Hormetic and Differential Apoptotic Effects of Bixin and Norbixin in Human Glioblastoma Cells (U87-MG)

Hormetic and Differential Apoptotic Effects of Bixin and Norbixin in Human Glioblastoma Cells (U87-MG) | 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 Hormetic and Differential Apoptotic Effects of Bixin and Norbixin in Human Glioblastoma Cells (U87-MG) Nicoli Dolores Gonçalves Correa, Sirlene da Silva Rodrigues, Carlos Roberto Jorge Soares, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8620168/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 Glioblastoma multiforme (GBM) is an aggressive, treatment-resistant brain tumor with limited therapeutic success. Natural apocarotenoids, such as Bixin (lipophilic) and Norbixin (hydrophilic), derived from annatto ( Bixa orellana ), have demonstrated anticancer potential, but their effects on glioblastoma remain largely unexplored. This study evaluated, under in vitro conditions and for the first time, the cytotoxic and pro-apoptotic activities of Bixin and Norbixin against the human glioblastoma cell line U87-MG. Molecular characterization was performed using FTIR and 1 H-NMR spectroscopy, and reactivity was estimated via Frontier Molecular Orbitals (FMO) analysis. Cell viability was quantified using the tetrazolium-based colorimetric assay (MTS), and apoptosis was monitored via phase-contrast microscopy to determine IC 50 values and evaluate morphological changes. Bixin exhibited significantly higher potency, displaying a distinctive biphasic dose-response pattern consistent with a hormetic effect at low concentrations. Bixin yielded IC 50 values of 231.9 µM (viability) and 130.1 µM (apoptosis), indicating that apoptotic signaling precedes metabolic viability loss (AIIC 50 < MTSIC 50 ). Norbixin showed IC 50 of 532.9 µM for viability; 400.1 µM for apoptosis and a slower apoptotic induction profile. Morphological analyses corroborated these findings, confirming classic apoptotic features (e.g., cell shrinkage and membrane blebbing), particularly under cytotoxic concentrations, and were more pronounced in Bixin-treated cells. These results reveal distinct cytotoxic and apoptotic profiles between the two apocarotenoids, driven by their structural and physicochemical differences (lipophilicity vs. hydrophilicity). Collectively, the findings support Bixin and Norbixin as promising molecular scaffolds for further investigation, rather than therapeutic agents per se, in the context of carotenoid-based strategies targeting malignant glioma cells, warranting further mechanistic and in vivo validation. Cancer Biology Biological Chemistry Natural Product Chemistry Human glioblastoma Bixin Norbixin Cytotoxicity Apoptosis Hormesis Redox modulation U87MG cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Glioblastoma (GBM) represents the most aggressive and universally fatal primary malignancy of the central nervous system, characterized by rapid proliferation, diffuse infiltration, and marked resistance to conventional treatment modalities such as radiotherapy and alkylating chemotherapy. Despite current multimodal therapeutic approaches, the median overall survival for GBM patients remains dismal, at approximately 15 months, underscoring an urgent and critical demand for novel and efficacious therapeutic strategies [ 1 ]. In this context, natural products with modulatory effects on redox homeostasis and minimal systemic toxicity have garnered significant interest as potential adjuvants or alternatives in cancer therapy. Among these, Bixin and Norbixin, apocarotenoids derived from the seeds of Bixa orellana (annatto), have emerged as promising candidates due to their diverse biological activities relevant to oncological applications. Bixin has demonstrated notable antineoplastic effects across various experimental cancer models. In human melanoma cells (A2058), Bixin not only inhibited proliferation and migration but also induced apoptosis and G2/M cell cycle arrest, while potentiating the cytotoxicity of dacarbazine. These biological activities were mechanistically linked in previous studies to the induction of reactive oxygen species (ROS) and enhanced lipid peroxidation [ 2 ]. Furthermore, cis -Bixin, a geometric isomer, exhibited cytotoxicity across multiple tumor cell lines including lung (A549), prostate (PC3), breast (MCF7), colon (HCT-116), and thyroid (DRO), with half-maximal inhibitory concentrations (IC 50 ) ranging between 10 and 50 µM. Its cytotoxic mechanism has been proposed to involve ROS generation concomitant with inhibition of the thioredoxin (Trx) and thioredoxin reductase (TrxR) system, critical regulators of cellular redox homeostasis [ 3 ]. Supporting these findings, in silico molecular docking studies have suggested Bixin’s potential interactions with key oncogenic targets such as BRAF, MMP9, and TP53, all of which are frequently mutated or dysregulated in diverse cancer types [ 4 ]. Conversely, Norbixin, the water-soluble dicarboxylic acid derivative of Bixin, has been primarily investigated in non-oncological contexts. In experimental models of retinal degeneration, Norbixin exhibited pronounced neuroprotective properties, effectively preserving photoreceptor integrity and visual function through antioxidant mechanisms that attenuate oxidative damage and lipofuscin accumulation [ 5 ]. Recent investigations have further expanded the understanding of Norbixin’s biological effects through the development of its synthetic derivatives. Specifically, the Norbixin amide conjugate BIO203 demonstrated potent anti-inflammatory and antioxidative properties in preclinical models of retinal degeneration, preserving photoreceptor viability and reducing oxidative stress–induced cellular injury [ 6 ]. Collectively, these results highlight the context-dependent biological activities of Norbixin and its analogs, suggesting that their efficacy and mode of action may vary significantly depending on tissue type and the local redox environment, with limited data currently available for glioblastoma models. Pharmacokinetic studies in humans have established the systemic bioavailability of both Bixin and Norbixin following oral administration of annatto-based formulations. Notably, Norbixin exhibits higher plasma concentrations and prolonged persistence relative to Bixin, which is more rapidly cleared [ 7 ]. Furthermore, Bixin, but not Norbixin, has been reported to induce cytochrome P450 enzymes CYP1A1 and CYP1A2 in HepG2 hepatocarcinoma cells highlighting functional differences in metabolic interactions and gene regulation between these structurally related apocarotenoids [ 8 ]. Emerging evidence underscores the centrality of oxidative stress, mitochondrial dysfunction, and dysregulated antioxidant defenses in glioblastoma progression and therapeutic resistance. GBM cells display elevated basal levels of reactive oxygen species (ROS), which paradoxically sustain tumorigenic signaling while activating adaptive antioxidant mechanisms that confer resistance to conventional therapies. Therapeutic strategies that perturb this fragile redox equilibrium, either by amplifying ROS generation beyond tolerable thresholds or by suppressing endogenous antioxidant systems, have shown promise in experimental models in selectively inducing glioma cell death. Given Bixin’s established capacity to modulate redox-sensitive pathways such as the Trx/TrxR system, Nrf2-mediated transcription, and mitochondrial oxidative phosphorylation, it emerges as a compelling candidate for exploratory investigation in glioblastoma cells. Norbixin, although structurally similar, is hypothesized to exhibit divergent activity due to its enhanced polarity, differential membrane permeability, and potential for distinct subcellular distribution, which necessitates comparative evaluation in controlled in vitro settings. Beyond their redox-modulating properties, both Bixin and Norbixin possess physicochemical features that enable multimodal characterization, integrating chemical, structural, and biological dimensions. Advanced analytical techniques, including nuclear magnetic resonance (NMR) and Fourier-transform infrared spectroscopy (FTIR), provide detailed insights into molecular integrity and conformational characteristics. Correlating these precise physicochemical data with cellular assays, such as dose–response cytotoxicity evaluations in U87MG glioblastoma cells, facilitates the establishment of structure–activity relationships and the development of preliminary mechanistic hypotheses. The present study therefore employs a comprehensive approach to characterize Bixin and Norbixin and to evaluate their differential cytotoxic and pro-apoptotic potential against glioblastoma, aiming to provide foundational in vitro evidence to support future mechanistic and translational studies. 