modifications have yielded 3-bromcarotol ether, carotol aldehyde, 4-phenoxy carotol, and daucene (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), ketone mixtures (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e), and carota-1,4-dienaldehyde (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). To date, no carotol metabolites of human or mammalian origin have been reported.\u003c/p\u003e \u003cp\u003eThis study investigates the microbial biotransformation of carotol to generate novel derivatives in quantities sufficient for cytotoxicity assessment, with the aim of supporting their potential therapeutic applications. Furthermore, molecular docking analyses were performed to compare the binding interactions of the newly formed metabolites and carotol with NADPH oxidase, an enzyme implicated as a therapeutic target in cancer treatment strategies.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eSubstrate Preparation\u003c/h2\u003e \u003cp\u003ePure carotol (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) was obtained from carrot essential oil as previously described (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Briefly, therapeutic-grade carrot essential oil was purchased from Edens Garden (1322 Calle Avanzado, San Clemente, CA). Successive chromatographic purification yielded a carotol-rich fraction, which was further purified using preparative high-performance liquid chromatography (HPLC) to obtain pure carotol.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eMicroorganisms\u003c/h3\u003e\n\u003cp\u003eThe microbial cultures were sourced from the American Type Culture Collection (ATCC, Rockville, MD, USA), the Northern Regional Research Laboratories (NRRL), and Assiut University Mycology Center (AUMC). The following microorganisms were screened for their ability to catalyze the biotransformation of carotol: \u003cem\u003eAbsidia coerulea\u003c/em\u003e ATCC 6647, \u003cem\u003eFusarium\u003c/em\u003e sp. ATCC 11599, \u003cem\u003ePhanerochaete chrysosporium\u003c/em\u003e ATCC 24725, \u003cem\u003eRhizopus oryzae\u003c/em\u003e (syn. \u003cem\u003eR. nigricans\u003c/em\u003e) ATCC 34121, \u003cem\u003eRhizopus stolonifer\u003c/em\u003e ATCC 6227a, \u003cem\u003eAspergillus flavus\u003c/em\u003e AUMC 4787, \u003cem\u003eAspergillus niger\u003c/em\u003e NRRL 599, \u003cem\u003eCunninghamella blacksleeana\u003c/em\u003e AUMC, \u003cem\u003eCunninghamella echinulata\u003c/em\u003e NRRL 132, \u003cem\u003eMucor\u003c/em\u003e sp. AUMC, \u003cem\u003eRhizopus\u003c/em\u003e sp. ATCC 36060, \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e (Baker\u0026rsquo;s yeast), \u003cem\u003eCladosporium\u003c/em\u003e sp., \u003cem\u003eCordyceps sinensis\u003c/em\u003e AUMC, \u003cem\u003eBotrytis allii\u003c/em\u003e ATCC 9435, \u003cem\u003eStreptomyces griseus\u003c/em\u003e ATCC 13968, \u003cem\u003eStreptomyces spheroids\u003c/em\u003e ATCC 23965. Stock cultures were maintained on agar slants using media recommended by ATCC and stored at 4\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGeneral Experimental Procedure\u003c/h2\u003e \u003cp\u003eIR spectra were recorded as a chloroform film using Jasco FT/IR-4100 type A Spectrophotometer. The \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectra were obtained on a Bruker Avance II-600 spectrometer operating at 600 and 150 MHz, respectively. Both \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectra were recorded in CDCl\u003csub\u003e3\u003c/sub\u003e, and the chemical shift values were expressed in \u003cem\u003ed\u003c/em\u003e (ppm) relative to the internal standard TMS. For the \u003csup\u003e13\u003c/sup\u003eC NMR spectra, spectral editing was determined by DEPT. 2D NMR data were obtained using the standard pulse sequence of the Bruker Avance II-600 for COSY, HSQC and HMBC. EIHRMS analysis were carried out on a Thermo Scientific High-Resolution GC/MS-DFS (Double Focusing Sector) mass spectrometer. Column chromatography was carried out on Silica gel 60 (230\u0026ndash;400 mesh ASTM, Merck). TLC analysis was carried out on silica gel 60 F254 (Merck) plates. Compounds were detected by UV and \u003cem\u003ep\u003c/em\u003e-anisaldehyde/ H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e spraying reagent followed by heating at 105\u0026deg;C for 1\u0026ndash;2 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparative HPLC\u003c/h2\u003e \u003cp\u003eA Waters preparative HPLC system, equipped with a 2998 Photodiode Array detector, a 2545 quaternary gradient module, a FlexInject manual dual injector, and a Fraction Collector III, was used to isolate pure carotol. The separation was performed on a Waters XBridge\u0026trade; Prep C18 (10 \u0026times; 150 mm, 5 \u0026micro;m) column using isocratic elution with 30% methanol in acetonitrile at a flow rate of 1.7 mL/min. The detection wavelength was set at 218 nm. The sample was prepared by dissolving 100 \u0026micro;L of carotol-rich fraction in 1.5 mL of 30% methanol in acetonitrile, filtered through a 0.1 \u0026micro;m PTFE membrane, and injected into the system.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eScreening Procedure\u003c/h3\u003e\n\u003cp\u003eThe microbial screening was conducted following a standard two-stage protocol (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Fermentation medium alpha was prepared using 2% glucose, 0.5% yeast extract, 0.5% peptone, 0.5% NaCl, and 0.5% K₂HPO₄ in distilled water, sterilized at 121\u0026deg;C for 15 minutes. In Stage I, 250-mL culture flasks containing 50 mL of sterile medium alpha were inoculated with the selected microbial strains and incubated at 150 rpm for 72 hr at room temperature. Stage II involved transferring 5 mL of the Stage I culture to fresh medium, followed by 24 hr of incubation before substrate addition. Carotol (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) was prepared as a 15% solution in \u003cem\u003eN,N\u003c/em\u003e-dimethylformamide (DMF) and added to Stage II cultures at a final concentration of 0.5 mg/mL, then the cultures were incubated at 5 \u0026ordm;C for two weeks.\u003c/p\u003e \u003cp\u003eControl groups included substrate control where sterile medium with carotol (5 mg/100 \u0026micro;L DMF) incubated without microorganisms at 5 \u0026ordm;C. However, in culture controls microorganisms were grown under identical conditions without carotol. After two weeks, cultures were harvested and analyzed via thin-layer chromatography (TLC).\u003c/p\u003e\n\u003ch3\u003ePreparative-Scale Fermentation\u003c/h3\u003e\n\u003cp\u003eSince \u003cem\u003eAbsidia coerulea\u003c/em\u003e ATCC 6647 exhibited clear capability to metabolize carotol, it was cultured in five 250-mL flasks containing 50 mL of medium alpha (Stage I). A total of 304 mg of carotol, dissolved in 450 \u0026micro;L DMF, was distributed among nine 1-L flasks containing 24-hr-old Stage II cultures. After 14 days, the cultures were analyzed using TLC with an \u003cem\u003en\u003c/em\u003e-hexane\u0026ndash;acetone (8.5:1.5) solvent system. Visualization with \u003cem\u003ep\u003c/em\u003e-anisaldehyde/H₂SO₄ revealed the transformation of carotol (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) into three polar metabolites.