2. Materials and Methods 2.1 Plant Material and Sample Preparation The seeds and pods of Bixa orellana were collected in Manaus, Amazonas State, Brazil, at geographic coordinates 3°06′ S, 60°01′ W, and an altitude of 92 m. Following collection, the fruits were washed with distilled water and dried in a convection oven at 30°C for 3 days. Subsequently, 100 g of Bixa orellana seeds were defatted by washing with 400 mL of n -hexane ( ≥ 98%, Sigma-Aldrich) under constant magnetic stirring at room temperature (RT, 25°C) for 1 h. The mixture was filtered through Whatman paper, and this procedure was repeated twice to ensure the complete removal of the lipid fraction (rich in tocotrienols and geranylgeraniol) and other potential impurities. The defatted seeds were then dried under vacuum in a desiccator for 48 h at RT. 2.2 Synthesis and Purification of Apocarotenoids 2.2.1 Bixin Extraction and Recrystallization Bixin was obtained via solid–liquid extraction. Defatted Bixa orellana seeds (25 g) were soaked in 50 mL of anhydrous ethanol (( ≥ 99.5%, Sigma-Aldrich) for 24 h in the dark. The mixture was filtered (Whatman paper), and the filtrate was stored at 4°C overnight to induce Bixin crystallization. The resulting Bixin crystals were collected by a second filtration. Recrystallization was performed using anhydrous ethanol. The crude crystals were dissolved in anhydrous ethanol in a 250 mL beaker at RT, filtered, and the filtrate was stored for 24 h at 4°C. The purified Bixin crystals were collected by filtration, suspended in phosphate-buffered saline (PBS, pH 7.4) under magnetic stirring for 15 min, and subsequently lyophilized using a freeze dryer (Thermo Electron Corporation/Modulyod) for 48 h at − 32°C under vacuum (500 µATM). The resulting powders were sealed and stored protected from light at 4°C. 2.2.2 Norbixin Hemisynthesis Norbixin was obtained by base hydrolysis (hemisynthesis) of Bixin present in the seeds. Bixa orellana seeds (100 g) were soaked in 200 mL of an ethanolic ammonia–water mixture (composed of 70 g of 10% v/v ammonium hydroxide (Sigma-Aldrich, 33%) solution and 70 g of anhydrous ethanol). The seeds were maintained under constant magnetic stirring in the ammoniacal solution at RT for 1 h, promoting the hydrolysis of the Bixin methyl ester group to form the Norbixin carboxylate salt. The ethanolic ammonia–water mixture was filtered and then acidified to pH 4.0 using 85% phosphoric acid (Sigma Aldrich). The solution was then slowly cooled to between − 5°C and 0°C over 12 h to promote Norbixin crystallization. The crystallized Norbixin was collected by filtration, thoroughly washed with phosphate-buffered saline (PBS, pH 7.4), and subsequently lyophilized. The final powders were sealed and stored protected from light at 4°C. 2.3 Physicochemical Characterization The purity and structure of crystalized Bixin and Norbixin were assessed using UV-Vis spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and proton ( 1 H) nuclear magnetic resonance (NMR) spectroscopy. These analyses were performed to confirm the chemical identity and integrity of the compounds, without implying direct biological efficacy. 2.3.1 UV-Vis Spectroscopy UV–Vis spectra of Bixin and Norbixin were recorded between 300 nm and 600 nm at RT using a Varian Cary 50 spectrophotometer. Stock solutions (0.6 g/L in anhydrous ethanol) were diluted appropriately (e.g., 2 µL stock to 3 mL total volume) in a quartz cuvette. 2.3.2 NMR Spectroscopy 1 H and spectra were acquired on a Bruker DPX-400 Avance spectrometer (400 MHz) at RT. Deuterated chloroform (CDCl₃, ≥ 99.5%, Sigma-Aldrich) was used as the solvent. Samples were dissolved in 0.55 mL of CDCl₃ and transferred to 5 mm NMR tubes. One-dimensional 1 H experiments utilized a 5 mm dual 1 H direct detection probe, a 9.6 µs π/2 pulse, a 16 k points for induction decay (acquisition time 2.5 s), eight scans, two dummy scans, a 3 s recycling delay, and a spectral window of 10 ppm. Chemical shifts (δ) are reported in parts per million (ppm) relative to the internal standard tetramethylsilane (TMS) and the residual solvent signal (CDCl 3 , δH 7.26). Spectral data analysis was performed using Master New version 9.0. These measurements provide structural confirmation but do not alone predict cellular effects. 2.3.3 FT-IR Spectroscopy ATR-FTIR spectra were obtained using an Shimadzu IR Tracer 100 spectrometer in Attenuated Total Reflection (ATR) mode, utilizing a ZnSe crystal over the 4000–500 cm⁻¹ wavenumber range. Spectra were averaged from 64 scans with a resolution of 4 cm⁻¹. Interpretation of functional groups was intended for chemical characterization rather than efficacy prediction. 2.4 Biological Assays 2.4.1 Cell Culture The U87-MG human glioblastoma cell line (BCRJ code 0241, Rio de Janeiro, Brazil) was cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were maintained in a humidified atmosphere with 5% CO 2 at 37°C. The culture medium was refreshed every 2 days. Standard cell culture conditions were applied, and all experiments were exploratory in nature. 2.4.2 Cell Viability (MTS) Assay and Apoptotic Effects Cellular viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (ref. M5655, Sigma-Aldrich). U87-MG cells (5,000 cells/well) were seeded into 96-well plates (ref. 655090, Greiner). After 24 h (at ≈ 40% confluency), the culture medium was replaced, and cells were treated with different concentrations of Bixin and Norbixin for 48 h under standard incubation conditions. To mitigate interference from the colored compounds, the treatment medium was aspirated and replaced with fresh medium prior to the MTS assay. Subsequently, the MTS reagent was dissolved in culture medium and added to each well at a final concentration of 1.09 mM. Cells were incubated for 2 h at 37°C. The reduction of the MTS tetrazolium compound to a soluble formazan product by mitochondrial dehydrogenases was quantified by measuring absorbance at 490 nm using a microplate reader (Thermo Electron Corporation/Multiskan EX). Background subtraction was performed using wells containing medium but no cells. Results are expressed as the percentage of viable cells relative to the untreated control. These experiments provide preliminary information on cell response and do not establish clinical relevance. All experiments were performed in triplicate. 2.4.3 Morphological Analysis and Apoptotic Index (AI) Morphological changes and the Apoptotic Index ( AI ) were evaluated using digital image analysis via inverted phase-contrast microscopy. U87-MG cells (1.10 4 cells/well) were seeded in 96-well plates and incubated for 48 h with Bixin or Norbixin at concentrations ranging from 100 µM to 800 µM. Dimethyl sulfoxide (DMSO, 1.5%) served as the vehicle control. After incubation, the medium was removed, and cells were washed once with PBS. Digital micrographs were captured under a Nikon Eclipse TS100 inverted phase-contrast microscope (100 x magnification) using an integrated digital camera. Images from at least five randomly selected fields per well were acquired for each condition. Apoptotic cells were identified based on characteristic morphological features, including cell shrinkage, membrane blebbing, and the formation of apoptotic bodies. The total number of cells ( \(\:{N}_{total}\) ) and the number of apoptotic cells ( \(\:{N}_{Apoptotic}\) ) were manually counted using the Cell Counter plugin in ImageJ National Institutes of Health, Bethesda, MD, USA). The AI was calculated using the following equation: $$\:AI\left(\%\right)=\left(\frac{{N}_{Apoptotic}}{{N}_{total}}\right).100$$ The mean AI was determined from three independent experiments, each performed in triplicate. These measurements are exploratory and aimed at understanding potential cellular effects in vitro. Statistical analysis All data are expressed as the mean ± standard deviation (SD) from three independent experiments, each performed in triplicate. Normality was assessed using the Shapiro–Wilk test. Statistical significance was determined using Student’s t -test (for two groups) or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (for multiple groups) for normally distributed data. For non-normally distributed data, the Kruskal–Wallis test followed by Dunn’s multiple comparison test was applied. A p -value of < 0.05 was considered statistically significant. Statistical analyses and graph generation were performed using Python (version 3.12) with the libraries pandas (v2.2), numpy (v1.26), scipy (v1.13), statsmodels (v0.14), scikit-posthocs (v0.9), matplotlib (v3.9), and seaborn (v0.13). Representative images shown in the figures were selected to closely reflect the mean values of the respective groups accurately. Statistical conclusions are limited to the experimental context and do not imply translational or clinical outcomes. 