\u003c/p\u003e \u003cp\u003eEach culture flask was filtered, and the filtrates were extracted with chloroform (1.5 L \u0026times; 3). The combined extracts were dried over anhydrous Na₂SO₄ and evaporated to dryness under reduced pressure at 38\u0026deg;C, yielding a brownish residue (525 mg). Purification was performed using flash silica gel column chromatography (68 g, 2.7 \u0026times; 33 cm, 230\u0026ndash;400 mesh) with CHCl₃\u0026ndash;acetone (9.3:0.7) as the eluent. Fractions were collected (25 mL each) and pooled together based on TLC analysis.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSingle Crystal X-Ray Diffraction Analysis\u003c/h2\u003e \u003cp\u003eSingle crystals of CM1 used in this study were obtained via slow solvent evaporation. X-ray diffraction data were collected at 150 K using a Bruker X8 Prospector diffractometer equipped with Cu-Kα radiation. Reflection frames were integrated using the Bruker SAINT software package with a narrow-frame algorithm. The crystal structure was solved with the SHELXTL software suite and refined using SHELXL-2019/3. All non-hydrogen atoms were refined anisotropically, while hydrogen atoms were placed in calculated positions and refined using a riding model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxicity Evaluation\u003c/h2\u003e \u003cp\u003eThe cytotoxic activities of pure carotol (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) and its metabolites were evaluated against human liver (HepG-2), colon (HCT-116), breast (MCF-7), and lung (A-549) carcinoma cell lines, as well as normal human lung fibroblasts (MRC-5), using a standard cell viability (MTT) assay (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). The assay was conducted at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt.\u003c/p\u003e \u003cp\u003eCancer and normal cells were seeded into 96-well plates at a density of 5 \u0026times; 10⁴ cells per well and incubated for 24 hours prior to treatment. Test compounds were added in triplicate at twelve different concentrations. Vehicle controls (media only or media containing 0.5% DMSO) were included. After a 24-hour incubation period, cell viability was assessed via the MTT assay.\u003c/p\u003e \u003cp\u003eBriefly, the media were replaced with 100 \u0026micro;L of fresh RPMI-1640 medium, followed by the addition of 10 \u0026micro;L of a 12 mM MTT stock solution (5 mg/mL in PBS) to each well, including untreated controls. Plates were incubated at 37\u0026deg;C in a 5% CO₂ atmosphere for 4 hours. Subsequently, 85 \u0026micro;L of medium was removed from each well, and 50 \u0026micro;L of DMSO was added to dissolve the resulting formazan crystals. The plates were thoroughly mixed and incubated for an additional 10 minutes at 37\u0026deg;C. Absorbance was measured at 590 nm using a microplate reader (SunRise, TECAN Inc., USA). Cell viability percentages were calculated, and IC₅₀ values (\u0026micro;M) were determined from dose-response curves using GraphPad Prism software (San Diego, CA, USA) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDocking Study\u003c/h2\u003e \u003cp\u003eThe crystal structure of human NADPH oxidase was downloaded from protein data bank (pdb code\u0026thinsp;=\u0026thinsp;8gz3) (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). The structure was published in 2022, with resolution\u0026thinsp;=\u0026thinsp;3.3 \u0026Aring;. Molecular docking was done using Molecular Operating Environment (MOE 2022.02) package license purchased from Chemical Computing Group Inc., Sherbooke St, Montreal, QC, Canada. Triangle matcher was used as a placement method. London ΔG scoring method was applied to estimate the free energy of binding (Kcal/mol). Root mean square deviation RMSD of the docked ligands was computed to measure the deviation from the co-crystalized ligand. The root mean square fluctuations RMSF was computed to compare between carotol and its metabolites.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003ePreparative HPLC fractionation resulted in the isolation of two major compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), designated as Car-1 (t\u003csub\u003eR\u003c/sub\u003e = 5.9 min) and Car-2 (t\u003csub\u003eR\u003c/sub\u003e = 7.5 min). Spectral analysis confirmed that Car-2 corresponds to pure carotol.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWith the exception of a single reference (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), a comprehensive literature search revealed no prior documentation or investigation detailing either microbial or mammalian metabolic pathways of carotol. Although carotol has been previously investigated for its biological activities, including cytotoxic and antimicrobial properties, its metabolic fate, particularly \u003cem\u003evia\u003c/em\u003e microbial biotransformation, remains largely unexplored. The current findings thus provide novel insights into the metabolism of carotol, laying the foundation for future pharmacokinetic and bioactivity studies, and underscoring the value of microbial systems in elucidating the biotransformation pathways of bioactive sesquiterpenes.\u003c/p\u003e \u003cp\u003eThe metabolic capabilities of seventeen microbial cultures were assessed for carotol biotransformation, and \u003cem\u003eAbsidia coerulea\u003c/em\u003e ATCC 6647 exhibited significant metabolic activity. Consequently, this strain was selected for preparative-scale fermentation, yielding three metabolites: CM1, CM2, and CM3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The metabolites were purified using column chromatography, with multiple fractions collected and analyzed by TLC. Fractions 19, 21, and 25 were identified as promising based on their TLC profiles compared to the culture control.\u003c/p\u003e \u003cp\u003eFraction 19 (123 mg) was subjected to further purification over a silica gel 60 column (17 g, 30 \u0026times; 1.5 cm) with toluene-acetone (9:1) as the mobile phase. Fractions (5 mL each) were collected and analyzed by TLC using the same solvent system. Similar fractions were pooled, affording a pure, crystalline, non-UV-active compound, designated CM1 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) (104.3 mg, 30% yield), with R\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e=026 (toluene-acetone; 8.5:1.5).\u003c/p\u003e \u003cp\u003eLikewise, fraction 25 (36.8 mg), after further purification yielded 24.7 mg (6.64% yield) of pure, non-UV-active amorphous powder, designated CM2 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) with an R\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e value\u0026thinsp;=\u0026thinsp;0.27 (toluene-acetone; 8.5:1.5).