3. Results 3.1 UV–Vis Spectroscopy and Electronic Band Gap The Ultraviolet-Visible (UV–Vis) absorption spectra of Bixin and Norbixin in ethanol (Fig. 1 ) exhibited the characteristic three-band pattern typical of conjugated polyenes [ 9 – 10 ]. The maxima for Bixin were recorded at 429 nm, 450 nm, and 508 nm, and for Norbixin at 445 nm, 470 nm, and 500 nm, consistent with literature [ 11 – 12 ]. These features confirm the presence of an extended \(\:\) -conjugated system, where electronic transitions correspond to π→π* excitations. These spectroscopic features confirm molecular structure and electronic properties, serving as a physicochemical basis for subsequent cellular experiments. The observed spectral shifts relative to published values are likely due to solvatochromic effects and partial cis – trans isomerization in bixin, and the increased molecular polarity and modulation of conjugation due to de-esterification in Norbixin [ 11 , 12 ]. From a molecular orbital perspective, the π→π* transitions correspond to excitations between the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO). The optical band gaps (E g ) were determined from the linear region of Tauc plots (Fig. 2 ), yielding 2.56 eV for Bixin and 2.36 eV for Norbixin. Using a reference HOMO energy of -5.6 eV [ 15 ], the estimated LUMO energies were − 3.04 eV and − 3.24 eV, respectively. The narrower band gap in Norbixin, attributed to its extended \(\:\) -conjugation and higher polarity, indicates easier electronic excitation, which is relevant from a physicochemical perspective for potential redox activity in vitro [ 16 – 18 ]. 3.2 NMR Analysis and Conversion De gree The 1 H NMR spectrum of Bixin (Fig. 3 ) confirmed its conjugated diester structure, showing characteristic resonances for methoxy protons (OCH 3 ) at δ 3.80 ppm, aliphatic protons at δ 1.25 ppm and δ 1.55–1.75 ppm, allylic protons at δ 1.95–2.10 ppm, and the polyene system (CH = CH) spanning δ 5.05–6.95 ppm [ 12 , 19 – 20 ]. The 1 H NMR spectrum of Norbixin confirmed the intended hydrolysis, showing a marked decrease in the intensity of the methoxy signal at δ 3.80 ppm, signifying the cleavage of the methyl ester group(s). The persistence of the olefinic signals (δ 5.85–6.95 ppm) confirmed the preservation of the conjugated polyene backbone. Quantitative analysis of the normalized methoxy resonance intensity (δ 3.80 ppm) revealed a conversion degree of approximately 75%. This composition was used as obtained for exploratory cell-based assays, providing a chemically defined mixture for in vitro evaluation. This high conversion confirms the predominant formation of the monocarboxylic monoester Norbixin, providing a chemically defined mixture for exploratory in vitro assays. This mixture (75% Norbixin, 25 residual Bixin) was deemed suitable for cell-based assays, as the combination of these redox-active species may provide enhanced reactive oxygen species (ROS) quenching capacity, which is particularly relevant in the glioblastoma context [ 21 – 23 ]. 3.3 FTIR Analyses The FTIR spectra of the Bixin and Norbixin (Fig. 4 ) highlighted the structural difference resulting from the hemisynthesis. Bixin exhibited a strong band at 1710 cm − 1 , corresponding to the C = O stretching of the ester carbonyl (C–O–C vibrations between 1200–1155 cm − 1 ), confirming its esterified form. Norbixin showed characteristic broad absorption bands in the 3750–3400 cm − 1 region (O–H stretching) and C = O signals in the 1670 cm − 1 range (terminal carboxyls), confirming the presence of carboxylic acid (COOH) groups. Both compounds showed C = C stretching bands in the 1530–1430 cm − 1 region, confirming the integrity of the polyene conjugated system. These FTIR data further support the chemical identity and purity of the compounds used in subsequent in vitro assays. 3.4 Differential Cytotoxicity and Apoptotic Induction 3.4.1 Cytotoxicity Assessment (MTS Assay) Dose–response analysis of U87-MG glioblastoma cells revealed a significant differential cytotoxic effect between the two apocarotenoids (Fig. 5 ). Bixin displayed pronounced cytotoxicity, with an IC 50 of 231.9 ± 8.3 µM. Notably, cell viability exhibited a transient increase, reaching a maximum of 145.5% in the concentration range of 100 µM to 175 µM before the onset of the cytotoxic effect observed at 200 µMand above. The steep Hill slope (14.7 ± 7.3) suggests a rapid, cooperative mechanism of action following this initial peak. Norbixin (Fig. 5 ) exhibited lower potency, with an IC 50 of 532.9 ± 17.2 µM (more than double that of Bixin), and a shallower Hill slope (5.94 ± 0.85), consistent with a more gradual and less abrupt reduction in cell viability. These results indicate that the lipophilicity conferred by the esterified structure of Bixin significantly enhances its cytotoxic potency against U87-MG cells, likely by facilitating faster cellular uptake and accumulation. 3.4.2 Morphological Changes and Apoptotic Index Apoptosis-related morphological alterations were assessed by inverted phase-contrast microscopy (Figs. 7 and 8 ). At cytotoxic concentrations, cells treated with both compounds displayed hallmark apoptotic features, including cytoplasmic condensation, cell rounding, membrane blebbing, and the formation of apoptotic bodies [ 24 – 25 ]. Quantification via the Apoptotic Index (AI) confirmed a differential mechanism of action between the apocarotenoids (Fig. 9 ). For bixin, the IC 50 for apoptotic induction (≈130.1 µM) was markedly lower than the IC 50 for viability reduction (MTSIC 50 = 231.9 µM). This AIIC 50 < MTSIC 50 relationship strongly indicates that apoptosis is an early and predominant mechanism of Bixin-induced cell death in U87-MG cells. In contrast, Norbixin exhibited considerably higher IC50 values for both apoptosis (400.1 µM) and viability (532.9 µM), consistent with a slower and less potent apoptotic response. These findings describe relative differences in apoptotic induction within this cell culture model and support a structure–activity relationship, whereby the higher lipophilicity and esterified nature of Bixin may facilitate its diffusion and interaction with intracellular targets (e.g., mitochondria), potentially contributing to increased apoptotic signaling compared to the more polar Norbixin [ 26 ]. Consequently, Bixin may serve as a promising scaffold for exploring pro-apoptotic activity in glioblastoma cells in vitro, highlighting the influence of molecular structure on cellular uptake and redox activity in a controlled experimental model. 4. Discussion Glioblastoma multiforme (GBM) remains one of the most therapeutically challenging malignancies. Despite the current standard-of-care treatments, GBM is characterized by high rates of recurrence and poor overall survival [ 27 – 28 ]. The limited long-term efficacy of conventional therapies is largely attributed to factors such as intrinsic multidrug resistance (MDR), often mediated by ATP-binding cassette (ABC) transporters [ 29 – 30 , 38 ], and significant treatment-related neurotoxicity. Consequently, the investigation of adjuvant or complementary therapeutic strategies, particularly those derived from natural products with minimal systemic toxicity, represents a promising avenue for exploratory investigation. Our study aimed to address a significant gap in the literature by providing the first comparative evaluation of the cytotoxic and pro-apoptotic potential of Bixin and Norbixin in the human U87-MG glioblastoma cell line. Bixin has previously demonstrated broad antineoplastic effects across various in vitro models, including melanoma and lung cancer, often involving mechanisms linked to cell cycle arrest and apoptosis [ 31 – 37 ]. The present findings extend these observations to a glioblastoma cell model under controlled in vitro conditions. The observed biological activity finds a strong rationale in the compounds' electronic structure. The UV–Vis analysis revealed a smaller HOMO–LUMO energy gap (E g ) for Norbixin (2.36 eV) compared to Bixin (2.56 eV) (Figs. 1 – 2 ). This narrow band gap significantly enhances the apocarotenoids' propensity to participate in electron transfer processes, a fundamental requirement for the generation and scavenging of ROS [ 39 – 40 ]. From a physicochemical perspective, this dual capability may be relevant for modulating redox balance in cancer cell models, where molecules may act as pro-oxidants under specific conditions. The high conjugation and physicochemical differences, corroborated by FTIR and NMR analyses (Figs. 3 – 4 ), thus provide a molecular basis for hypothesizing selective redox modulation in U87-MG cells. Our dose–response evaluation established a clear structure–activity relationship (SAR) driven by the differential polarity of the apocarotenoids. Bixin exhibited significantly higher cytotoxic potency (IC 50 = 231.9 µM or 91.5 g/mL) compared to norbixin (IC 50 = 532.9 µM or 202.79 g/mL) (Figs. 5 – 6 ). Based on established criteria for natural product activity [ 41 – 43 ], Bixin is classified as moderately active, while Norbixin falls into the weakly active range against U87-MG cells. These classifications apply specifically to in vitro screening contexts. The superior efficacy of Bixin is primarily attributed