\u003c/p\u003e \u003cp\u003eMoreover, fraction 21 (30.5 mg) was purified using a silica gel column (15 \u0026times; 1.4 cm, 6.4 g) and eluted with toluene-acetone (8.7:1.3). Fractions (2 mL each) were collected and monitored by TLC using the same mobile phase. Similar fractions were pooled, yielding CM3 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) (11.5 mg, 3.28% yield) as a pure, non-UV-active amorphous powder. The R\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e value was 0.27 in toluene-acetone (9:1).\u003c/p\u003e \u003cp\u003eCM1 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) structure was characterized based on detailed spectroscopic analysis. Its molecular formula was established as C₁₅H₂₆O₃, supported by the molecular ion peak at \u003cem\u003em/z\u003c/em\u003e 254.1873, along with its \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR data (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The \u003csup\u003e13\u003c/sup\u003eC NMR spectrum exhibited 15 resonances, categorized as three singlets, four doublets, four triplets, and four quartets. Compared to carotol, CM1 lacked the characteristic olefinic signals observed at δ\u003csub\u003eC\u003c/sub\u003e 138.8 (singlet) and δ\u003csub\u003eC\u003c/sub\u003e 122.3 (doublet), indicating the absence of the double bond present in the parent compound.\u003c/p\u003e \u003cp\u003eConversely, CM1 displayed four oxygenated aliphatic carbon signals at δ\u003csub\u003eC\u003c/sub\u003e 71.5 (doublet, C-3), δ\u003csub\u003eC\u003c/sub\u003e 85.6 (singlet, C-4), δ\u003csub\u003eC\u003c/sub\u003e 92.6 (singlet, C-7), and δ\u003csub\u003eC\u003c/sub\u003e 73.3 (doublet, C-9). Signal assignments were confirmed through 2D NMR experiments (HSQC and HMBC), which enabled unambiguous correlation of proton and carbon resonances. Notably, several of these chemical shifts closely matched those reported for daucol (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), a major sesquiterpene constituent of carrot seed oil (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The identity and absolute configuration of CM1 were further confirmed by single crystal X-ray diffraction.\u003c/p\u003e \u003cp\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\u003e\u003csup\u003e1\u003c/sup\u003eH NMR spectroscopic data (600 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) of carotol and its metabolites.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e#\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eẟ\u003csub\u003eH,\u003c/sub\u003e multiplicity (\u003cem\u003eJ\u003c/em\u003e in Hz)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarotol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCM1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCM2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCM3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.73, dd (12.1, 8.4)\u003c/p\u003e \u003cp\u003e2.29, d (16.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.22, dd (12.0, 12.0)\u003c/p\u003e \u003cp\u003e1.75, dd (12.9, 6.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.79, dd (12.2, 5.6)\u003c/p\u003e \u003cp\u003e2.27, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.39, m\u003c/p\u003e \u003cp\u003e1.55, dd (12.0, 10.2)\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\u003e5.35, d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.76, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.85, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.62, d (10.2)\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\u003e2.10, dd (5.4, 5.4)\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.34, dd (5.4, 1.2)\u003c/p\u003e \u003cp\u003e1.85, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.35, m\u003c/p\u003e \u003cp\u003e1.92, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.46, m\u003c/p\u003e \u003cp\u003e1.88, m\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\u003e1.65, m\u003c/p\u003e \u003cp\u003e1.97, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.54, ddd (13.0, 13.0, 4.8)\u003c/p\u003e \u003cp\u003e2.14, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.25, m\u003c/p\u003e \u003cp\u003e1.78, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.68, m\u003c/p\u003e \u003cp\u003e1.74, m\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\u003e1.82, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.85, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.45, d (8.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.47, m\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\u003e1.52, m\u003c/p\u003e \u003cp\u003e1.67, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.47, ddd (7.2, 7.2, 0.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.56, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.45, m\u003c/p\u003e \u003cp\u003e1.63, m\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\u003e1.32, m\u003c/p\u003e \u003cp\u003e1.58, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.45, dd (13.2, 0.6)\u003c/p\u003e \u003cp\u003e1.80, ddd (13.8, 7.2, 0.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.60, m\u003c/p\u003e \u003cp\u003e1.65, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.47, m\u003c/p\u003e \u003cp\u003e1.93, m\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\u003e0.96, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.36, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.38, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.01, s\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\u003e1.68, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.30, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.37, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.24, s\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\u003e1.83, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.10, m\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.64, m\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.02, d (6.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.04, d (6.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.47, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.98, d (6.6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.97, d (6.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.17, d (6.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.48, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.90, d (6.6)\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\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\u003e\u003csup\u003e13\u003c/sup\u003eC NMR spectroscopic data (150 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) of carotol and its metabolites.