to its esterified and highly lipophilic nature. This increased lipophilicity may facilitate passive diffusion across the lipid bilayer of the plasma and mitochondrial membranes, leading to faster intracellular accumulation and potentially more direct interaction with apoptotic regulators compared to the more polar, dicarboxylated Norbixin. Furthermore, Bixin exhibited a steep Hill slope (14.7 ± 7.3), indicative of a highly cooperative or rapid, all-or-nothing cytotoxic mechanism once the threshold concentration is reached. Conversely, Norbixin's shallower Hill slope (5.94 ± 0.85) suggests a more gradual reduction in cell viability under the experimental conditions employed. A critical finding in the Bixin dose-response curve was the transient increase in cell viability in the concentration range of 100 µM to 175 µM, peaking at 145.5% viability, before the onset of cytotoxicity at 200 µM and higher. This biphasic pattern is consistent with a hormetic effect, as defined in vitro [ 44 ], where a low dose of an otherwise toxic or stressful agent elicits a beneficial or stimulatory response, while high doses are inhibitory. This hormetic behavior was observed exclusively in vitro and should be interpreted within this experimental framework. At these non-cytotoxic concentrations (100–175 µM), we propose that Bixin, a known antioxidant carotenoid, functions as a mild stressor. This subtle perturbation likely triggers an adaptive stress response in U87-MG cells, often mediated through the Nrf2/Keap1 signaling pathway. Nrf2 activation results in the transcriptional upregulation of cytoprotective genes, including Phase II detoxification enzymes and antioxidant proteins (e.g., HO-1 and NQO1). This adaptive supercompensation mechanism will enhances the cell's endogenous defense capacity, leading to an apparent increase in cell viability or proliferation. Such mechanisms are proposed based on existing literature and were not directly measured in this study. The transition from the hormetic (stimulatory) phase to the cytotoxic (inhibitory) phase above 200 µM suggests a saturation of the cellular adaptive capacity. At these higher concentrations, the inherent pro-oxidant activity of Bixin, particularly when accumulated in the mitochondrial membrane due to its lipophilicity, may become dominant. The resulting mitochondrial dysfunction, increased reactive oxygen species (ROS) generation, and collapse of the membrane potential are plausible contributors, to the commitment of cell apoptosis via the intrinsic pathway. The ability of Bixin to traverse cell membranes effectively is, therefore, hypothesized to be relevant for both its hormetic effects at low concentrations and its potent cytotoxic action at high concentrations. In stark contrast, the Norbixin dose-response curve did not show a significant increase in cell viability at low concentrations, failing to elicit the characteristic hormetic effect. Norbixin's lack of a hormetic phase is most likely attributable to its hydrophilic nature. Being highly water-soluble, Norbixin may have reduced capacity to cross the lipophilic cell and mitochondrial membranes to reach and trigger intracellular sensors (like Keap1/Nrf2) or induce the subtle mitochondrial stress required to activate the adaptive response. Consequently, Norbixin's action is predominantly inhibitory, and occurred at higher concentrations, further supporting the role of lipophilicity in modulating cellular responses in this model system. Although cytotoxic potency is modest compared to conventional chemotherapeutics, the IC 50 values suggest that these apocarotenoids may serve as exploratory scaffolds for selective, mechanism-based cellular effects in vitro. Such a role remains speculative and requires validation in additional experimental models. The identification of a hormetic window for Bixin is nonetheless relevant for experimental design, highlighting the importance of dose selection in studies involving redox-active compounds. To further elucidate the MOA, we conducted morphological and quantitative analyses of apoptosis (Figs. 7 – 9 ). Both compounds induced classic hallmark apoptotic features (cell shrinkage, membrane blebbing, etc.), consistent with programmed cell death [ 45 ]. Crucially, the comparative assay revealed that for Bixin, the IC 50 for apoptotic induction (≈ 130.1 µM) was substantially lower than the IC 50 for metabolic viability reduction (231.9 µM). This AIIC 50 < MTSIC 50 relationship confirms that apoptosis is the early and predominant event in the cytotoxic cascade of Bixin, preceding the loss of general metabolic function. Conversely, Norbixin required higher concentrations for both effects, suggesting a slower and less efficient activation of apoptotic pathways. Mechanistically, this pro-apoptotic effect may involve mitochondrial perturbation and ROS generation, as suggested in the literature [ 46 ]. Carotenoids, particularly those with higher lipophilicity, are known to integrate into cellular membranes and modulate Bcl-2 family proteins, thereby enhancing caspase-dependent apoptosis [ 47 ]. While consistent with existing literature, these mechanisms were not directly assessed in the present study. The pronounced difference between Bixin and Norbixin underscores how subtle structural modifications, such as the ester hydrolysis, can profoundly influence the bioavailability, subcellular distribution, and resulting cytotoxic and pro-apoptotic efficacy. Conclusions This study provides, to the best of our knowledge, the first evidence that the natural apocarotenoids Bixin and Norbixin exert differential cytotoxic and pro-apoptotic effects in U87-MG human glioblastoma cells. Bixin exhibited significantly greater potency, displaying a characteristic biphasic dose-response profile indicative of a hormetic effect at low concentrations, followed by robust cytotoxicity at high concentrations. The IC 50 for apoptosis was markedly lower than that for overall metabolic viability, indicating that programmed cell death is an early and prominent event associated with its cytotoxicity. In contrast, Norbixin showed weaker and slower apoptotic induction, consistent with its higher polarity and reduced lipophilicity. These findings underscore the critical role of molecular structure and lipophilicity in influencing cellular uptake and apoptotic efficiency within this experimental model. Overall, Bixin emerges as a promising molecular scaffold for further in vitro investigation, rather than a therapeutic agent, in the rational design of carotenoid-derived compounds targeting aggressive glioma cells. Additional mechanistic studies and in vivo validation are required to further explore its mode of action and potential relevance in neuro-oncological research. Declarations Credit authorship contribution statement Alvaro Antonio Alencar de Queiroz: Writing – review & editing, Writing – original draft, Software, Investigation, Visualization, Formal analysis, Conceptualization. Carlos Roberto Jorge Soares: Writing – review, Investigation, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Formal analysis, Data curation, Conceptualization. Nicoli Dolores Gonçalves Correa: Investigation, Methodology, Data curation, Formal analysis, Validation. Sirlene da Silva Rodrigues : Investigation, Methodology, Validation, Resources. Funding This research was partially supported by National Council for Scientific and Technological Development - CNPq (PQ 307047/2023-7/ DT 302970/2025-8) and by Coordination for the Improvement of Higher Education Personnel-CAPES. Data availability Not applicable. Declaration of Competing Interest The authors declare that they have no conflicts of interest that could have influenced the work reported in this study. Declaration of generative AI and AI-assisted technologies in the manuscript preparation process The authors declare that no generative artificial intelligence (AI) or AI-assisted technologies were used in the preparation of this manuscript. References Sipos D et al (2025) Glioblastoma: clinical presentation, multidisciplinary management, and long-term outcomes. Cancers 17(1):146 de Oliveira J Jr. et al (2019) Bixin, an apocarotenoid isolated from Bixa orellana L., sensitizes human melanoma cells (A2058) to dacarbazine-induced apoptosis through ROS-mediated cytotoxicity. 