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e#\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eẟc, multiplicity \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarotol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCM1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCM2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCM3\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\u003e49.3, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.8, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.5, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e47.5, s\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\u003e38.8, t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.8, t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.4, t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.7, t\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\u003e122.3, d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.5, d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71.1, d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75.4, d\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\u003e138.8, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85.6, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.4, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75.5, s\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\u003e29.6, t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.2, t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.1, t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35.5, t\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\u003e34.6, t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.7, t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.8, t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.3, t\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\u003e84.8, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e92.6, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e91.9, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e83.6, s\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\u003e52.7, d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58.8, d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60.1, d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56.6, d\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\u003e24.6, t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e73.3, d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74.3, d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.5, t\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\u003e39.6, t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.4, t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e43.7, t\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34.1, t\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\u003e21.7, q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.1, q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.0, q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.9, q\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\u003e25.2, q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.1, q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.3, q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.4, q\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\u003e27.8, d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.4, d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74.1, s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28.9, d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.3, q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26.0, q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.1, q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23.5, q\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.6, q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.7, q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.2, q\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.8, q\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\u003e \u003csup\u003ea\u003c/sup\u003e Carbon multiplicities were determined by DEPT 135\u0026deg;.\u003c/p\u003e \u003cp\u003eThe crystal structure of CM1 obtained from single crystal diffraction analysis is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and important crystallographic parameters of this crystal are provided in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\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\u003eSummary on the various crystallographic parameters of the compound \u003cb\u003eCM1\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eValue\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChemical formula\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eM\u003c/em\u003e\u003csub\u003er\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e286.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrystal system, space group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOrthorhombic, \u003cem\u003eP\u003c/em\u003e2\u003csub\u003e1\u003c/sub\u003e2\u003csub\u003e1\u003c/sub\u003e2\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature (K)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ea\u003c/em\u003e, \u003cem\u003eb\u003c/em\u003e, \u003cem\u003ec\u003c/em\u003e (\u0026Aring;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.4762 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), 15.7204 (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), 15.9679 (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα, β, γ (\u0026deg;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90, 90, 90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e (\u0026Aring;\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1625.67 (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eZ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRadiation type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCu \u003cem\u003eK\u003c/em\u003eα\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026micro; (mm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrystal size (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.21 \u0026times; 0.19 \u0026times; 0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiffractometer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBruker \u003cem\u003eAPEX\u003c/em\u003e-II CCD\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsorption correction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMulti-scan\u003c/p\u003e \u003cp\u003e\u003cem\u003eSADABS2016\u003c/em\u003e/2 - Bruker AXS area detector scaling and absorption correction\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eT\u003c/em\u003e\u003csub\u003emin\u003c/sub\u003e, T\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.86, 0.