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J Biomol Struct Dyn 42(22):12244–12258 Lima IT et al (2022) Organic electronics from nature: computational investigation of the electronic and optical properties of the isomers of bixin and norbixin present in the achiote seeds. Molecules 27(7):2138 Atjanasuppat K et al (2009) In vitro screening for antihelminthic and antitumour activity of ethnomedicinal plants from Thailand. J Ethnoparmacol 123(3):475–482 Omoruyi SI (2025) Investigating the antiproliferative properties of Amaryllidaceae plant species and their bioactive compounds on brain tumour cell lines. Phytomedicine 25:787–799 Kangra K et al (2025) Incredible use of plant-derived bioactives as anticancer agents. RSC Adv 15:1721–1746 Calabrese EJ, Mattson MP (2017) How does hormoesis impact biology, toxicology and medicine? NPJ Aging Mech. Dis 9(1):13 Kumar Y, Phaniendra A, Periyasamy L (2018) Bixin triggers apoptosis of human hep3b hepatocellular carcinoma cells: an insight to molecular and in silico approach. Nutr Cancer 70(6):971–983 Shin J et al (2020) Pro-oxidant actions of carotenoids in triggering apoptosis of cancer cells: a review of emerging evidence. Antioxid (Basel) 9(6):532 Baeza-Morales A et al (2024) The antitumour mechanisms of carotenoids: a comprehensive review. Antioxid (Basel) 13(9):1060 Additional Declarations The authors declare no competing interests. Supplementary Files GRAPHICALABSTRACT.png Graphical Abstract: From the seeds of Bixa orellana , the annatto apocarotenoids bixin and norbixin were isolated and their morphology illustrated through SEM imaging. When applied to U87MG glioblastoma cells, these compounds prompted clear apoptotic features under optical microscopy. As concentrations increased, apoptosis rose markedly—particularly in cells exposed to bixin—highlighting its stronger pro-apoptotic potential. 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06:08:05","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115764,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8620168/v1/9ee5ea8ea9d5f9693cd5ef7f.html"},{"id":100543612,"identity":"666e32a9-5727-4893-a9a0-59862123b5e9","added_by":"auto","created_at":"2026-01-19 06:08:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":62780,"visible":true,"origin":"","legend":"\u003cp\u003eUV–Vis absorption spectra of Bixin and Norbixin (250 mM) recorded in ethanol at 25 °C.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8620168/v1/88ad42917838a84919b49e5d.png"},{"id":100548670,"identity":"6957ffc0-cafc-43c8-934d-c35e0c99b62f","added_by":"auto","created_at":"2026-01-19 08:20:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":63197,"visible":true,"origin":"","legend":"\u003cp\u003eTauc plots for determining the optical band gap (Eg) of Bixin (blue) and Norbixin (green). The analyses were performed using ethanol (250 mM) at 25 °C. Shaded regions denote the linear segments used for extrapolation via R\u003csup\u003e2\u003c/sup\u003e-maximized linear regression (R\u003csup\u003e2\u003c/sup\u003e = 0.9987). The resulting optical band gaps are Eg = 2.56 eV for Bixin and Eg = 2.36 eV for Norbixin.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8620168/v1/9b96d272d252fe19f638c93b.png"},{"id":100543619,"identity":"0f4a7749-cd2e-4816-b0f2-c7d05633de50","added_by":"auto","created_at":"2026-01-19 06:08:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":354465,"visible":true,"origin":"","legend":"\u003cp\u003eComparative Proton Nuclear Magnetic Resonance (\u003csup\u003e1\u003c/sup\u003eH-NMR) spectra of Bixin and Norbixin. Spectra were recorded in CDCl\u003csub\u003e3\u003c/sub\u003e (Deuterated Chloroform) at 500 MHz and ambient temperature (25 °C), confirming the preservation of the conjugated polyene backbone and the differential functional groups following Norbixin hemisynthesis.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8620168/v1/776c1b337569de5f5b7c102d.png"},{"id":100548864,"identity":"2f803fae-90d5-4abd-ab1e-3b95e9d83632","added_by":"auto","created_at":"2026-01-19 08:21:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":50112,"visible":true,"origin":"","legend":"\u003cp\u003eComparative Fourier-transform infrared (FTIR) spectra of Bixin (blue) and Norbixin (red).The analysis was performed at ambient temperature (25 °C), highlighting the structural differences, specifically the characteristic C=O stretching bands confirming the ester group (Bixin) versus the carboxylic acid group (Norbixin).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8620168/v1/720ddc2823ab78b6c3ffadc2.png"},{"id":100548750,"identity":"5fe5e49f-a4db-4d68-9078-96fd8d01124f","added_by":"auto","created_at":"2026-01-19 08:20:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":50812,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic effect of Bixin on U87-MG human glioblastoma cells. Cells were treated with varying concentrations of Bixin for 48 h. Cell viability was quantified using the MTS assay. Data are expressed as the mean percentage of viable cells \u003cu\u003e+\u003c/u\u003e standard deviation (SD) from three independent experiments performed in triplicate. Statistical analysis indicates significant difference across all treatment groups compared to the untreated control (p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8620168/v1/82fc4c4d98665ee502c2dba4.png"},{"id":100543624,"identity":"fa301a7a-2f7e-47f7-b579-da23be8aa149","added_by":"auto","created_at":"2026-01-19 06:08:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":52586,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic effect of Norbixin on U87-MG human glioblastoma cells. Cells were treated with varying concentrations of Norbixin for 48 h. Cell viability was quantified using the MTS assay. Data are expressed as the mean percentage of viable cells \u003cu\u003e+\u003c/u\u003e standard deviation (SD) from three independent experiments performed in triplicate. Statistical analysis indicates significant difference across all treatment groups compared to the untreated control (p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8620168/v1/369609b8f63e5fd3180ce4c7.png"},{"id":100543625,"identity":"f565f6d9-b383-4b4c-9110-670b9eb43b0b","added_by":"auto","created_at":"2026-01-19 06:08:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":732683,"visible":true,"origin":"","legend":"\u003cp\u003eBixin-induced morphological changes and apoptosis in U87-MG human glioblastoma cells. Cells were treated for 48 h with increasing concentrations of Bixin: 0 mM (A), 100 mM (B), 200 mM (C), 250 mM (D), 300 mM (E), and 400 mM (F). Images were acquired using an inverted phase-contrast microscope at 100 x magnification. Black arrows indicate hallmark apoptotic features (e.g., cell shrinkage, membrane blebbing).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8620168/v1/18cc3c5f30730bf8913e9beb.png"},{"id":100549467,"identity":"14202a84-452d-4cc4-b5a0-1b4a8656b803","added_by":"auto","created_at":"2026-01-19 08:23:23","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":833869,"visible":true,"origin":"","legend":"\u003cp\u003eNorbixin-induced morphological changes and apoptosis in U87-MG human glioblastoma cells.Cells were treated for 48 h with increasing concentrations of Norbixin: 0 mM (A), 200 mM (B), 300 mM (C), 400 mM (D), 600 mM (E), and 800 mM (F). Images were acquired using an inverted phase-contrast microscope at 100 x magnification. Black arrows indicate hallmark apoptotic features (e.g., cell rounding, partial detachment).\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8620168/v1/7f824a4344aa5a9b423e8bcf.png"},{"id":100549132,"identity":"70c8d653-be1a-48ed-8ccb-83eb76411a17","added_by":"auto","created_at":"2026-01-19 08:22:31","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":56011,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential dose-dependent induction of apoptosis in U87-MG human glioblastoma cells following 48 h of exposure to Bixin and Norbixin. The IC\u003csub\u003e50\u003c/sub\u003e values determined from this analysis provide the Apoptotic Index (AI), which is compared to metabolic viability (MTS assay) in the Discussion. The 0 mM concentration represents the untreated control group.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-8620168/v1/17931d71ed000454d08d2132.png"},{"id":100554872,"identity":"87128249-fec3-4acd-9554-a3bcc67dbb84","added_by":"auto","created_at":"2026-01-19 08:39:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3546203,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8620168/v1/b896db91-4f1e-42a3-b85e-267ea6181165.pdf"},{"id":100543615,"identity":"88a59ef4-57a2-4cf1-bbfc-494efade5886","added_by":"auto","created_at":"2026-01-19 06:08:04","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":325246,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract: From the seeds of \u003cem\u003eBixa orellana\u003c/em\u003e, the annatto apocarotenoids bixin and norbixin were isolated and their morphology illustrated through SEM imaging. When applied to U87MG glioblastoma cells, these compounds prompted clear apoptotic features under optical microscopy. As concentrations increased, apoptosis rose markedly—particularly in cells exposed to bixin—highlighting its stronger pro-apoptotic potential.\u003c/p\u003e","description":"","filename":"GRAPHICALABSTRACT.png","url":"https://assets-eu.researchsquare.com/files/rs-8620168/v1/1bc87986e2e1926e64129d5b.png"},{"id":100543614,"identity":"9fc5c211-d083-4f7c-8661-0e1b58fd162a","added_by":"auto","created_at":"2026-01-19 06:08:04","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":21284,"visible":true,"origin":"","legend":"\u003cp\u003eAbout this Prepriny\u003c/p\u003e","description":"","filename":"Aboutthispreprint.docx","url":"https://assets-eu.researchsquare.com/files/rs-8620168/v1/ab502fba1a90f1baab7608fc.