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of measured, independent \u0026amp; observed [\u003cem\u003eI\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;2σ(\u003cem\u003eI\u003c/em\u003e)] reflections\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10554, 2864, 2713\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR\u003c/em\u003e\u003csub\u003eint\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.037\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(sin θ/λ)\u003csub\u003emax\u003c/sub\u003e (\u0026Aring;\u003csup\u003e\u0026minus;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.596\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR\u003c/em\u003e[F\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;2σ(F\u003csup\u003e2\u003c/sup\u003e)], wR(F\u003csup\u003e2\u003c/sup\u003e), S\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.037, 0.099, 1.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of reflections\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2864\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of parameters\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e189\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH-atom treatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConstrained\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔρ\u003csub\u003emax\u003c/sub\u003e, Δρ\u003csub\u003emin\u003c/sub\u003e (e \u0026Aring;\u003csup\u003e\u0026minus;3\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.13, \u0026minus;\u0026thinsp;0.20\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\u003eCM1 crystallizes in the orthorhombic crystal system, space group \u003cem\u003eP\u003c/em\u003e2\u003csub\u003e1\u003c/sub\u003e2\u003csub\u003e1\u003c/sub\u003e2\u003csub\u003e1\u003c/sub\u003e. Methanol molecules are incorporated into the crystal lattice as space-filling solvents, stabilizing the crystal structure through non-bonding interactions with adjacent CM1 molecules. The crystal structure of CM1 reveals the presence of six chiral carbon atoms in the molecule. These chiral centers include C-1 (\u003cem\u003eR\u003c/em\u003e), C-3 (\u003cem\u003eS\u003c/em\u003e), C-4 (\u003cem\u003eS\u003c/em\u003e), C-7 (\u003cem\u003eS\u003c/em\u003e), C-8 (\u003cem\u003eS\u003c/em\u003e), and C-9 (\u003cem\u003eS\u003c/em\u003e), which are color-labeled in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Thus, CM1 identity was established as 9α-hydroxydaucol (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the other hand, CM2 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) was determined to have the molecular formula C₁₅H₂₆O₄, as established by its molecular ion peak at \u003cem\u003em/z\u003c/em\u003e 270.1826, supported by its \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR data (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The \u003csup\u003e13\u003c/sup\u003eC NMR spectrum displayed 15 carbon resonances, comprising four singlets, three doublets, four triplets, and four quartets. Five of these resonances were clearly in the oxygenated aliphatic region. Comparison with the NMR data of CM1 revealed the presence of an additional oxygenated carbon in CM2, resonating at δ\u003csub\u003eC\u003c/sub\u003e 74.1 as a singlet, and the absence of the doublet observed in CM1 at δ\u003csub\u003eC\u003c/sub\u003e 29.4 (assigned to C-13). This suggested that the additional oxygen functionality in CM2 was located at C-13. This assignment was further supported by downfield chemical shifts of the adjacent methyl carbons, C-14 (δ\u003csub\u003eC\u003c/sub\u003e 31.1, Δ\u0026thinsp;=\u0026thinsp;4.9 ppm) and C-15 (δ\u003csub\u003eC\u003c/sub\u003e 30.2, Δ\u0026thinsp;=\u0026thinsp;7.5 ppm), indicating deshielding due to proximity to the oxygenated center. The remaining spectral data were consistent with those observed for CM1 (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Based on these findings, the structure of CM2 was established as 9α,13-dihydroxydaucol (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLikewise, CM3 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) was shown to have a molecular formula of C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e based on its HRMS that shows the molecular ion peak at \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e 256.2032 and its \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR data. Again, there were 15 carbon resonances distributed as three singlets, three doublets, five triplets, and four quarters. Of these resonances, three appear in the oxygenated aliphatic region at δ\u003csub\u003eC\u003c/sub\u003e 75.4 (doublet, C-3), δ\u003csub\u003eC\u003c/sub\u003e 75.5 (singlet, C-4), and δ\u003csub\u003eC\u003c/sub\u003e 83.6 (singlet, C-7). Comparison of the \u003csup\u003e13\u003c/sup\u003eC NMR data of CM3 with those published for daucol indicates that CM3 is a diol derivative resulting from the cleavage of the daucol ether linkage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Notably, CM3 is confirmed as a true microbial metabolite, as pure carotol was used as the sole substrate in the microbial cultures. The unambiguous assignment of all \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC resonances was aided through DEPT 135\u0026deg;, HSQC, and HMBC experiments.\u003c/p\u003e \u003cp\u003eCM1 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e): colorless prims (MeOH): mp 122\u0026ndash;124\u0026deg;C; IR (neat) υ\u003csub\u003emax\u003c/sub\u003e 3434 (OH), 2935 (saturated C-H) cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003csup\u003e1\u003c/sup\u003eH NMR (CDCl\u003csub\u003e3\u003c/sub\u003e, 600 MHz) see Table\u0026nbsp;1; \u003csup\u003e13\u003c/sup\u003eC NMR (CDCl\u003csub\u003e3\u003c/sub\u003e, 150 MHz) see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; EIHRMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e 254.1873 [M]\u003csup\u003e+\u003c/sup\u003e (calcd for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e 254.1882).\u003c/p\u003e \u003cp\u003eCM2 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e): colorless amorphous powder; IR (neat) υ\u003csub\u003emax\u003c/sub\u003e 3386 (OH), 2935 (saturated C-H) cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003csup\u003e1\u003c/sup\u003eH NMR (CDCl\u003csub\u003e3\u003c/sub\u003e, 600 MHz) see Table\u0026nbsp;1; \u003csup\u003e13\u003c/sup\u003eC NMR (CDCl\u003csub\u003e3\u003c/sub\u003e, 150 MHz) see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; HRMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e 270.1826 [M]\u003csup\u003e+\u003c/sup\u003e (calcd for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e 270.1831).