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eHormetic and Differential Apoptotic Effects of Bixin and Norbixin in Human Glioblastoma Cells (U87-MG)\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGlioblastoma (GBM) represents the most aggressive and universally fatal primary malignancy of the central nervous system, characterized by rapid proliferation, diffuse infiltration, and marked resistance to conventional treatment modalities such as radiotherapy and alkylating chemotherapy. Despite current multimodal therapeutic approaches, the median overall survival for GBM patients remains dismal, at approximately 15 months, underscoring an urgent and critical demand for novel and efficacious therapeutic strategies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In this context, natural products with modulatory effects on redox homeostasis and minimal systemic toxicity have garnered significant interest as potential adjuvants or alternatives in cancer therapy. Among these, Bixin and Norbixin, apocarotenoids derived from the seeds of \u003cem\u003eBixa orellana\u003c/em\u003e (annatto), have emerged as promising candidates due to their diverse biological activities relevant to oncological applications.\u003c/p\u003e \u003cp\u003eBixin has demonstrated notable antineoplastic effects across various experimental cancer models. In human melanoma cells (A2058), Bixin not only inhibited proliferation and migration but also induced apoptosis and G2/M cell cycle arrest, while potentiating the cytotoxicity of dacarbazine. These biological activities were mechanistically linked in previous studies to the induction of reactive oxygen species (ROS) and enhanced lipid peroxidation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Furthermore, \u003cem\u003ecis\u003c/em\u003e-Bixin, a geometric isomer, exhibited cytotoxicity across multiple tumor cell lines including lung (A549), prostate (PC3), breast (MCF7), colon (HCT-116), and thyroid (DRO), with half-maximal inhibitory concentrations (IC\u003csub\u003e50\u003c/sub\u003e) ranging between 10 and 50 \u0026micro;M. Its cytotoxic mechanism has been proposed to involve ROS generation concomitant with inhibition of the thioredoxin (Trx) and thioredoxin reductase (TrxR) system, critical regulators of cellular redox homeostasis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Supporting these findings, \u003cem\u003ein silico\u003c/em\u003e molecular docking studies have suggested Bixin\u0026rsquo;s potential interactions with key oncogenic targets such as BRAF, MMP9, and TP53, all of which are frequently mutated or dysregulated in diverse cancer types [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConversely, Norbixin, the water-soluble dicarboxylic acid derivative of Bixin, has been primarily investigated in non-oncological contexts. In experimental models of retinal degeneration, Norbixin exhibited pronounced neuroprotective properties, effectively preserving photoreceptor integrity and visual function through antioxidant mechanisms that attenuate oxidative damage and lipofuscin accumulation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Recent investigations have further expanded the understanding of Norbixin\u0026rsquo;s biological effects through the development of its synthetic derivatives. Specifically, the Norbixin amide conjugate BIO203 demonstrated potent anti-inflammatory and antioxidative properties in preclinical models of retinal degeneration, preserving photoreceptor viability and reducing oxidative stress\u0026ndash;induced cellular injury [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Collectively, these results highlight the context-dependent biological activities of Norbixin and its analogs, suggesting that their efficacy and mode of action may vary significantly depending on tissue type and the local redox environment, with limited data currently available for glioblastoma models.\u003c/p\u003e \u003cp\u003ePharmacokinetic studies in humans have established the systemic bioavailability of both Bixin and Norbixin following oral administration of annatto-based formulations. Notably, Norbixin exhibits higher plasma concentrations and prolonged persistence relative to Bixin, which is more rapidly cleared [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Furthermore, Bixin, but not Norbixin, has been reported to induce cytochrome P450 enzymes CYP1A1 and CYP1A2 in HepG2 hepatocarcinoma cells highlighting functional differences in metabolic interactions and gene regulation between these structurally related apocarotenoids [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEmerging evidence underscores the centrality of oxidative stress, mitochondrial dysfunction, and dysregulated antioxidant defenses in glioblastoma progression and therapeutic resistance. GBM cells display elevated basal levels of reactive oxygen species (ROS), which paradoxically sustain tumorigenic signaling while activating adaptive antioxidant mechanisms that confer resistance to conventional therapies.\u003c/p\u003e \u003cp\u003eTherapeutic strategies that perturb this fragile redox equilibrium, either by amplifying ROS generation beyond tolerable thresholds or by suppressing endogenous antioxidant systems, have shown promise in experimental models in selectively inducing glioma cell death. Given Bixin\u0026rsquo;s established capacity to modulate redox-sensitive pathways such as the Trx/TrxR system, Nrf2-mediated transcription, and mitochondrial oxidative phosphorylation, it emerges as a compelling candidate for exploratory investigation in glioblastoma cells. Norbixin, although structurally similar, is hypothesized to exhibit divergent activity due to its enhanced polarity, differential membrane permeability, and potential for distinct subcellular distribution, which necessitates comparative evaluation in controlled in vitro settings.\u003c/p\u003e \u003cp\u003eBeyond their redox-modulating properties, both Bixin and Norbixin possess physicochemical features that enable multimodal characterization, integrating chemical, structural, and biological dimensions. Advanced analytical techniques, including nuclear magnetic resonance (NMR) and Fourier-transform infrared spectroscopy (FTIR), provide detailed insights into molecular integrity and conformational characteristics. Correlating these precise physicochemical data with cellular assays, such as dose\u0026ndash;response cytotoxicity evaluations in U87MG glioblastoma cells, facilitates the establishment of structure\u0026ndash;activity relationships and the development of preliminary mechanistic hypotheses. The present study therefore employs a comprehensive approach to characterize Bixin and Norbixin and to evaluate their differential cytotoxic and pro-apoptotic potential against glioblastoma, aiming to provide foundational in vitro evidence to support future mechanistic and translational studies.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant Material and Sample Preparation\u003c/h2\u003e \u003cp\u003eThe seeds and pods of \u003cem\u003eBixa orellana\u003c/em\u003e were collected in Manaus, Amazonas State, Brazil, at geographic coordinates 3\u0026deg;06\u0026prime; S, 60\u0026deg;01\u0026prime; W, and an altitude of 92 m. Following collection, the fruits were washed with distilled water and dried in a convection oven at 30\u0026deg;C for 3 days.\u003c/p\u003e \u003cp\u003eSubsequently, 100 g of \u003cem\u003eBixa orellana\u003c/em\u003e seeds were defatted by washing with 400 mL of \u003cem\u003en\u003c/em\u003e-hexane (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026ge;\u003c/span\u003e\u0026thinsp;98%, Sigma-Aldrich) under constant magnetic stirring at room temperature (RT, 25\u0026deg;C) for 1 h. The mixture was filtered through Whatman paper, and this procedure was repeated twice to ensure the complete removal of the lipid fraction (rich in tocotrienols and geranylgeraniol) and other potential impurities. The defatted seeds were then dried under vacuum in a desiccator for 48 h at RT.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Synthesis and Purification of Apocarotenoids\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Bixin Extraction and Recrystallization\u003c/h2\u003e \u003cp\u003eBixin was obtained via solid\u0026ndash;liquid extraction. Defatted \u003cem\u003eBixa orellana\u003c/em\u003e seeds (25 g) were soaked in 50 mL of anhydrous ethanol ((\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026ge;\u003c/span\u003e\u0026thinsp;99.5%, Sigma-Aldrich) for 24 h in the dark. The mixture was filtered (Whatman paper), and the filtrate was stored at 4\u0026deg;C overnight to induce Bixin crystallization. The resulting Bixin crystals were collected by a second filtration.