\u003c/p\u003e \u003cp\u003eCM3 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e): gummy residue; IR (neat) υ\u003csub\u003emax\u003c/sub\u003e 3380 (OH), 2910 (saturated C-H) cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; \u003csup\u003e1\u003c/sup\u003eH NMR (CDCl\u003csub\u003e3\u003c/sub\u003e, 600 MHz) see Table\u0026nbsp;1; \u003csup\u003e13\u003c/sup\u003eC NMR (CDCl\u003csub\u003e3\u003c/sub\u003e, 150 MHz) see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; HRMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e 256.2032 [M]\u003csup\u003e+\u003c/sup\u003e (calcd for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e 256.2038).\u003c/p\u003e \u003cp\u003eThe cytotoxic effects of carotol and its metabolites were evaluated against several cancer cell lines (HepG2, HCT-116, MCF-7, and A549) as well as the normal human lung fibroblast cell line (MRC-5). Among the tested compounds, the parent molecule, carotol, exhibited the most potent cytotoxic activity, followed by its metabolites CM2, CM3, and CM1, in descending order of efficacy, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Notably, carotol and its metabolites demonstrated selectivity toward cancer cells over normal cells.\u003c/p\u003e \u003cp\u003ePrevious studies have reported the cytotoxicity of carrot seed essential oils from various geographical sources, along with pure carotol, using green monkey kidney epithelial cells (VERO) and human hypopharyngeal squamous cell carcinoma cells (FaDu). Moroccan and French essential oils exhibited comparable cytotoxic effects, while the Polish essential oil displayed lower activity. In contrast, pure carotol demonstrated moderate cytotoxicity on both VERO and FaDu cell lines, with no apparent selectivity (IC₅₀ = 39.7 \u0026micro;g/mL and 38.3 \u0026micro;g/mL, respectively) (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\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\u003eIC\u003csub\u003e50\u003c/sub\u003e values (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) of carotol along with its metabolites.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCell line\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M)\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarotol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCM1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCM2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCM3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHepG-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e52.34\u0026nbsp;\u0026plusmn; 4.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e220.74\u0026thinsp;\u0026plusmn;\u0026thinsp;12.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e154.45\u0026thinsp;\u0026plusmn;\u0026thinsp;9.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e195.53\u0026thinsp;\u0026plusmn;\u0026thinsp;10.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHCT-116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e25.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e339.38\u0026thinsp;\u0026plusmn;\u0026thinsp;15.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e180.64\u0026thinsp;\u0026plusmn;\u0026thinsp;9.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e226.38\u0026thinsp;\u0026plusmn;\u0026thinsp;11.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCF-7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e68.38\u0026nbsp;\u0026plusmn; 6.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e400.75\u0026thinsp;\u0026plusmn;\u0026thinsp;20.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e212.85\u0026thinsp;\u0026plusmn;\u0026thinsp;13.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e242.14\u0026thinsp;\u0026plusmn;\u0026thinsp;15.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA-549\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e28.65\u0026nbsp;\u0026plusmn; 2.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e225.38\u0026thinsp;\u0026plusmn;\u0026thinsp;13.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e138.21\u0026thinsp;\u0026plusmn;\u0026thinsp;9.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e205.24\u0026thinsp;\u0026plusmn;\u0026thinsp;10.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMRC-5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e175.61\u0026thinsp;\u0026plusmn;\u0026thinsp;10.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e466.17\u0026thinsp;\u0026plusmn;\u0026thinsp;20.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e247.62\u0026thinsp;\u0026plusmn;\u0026thinsp;11.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e316.36\u0026thinsp;\u0026plusmn;\u0026thinsp;15.17\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\u003e \u003csup\u003ea\u003c/sup\u003e HepG-2: hepatocellular carcinoma, HCT-116: colon carcinoma, MCF-7: breast carcinoma, A-549: lung carcinoma, MRC-5: normal human lung fibroblasts.\u003c/p\u003e \u003cp\u003eIt has been previously documented that sesquiterpene alcohols can act as inhibitors of NADPH oxidase, an enzyme implicated in oxidative stress and cancer progression. The inhibition of NADPH oxidase is regarded as a key mechanism contributing to the anticancer potential of this class of compounds (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In the present study, molecular docking simulations were performed to assess the binding interactions, free binding energy (ΔG), and binding stability of carotol and its metabolites with NADPH oxidase. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, carotol exhibited the most favorable binding profile, with the highest binding affinity (ΔG = \u0026minus;\u0026thinsp;5.65 kcal/mol) and the lowest RMSD value (1.35 \u0026Aring;), indicating a stable and specific interaction. CM2 followed with a ΔG of \u0026minus;\u0026thinsp;5.41 kcal/mol and an RMSD of 1.50 \u0026Aring;. In contrast, CM1 and CM3 showed lower binding affinities (ΔG = \u0026minus;\u0026thinsp;5.11 and \u0026minus;\u0026thinsp;5.16 kcal/mol, respectively), suggesting weaker interactions with the enzyme. These computational findings are in good agreement with the experimental cytotoxicity data presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and further support the hypothesis that carotol and CM2 exert anticancer effects, at least in part, through NADPH oxidase inhibition.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDocking results against NADPH oxidase (PBD\u0026thinsp;=\u0026thinsp;8gz3)\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=\"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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFree energy of binding\u003c/p\u003e \u003cp\u003e(Kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRMSD (\u0026Aring;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInteracted residues\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarotol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-5.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHis338, Trp361\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-5.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrp361\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCM2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-5.