\u003c/p\u003e \u003cp\u003eRecrystallization was performed using anhydrous ethanol. The crude crystals were dissolved in anhydrous ethanol in a 250 mL beaker at RT, filtered, and the filtrate was stored for 24 h at 4\u0026deg;C. The purified Bixin crystals were collected by filtration, suspended in phosphate-buffered saline (PBS, pH 7.4) under magnetic stirring for 15 min, and subsequently lyophilized using a freeze dryer (Thermo Electron Corporation/Modulyod) for 48 h at \u0026minus;\u0026thinsp;32\u0026deg;C under vacuum (500 \u0026micro;ATM). The resulting powders were sealed and stored protected from light at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Norbixin Hemisynthesis\u003c/h2\u003e \u003cp\u003eNorbixin was obtained by base hydrolysis (hemisynthesis) of Bixin present in the seeds. \u003cem\u003eBixa orellana\u003c/em\u003e seeds (100 g) were soaked in 200 mL of an ethanolic ammonia\u0026ndash;water mixture (composed of 70 g of 10% v/v ammonium hydroxide (Sigma-Aldrich, 33%) solution and 70 g of anhydrous ethanol). The seeds were maintained under constant magnetic stirring in the ammoniacal solution at RT for 1 h, promoting the hydrolysis of the Bixin methyl ester group to form the Norbixin carboxylate salt.\u003c/p\u003e \u003cp\u003eThe ethanolic ammonia\u0026ndash;water mixture was filtered and then acidified to pH 4.0 using 85% phosphoric acid (Sigma Aldrich). The solution was then slowly cooled to between \u0026minus;\u0026thinsp;5\u0026deg;C and 0\u0026deg;C over 12 h to promote Norbixin crystallization. The crystallized Norbixin was collected by filtration, thoroughly washed with phosphate-buffered saline (PBS, pH 7.4), and subsequently lyophilized. The final powders were sealed and stored protected from light at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Physicochemical Characterization\u003c/h2\u003e \u003cp\u003eThe purity and structure of crystalized Bixin and Norbixin were assessed using UV-Vis spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and proton (\u003csup\u003e1\u003c/sup\u003eH) nuclear magnetic resonance (NMR) spectroscopy. These analyses were performed to confirm the chemical identity and integrity of the compounds, without implying direct biological efficacy.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 UV-Vis Spectroscopy\u003c/h2\u003e \u003cp\u003eUV\u0026ndash;Vis spectra of Bixin and Norbixin were recorded between 300 nm and 600 nm at RT using a Varian Cary 50 spectrophotometer. Stock solutions (0.6 g/L in anhydrous ethanol) were diluted appropriately (e.g., 2 \u0026micro;L stock to 3 mL total volume) in a quartz cuvette.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 NMR Spectroscopy\u003c/h2\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH and spectra were acquired on a Bruker DPX-400 Avance spectrometer (400 MHz) at RT. Deuterated chloroform (CDCl₃, \u0026ge;\u0026thinsp;99.5%, Sigma-Aldrich) was used as the solvent. Samples were dissolved in 0.55 mL of CDCl₃ and transferred to 5 mm NMR tubes. One-dimensional \u003csup\u003e1\u003c/sup\u003eH experiments utilized a 5 mm dual \u003csup\u003e1\u003c/sup\u003eH direct detection probe, a 9.6 \u0026micro;s π/2 pulse, a 16 k points for induction decay (acquisition time 2.5 s), eight scans, two dummy scans, a 3 s recycling delay, and a spectral window of 10 ppm. Chemical shifts (δ) are reported in parts per million (ppm) relative to the internal standard tetramethylsilane (TMS) and the residual solvent signal (CDCl\u003csub\u003e3\u003c/sub\u003e, δH 7.26). Spectral data analysis was performed using Master New version 9.0. These measurements provide structural confirmation but do not alone predict cellular effects.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 FT-IR Spectroscopy\u003c/h2\u003e \u003cp\u003eATR-FTIR spectra were obtained using an Shimadzu IR Tracer 100 spectrometer in Attenuated Total Reflection (ATR) mode, utilizing a ZnSe crystal over the 4000\u0026ndash;500 cm⁻\u0026sup1; wavenumber range. Spectra were averaged from 64 scans with a resolution of 4 cm⁻\u0026sup1;. Interpretation of functional groups was intended for chemical characterization rather than efficacy prediction.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Biological Assays\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Cell Culture\u003c/h2\u003e \u003cp\u003eThe U87-MG human glioblastoma cell line (BCRJ code 0241, Rio de Janeiro, Brazil) was cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were maintained in a humidified atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. The culture medium was refreshed every 2 days. Standard cell culture conditions were applied, and all experiments were exploratory in nature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Cell Viability (MTS) Assay and Apoptotic Effects\u003c/h2\u003e \u003cp\u003eCellular viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (ref. M5655, Sigma-Aldrich). U87-MG cells (5,000 cells/well) were seeded into 96-well plates (ref. 655090, Greiner). After 24 h (at \u0026asymp; 40% confluency), the culture medium was replaced, and cells were treated with different concentrations of Bixin and Norbixin for 48 h under standard incubation conditions. To mitigate interference from the colored compounds, the treatment medium was aspirated and replaced with fresh medium prior to the MTS assay.\u003c/p\u003e \u003cp\u003eSubsequently, the MTS reagent was dissolved in culture medium and added to each well at a final concentration of 1.09 mM. Cells were incubated for 2 h at 37\u0026deg;C. The reduction of the MTS tetrazolium compound to a soluble formazan product by mitochondrial dehydrogenases was quantified by measuring absorbance at 490 nm using a microplate reader (Thermo Electron Corporation/Multiskan EX). Background subtraction was performed using wells containing medium but no cells. Results are expressed as the percentage of viable cells relative to the untreated control. These experiments provide preliminary information on cell response and do not establish clinical relevance. All experiments were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Morphological Analysis and Apoptotic Index (AI)\u003c/h2\u003e \u003cp\u003eMorphological changes and the Apoptotic Index \u003cb\u003e(\u003c/b\u003eAI\u003cb\u003e)\u003c/b\u003e were evaluated using digital image analysis via inverted phase-contrast microscopy. U87-MG cells (1.10\u003csup\u003e4\u003c/sup\u003e cells/well) were seeded in 96-well plates and incubated for 48 h with Bixin or Norbixin at concentrations ranging from 100 \u0026micro;M to 800 \u0026micro;M. Dimethyl sulfoxide (DMSO, 1.5%) served as the vehicle control. After incubation, the medium was removed, and cells were washed once with PBS.\u003c/p\u003e \u003cp\u003eDigital micrographs were captured under a Nikon Eclipse TS100 inverted phase-contrast microscope (100 x magnification) using an integrated digital camera. Images from at least five randomly selected fields per well were acquired for each condition. Apoptotic cells were identified based on characteristic morphological features, including cell shrinkage, membrane blebbing, and the formation of apoptotic bodies. The total number of cells (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{N}_{total}\\)\u003c/span\u003e\u003c/span\u003e) and the number of apoptotic cells (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{N}_{Apoptotic}\\)\u003c/span\u003e\u003c/span\u003e) were manually counted using the Cell Counter plugin in ImageJ National Institutes of Health, Bethesda, MD, USA). The AI was calculated using the following equation:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:AI\\left(\\%\\right)=\\left(\\frac{{N}_{Apoptotic}}{{N}_{total}}\\right).100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe mean AI was determined from three independent experiments, each performed in triplicate. These measurements are exploratory and aimed at understanding potential cellular effects in vitro.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from three independent experiments, each performed in triplicate. Normality was assessed using the Shapiro\u0026ndash;Wilk test. Statistical significance was determined using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test (for two groups) or one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test (for multiple groups) for normally distributed data. For non-normally distributed data, the Kruskal\u0026ndash;Wallis test followed by Dunn\u0026rsquo;s multiple comparison test was applied. A \u003cem\u003ep\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. Statistical analyses and graph generation were performed using Python (version 3.12) with the libraries \u003cem\u003epandas\u003c/em\u003e (v2.2), \u003cem\u003enumpy\u003c/em\u003e (v1.26), \u003cem\u003escipy\u003c/em\u003e (v1.13), \u003cem\u003estatsmodels\u003c/em\u003e (v0.14), \u003cem\u003escikit-posthocs\u003c/em\u003e (v0.9), \u003cem\u003ematplotlib\u003c/em\u003e (v3.9), and \u003cem\u003eseaborn\u003c/em\u003e (v0.13). Representative images shown in the figures were selected to closely reflect the mean values of the respective groups accurately. Statistical conclusions are limited to the experimental context and do not imply translational or clinical outcomes.