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGly359, His338\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCM3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-5.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThr362\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\u003eFurther analysis of the molecular docking interactions revealed distinct binding modes for carotol and its metabolites with NADPH oxidase (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Carotol formed a hydrogen bond with His338 and exhibited potential hydrophobic interactions with Trp361 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), suggesting a stable and favorable binding conformation. CM1 established a single hydrogen bond with the \u0026ndash;NH group of Trp361 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating a moderately stable interaction. Notably, CM2 was the only metabolite to exhibit an intramolecular hydrogen bond, which may contribute to enhanced binding stability. Additionally, CM2 formed two hydrogen bonds with Gly359 and His338 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), reinforcing its strong binding affinity. In contrast, CM3 interacted via a single hydrogen bond with Thr362 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), suggesting a comparatively weaker interaction. These distinct binding interactions further support the differential binding affinities observed in the docking scores and correlate well with the experimental cytotoxicity data.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the stability of binding interactions with NADPH oxidase, molecular dynamics (MD) simulations were performed over a 50 ns timescale for the best-docked complexes of carotol and its metabolites (CM1, CM2, and CM3). The resulting trajectories were analyzed to compute the root mean square fluctuations (RMSF) of NADPH oxidase residues in each complex, providing insight into local flexibility and binding stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Carotol exhibited the lowest RMSF values (blue trace), indicating a highly stable interaction with the enzyme throughout the simulation. CM2 displayed RMSF values comparable to those of carotol (orange trace), supporting its strong and stable binding. In contrast, CM1 (purple trace) showed higher fluctuations relative to carotol and CM2, suggesting a less stable interaction. The metabolite CM3 (green trace) demonstrated the highest residue fluctuations, with RMSF values exceeding 3.5 \u0026Aring; in certain regions, indicating the least stable binding among the tested compounds. These findings align well with the cytotoxicity results and further support the superior stability and potential biological activity of CM2 relative to the other metabolites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study successfully isolated pure carotol from carrot essential oil and evaluated its microbial biotransformation by diverse microorganisms. Among the strains tested, \u003cem\u003eAbsidia coerulea\u003c/em\u003e ATCC 6647 demonstrated significant metabolic activity, producing three distinct metabolites (CM1, CM2, and CM3). Structural elucidation of these metabolites was achieved through comprehensive spectroscopic analyses and single-crystal X-ray diffraction, confirming their identities as oxygenated sesquiterpene derivatives. Cytotoxicity assays revealed that carotol exhibited the strongest anticancer activity against multiple human carcinoma cell lines, with its metabolites showing moderate but selective cytotoxicity toward cancer versus normal cells. Molecular docking and dynamics simulations supported these findings, indicating that carotol and CM2 bind NADPH oxidase with higher affinity and stability, potentially underpinning their anticancer mechanisms through enzyme inhibition. This work provides novel insights into the microbial metabolism of carotol, a bioactive sesquiterpene with promising anticancer properties, and highlights the utility of microbial biotransformation as a tool for generating and characterizing novel derivatives. Future studies are warranted to further explore the pharmacokinetics, \u003cem\u003ein vivo\u003c/em\u003e efficacy, and safety profiles of carotol metabolites, ultimately contributing to the development of new therapeutic agents derived from natural products.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAPI: Active Pharmaceutical Ingredient\u003c/p\u003e\n\u003cp\u003eATCC: American Type Culture Collection\u003c/p\u003e\n\u003cp\u003eAUMC: Assiut University Mycology Center\u003c/p\u003e\n\u003cp\u003eBChE: Butyrylcholinesterase\u003c/p\u003e\n\u003cp\u003eCM1, CM2, CM3: Carotol metabolites 1, 2, and 3\u003c/p\u003e\n\u003cp\u003eCOX-2: Cyclooxygenase-2\u003c/p\u003e\n\u003cp\u003eCOSY: Correlation Spectroscopy\u003c/p\u003e\n\u003cp\u003eDEPT: Distortionless Enhancement by Polarization Transfer\u003c/p\u003e\n\u003cp\u003eDMF: \u003cem\u003eN,N\u003c/em\u003e-Dimethylformamide\u003c/p\u003e\n\u003cp\u003eEIHRMS: Electron Impact High-Resolution Mass Spectrometry\u003c/p\u003e\n\u003cp\u003eHPLC: High-Performance Liquid Chromatography\u003c/p\u003e\n\u003cp\u003eHSQC: Heteronuclear Single Quantum Coherence\u003c/p\u003e\n\u003cp\u003eHMBC: Heteronuclear Multiple Bond Correlation\u003c/p\u003e\n\u003cp\u003eIC₅₀: Half Maximal Inhibitory Concentration\u003c/p\u003e\n\u003cp\u003eIR: Infrared Spectroscopy\u003c/p\u003e\n\u003cp\u003eMD: Molecular Dynamic\u003c/p\u003e\n\u003cp\u003eMTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide\u003c/p\u003e\n\u003cp\u003eNRRL: Northern Regional Research Laboratories\u003c/p\u003e\n\u003cp\u003ePBS: Phosphate-Buffered Saline\u003c/p\u003e\n\u003cp\u003ePTFE: Polytetrafluoroethylene\u003c/p\u003e\n\u003cp\u003eRMSD: Root Mean Square Deviation\u003c/p\u003e\n\u003cp\u003eRMSF: Root Mean Square Fluctuations\u003c/p\u003e\n\u003cp\u003eTLC: Thin-Layer Chromatography\u003c/p\u003e\n\u003cp\u003eTMS: Tetramethylsilane\u003c/p\u003e\n\u003cp\u003eUV: Ultraviolet\u003c/p\u003e\n\u003cp\u003eXRD: X-Ray Diffraction\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpectroscopic and single crystal X-ray analyses were carried out at the Research Sector Projects Unit (RSPU), College of Science, Kuwait University, with support from grant numbers GS01/01 and GS01/03 (for spectral analyses), and GS03/08 (for X-ray analyses).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKYO and MMR conceived and designed the experiments; MMR, KYO and AA isolated carotol; HGS purified carotol; HGS and KYO performed the biotransformations; KYO and HGS analyzed the spectral data; MV performed single crystal X-ray analysis; MAK performed docking study; KYO interpreted the results; KYO and HGS wrote the paper; KYO administered and supervised the project. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpectral analyses were done at RSPU facilities, College of Science, Kuwait University. Cytotoxicity evaluations were carried out at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKYO: Department of Pharmaceutical Chemistry, College of Pharmacy, Kuwait University, Safat 13110, Kuwait.\u003c/p\u003e\n\u003cp\u003eHGS: Department of Pharmaceutical Chemistry, College of Pharmacy, Kuwait University, Safat 13110, Kuwait, and Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, Cairo, Egypt.\u003c/p\u003e\n\u003cp\u003eMAK: Department of Pharmaceutical Chemistry, College of Pharmacy, Kuwait University, Safat 13110, Kuwait. Current address: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Ain Helwan, Cairo 11795, Egypt.\u003c/p\u003e\n\u003cp\u003eMMR: Department of Biomolecular Sciences and National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS 38677, USA.\u003c/p\u003e\n\u003cp\u003eAA: National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, MS 38677, USA.\u003c/p\u003e\n\u003cp\u003eMV: Research Sector Project Units, College of Science, Kuwait University, Safat 13060, Kuwait.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 1D and 2D NMR spectra of carotol and its metabolites accompany this paper at https://doi.org/xxxxxxxxxxxxx.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSimon PW. Domestication, historical development, and modern breeding of carrot. 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Carrot seed essential oil-Source of carotol and cytotoxicity study. Industrial Crops and Products. 2016;92:109-15.\u003c/li\u003e\n\u003cli\u003eAlves-Silva J, Zuzarte M, Gonc\u0026cedil; alves M, Cavaleiro C, Cruz M, Cardoso S, Salgueiro L. New claims for wild carrot (Daucus carota ssp carota) essential oil. Evid Based Complement Altern Med. 2016.\u003c/li\u003e\n\u003cli\u003eJasicka-Misiak I, Lipok J, Nowakowska EM, Wieczorek PP, Młynarz P, Kafarski P. Antifungal activity of the carrot seed oil and its major sesquiterpene compounds. Z Naturforsch C J Biosci. 2004;59(11-12):791-6.\u003c/li\u003e\n\u003cli\u003eKhan I, Abourashed E. Leung\u0026rsquo;s Encyclopedia of common natural ingredients used in food, drugs, and cosmetics, 3rd ed John Wiley \u0026amp; Sons, Inc., Hoboken, US, NJ. 2010. p. 147\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eRadulovic N, \u0026ETH;ordevic N, Stojanovic-Radic Z. Volatiles of Balkan Endemic Daucus guttatus ssp: zahariadii and cultivated and wild-growing D. carota- a comparison study. Food Chem 2011;125:35-43.\u003c/li\u003e\n\u003cli\u003eMahboubi. M, Kazempour N, Mahboubi A. The efficacy of essential oils as natural preservatives in vegetable oil. J Diet Suppl 2014;11:334-46.\u003c/li\u003e\n\u003cli\u003eAli A, Radwan MM, Wanas AS, Khan IA. Repellent activity of carrot seed essential oil and its pure compound, carotol, against mosquitoes. Journal of the American Mosquito Control Association. 2018;34(4):272-80.\u003c/li\u003e\n\u003cli\u003eMusnaini M, Fransisca S, Leslie W. Effectiveness of cream formulation of carrot seed oil as anti-aging. International Journal of Health and Pharmaceutical (IJHP). 2022;3(2):331\u0026ndash;40.\u003c/li\u003e\n\u003cli\u003eManjunath C, Malpani A, Mahurkar N. 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Available from: https://doi.org/10.7554/eLife.83743.\u003c/li\u003e\n\u003cli\u003eMishra SK, Bae YS, Lee Y-M, Kim J-S, Oh SH, Kim HM. Sesquiterpene Alcohol Cedrol Chemosensitizes Human Cancer Cells and Suppresses Cell Proliferation by Destabilizing Plasma Membrane Lipid Rafts. Frontiers in Cell and Developmental Biology. 2021;Volume 8 - 2020.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Carotol, Absidia coerulea, Microbial biotransformation, Sesquiterpene alcohol, Hydroxydaucol, Carrot seed essential oil, Molecular docking, Anticancer activity","lastPublishedDoi":"10.21203/rs.3.rs-6893326/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6893326/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCarotol, a major sesquiterpene alcohol found in carrot essential oil, exhibits promising biological activities including cytotoxic effects against various cancer cell lines. Despite its bioactivity, the metabolic fate and biotransformation pathways of carotol remain largely unexplored, particularly through microbial systems that can offer novel insights into its structural modifications and potential pharmacological applications.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, seventeen microbial strains were screened for their ability to biotransform carotol, with \u003cem\u003eAbsidia coerulea\u003c/em\u003e ATCC 6647 identified as the most effective strain. Preparative-scale fermentation using this strain led to the isolation and purification of three metabolites (CM1, CM2, and CM3). Spectroscopic analysis, including 1D and 2D NMR, HRMS, and single crystal X-ray diffraction, elucidated the structures of these metabolites as 9α-hydroxydaucol (CM1), 9α,13-dihydroxydaucol (CM2), and a diol derivative of daucol (CM3). Cytotoxicity evaluation against human liver (HepG-2), colon (HCT-116), breast (MCF-7), and lung (A-549) carcinoma cell lines, alongside normal lung fibroblasts (MRC-5), revealed that carotol exhibited the highest anticancer activity followed by CM2, CM3, and CM1. Molecular docking studies against human NADPH oxidase demonstrated that carotol and CM2 have stronger binding affinities and more stable interactions compared to the other metabolites, suggesting NADPH oxidase inhibition as a possible mechanism for their anticancer effects.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study provides the first comprehensive microbial biotransformation pathway for carotol, leading to the identification of novel hydroxylated metabolites with varying cytotoxic activities. The findings highlight the potential of \u003cem\u003eAbsidia coerulea\u003c/em\u003e as a biocatalyst for producing bioactive carotol derivatives and underscore the relevance of NADPH oxidase inhibition in their anticancer mechanism. These results lay a foundation for future pharmacokinetic and drug development research involving carotol and its metabolites.\u003c/p\u003e","manuscriptTitle":"Microbial Biotransformation of the Sesquiterpene Carotol: Generation of Hydroxylated Metabolites with Potential Cytotoxic and Target-Specific Binding Activities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-19 14:07:42","doi":"10.21203/rs.3.rs-6893326/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"40127443-662e-44a0-914a-b00b153d2e50","owner":[],"postedDate":"June 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-10T17:08:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-19 14:07:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6893326","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6893326","identity":"rs-6893326","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}