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 UV\u0026ndash;Vis Spectroscopy and Electronic Band Gap\u003c/h2\u003e \u003cp\u003eThe Ultraviolet-Visible (UV\u0026ndash;Vis) absorption spectra of Bixin and Norbixin in ethanol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) exhibited the characteristic three-band pattern typical of conjugated polyenes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The maxima for Bixin were recorded at 429 nm, 450 nm, and 508 nm, and for Norbixin at 445 nm, 470 nm, and 500 nm, consistent with literature [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These features confirm the presence of an extended \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\)\u003c/span\u003e\u003c/span\u003e-conjugated system, where electronic transitions correspond to π\u0026rarr;π* excitations. These spectroscopic features confirm molecular structure and electronic properties, serving as a physicochemical basis for subsequent cellular experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe observed spectral shifts relative to published values are likely due to solvatochromic effects and partial \u003cem\u003ecis\u003c/em\u003e\u0026ndash;\u003cem\u003etrans\u003c/em\u003e isomerization in bixin, and the increased molecular polarity and modulation of conjugation due to de-esterification in Norbixin [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrom a molecular orbital perspective, the π\u0026rarr;π* transitions correspond to excitations between the Highest Occupied Molecular Orbital (HOMO) and the Lowest Unoccupied Molecular Orbital (LUMO). The optical band gaps (E\u003csub\u003eg\u003c/sub\u003e) were determined from the linear region of Tauc plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), yielding 2.56 eV for Bixin and 2.36 eV for Norbixin. Using a reference HOMO energy of -5.6 eV [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], the estimated LUMO energies were \u0026minus;\u0026thinsp;3.04 eV and \u0026minus;\u0026thinsp;3.24 eV, respectively. The narrower band gap in Norbixin, attributed to its extended \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\)\u003c/span\u003e\u003c/span\u003e-conjugation and higher polarity, indicates easier electronic excitation, which is relevant from a physicochemical perspective for potential redox activity in vitro [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.2 NMR Analysis and Conversion De\u003c/em\u003egree\u003c/h2\u003e \u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of Bixin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) confirmed its conjugated diester structure, showing characteristic resonances for methoxy protons (OCH\u003csub\u003e3\u003c/sub\u003e) at δ 3.80 ppm, aliphatic protons at δ 1.25 ppm and δ 1.55\u0026ndash;1.75 ppm, allylic protons at δ 1.95\u0026ndash;2.10 ppm, and the polyene system (CH\u0026thinsp;=\u0026thinsp;CH) spanning δ 5.05\u0026ndash;6.95 ppm [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of Norbixin confirmed the intended hydrolysis, showing a marked decrease in the intensity of the methoxy signal at δ 3.80 ppm, signifying the cleavage of the methyl ester group(s). The persistence of the olefinic signals (δ 5.85\u0026ndash;6.95 ppm) confirmed the preservation of the conjugated polyene backbone.\u003c/p\u003e \u003cp\u003eQuantitative analysis of the normalized methoxy resonance intensity (δ 3.80 ppm) revealed a conversion degree of approximately 75%. This composition was used as obtained for exploratory cell-based assays, providing a chemically defined mixture for in vitro evaluation. This high conversion confirms the predominant formation of the monocarboxylic monoester Norbixin, providing a chemically defined mixture for exploratory in vitro assays. This mixture (75% Norbixin, 25 residual Bixin) was deemed suitable for cell-based assays, as the combination of these redox-active species may provide enhanced reactive oxygen species (ROS) quenching capacity, which is particularly relevant in the glioblastoma context [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 FTIR Analyses\u003c/h2\u003e \u003cp\u003eThe FTIR spectra of the Bixin and Norbixin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) highlighted the structural difference resulting from the hemisynthesis. Bixin exhibited a strong band at 1710 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, corresponding to the C\u0026thinsp;=\u0026thinsp;O stretching of the ester carbonyl (C\u0026ndash;O\u0026ndash;C vibrations between 1200\u0026ndash;1155 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), confirming its esterified form. Norbixin showed characteristic broad absorption bands in the 3750\u0026ndash;3400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e region (O\u0026ndash;H stretching) and C\u0026thinsp;=\u0026thinsp;O signals in the 1670 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e range (terminal carboxyls), confirming the presence of carboxylic acid (COOH) groups. Both compounds showed C\u0026thinsp;=\u0026thinsp;C stretching bands in the 1530\u0026ndash;1430 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e region, confirming the integrity of the polyene conjugated system. These FTIR data further support the chemical identity and purity of the compounds used in subsequent in vitro assays.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Differential Cytotoxicity and Apoptotic Induction\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Cytotoxicity Assessment (MTS Assay)\u003c/h2\u003e \u003cp\u003eDose\u0026ndash;response analysis of U87-MG glioblastoma cells revealed a significant differential cytotoxic effect between the two apocarotenoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Bixin displayed pronounced cytotoxicity, with an IC\u003csub\u003e50\u003c/sub\u003e of 231.9\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;8.3 \u0026micro;M. Notably, cell viability exhibited a transient increase, reaching a maximum of 145.5% in the concentration range of 100 \u0026micro;M to 175 \u0026micro;M before the onset of the cytotoxic effect observed at 200 \u0026micro;Mand above. The steep Hill slope (14.7\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;7.3) suggests a rapid, cooperative mechanism of action following this initial peak.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNorbixin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) exhibited lower potency, with an IC\u003csub\u003e50\u003c/sub\u003e of 532.9\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;17.2 \u0026micro;M (more than double that of Bixin), and a shallower Hill slope (5.94\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.85), consistent with a more gradual and less abrupt reduction in cell viability. These results indicate that the lipophilicity conferred by the esterified structure of Bixin significantly enhances its cytotoxic potency against U87-MG cells, likely by facilitating faster cellular uptake and accumulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2 Morphological Changes and Apoptotic Index\u003c/h2\u003e \u003cp\u003eApoptosis-related morphological alterations were assessed by inverted phase-contrast microscopy (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e). At cytotoxic concentrations, cells treated with both compounds displayed hallmark apoptotic features, including cytoplasmic condensation, cell rounding, membrane blebbing, and the formation of apoptotic bodies [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Quantification via the Apoptotic Index (AI) confirmed a differential mechanism of action between the apocarotenoids (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e). For bixin, the IC\u003csub\u003e50\u003c/sub\u003e for apoptotic induction (\u0026asymp;130.1 \u0026micro;M) was markedly lower than the IC\u003csub\u003e50\u003c/sub\u003e for viability reduction (MTSIC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;231.9 \u0026micro;M). This AIIC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;MTSIC\u003csub\u003e50\u003c/sub\u003e relationship strongly indicates that apoptosis is an early and predominant mechanism of Bixin-induced cell death in U87-MG cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, Norbixin exhibited considerably higher IC50 values for both apoptosis (400.1 \u0026micro;M) and viability (532.9 \u0026micro;M), consistent with a slower and less potent apoptotic response. These findings describe relative differences in apoptotic induction within this cell culture model and support a structure\u0026ndash;activity relationship, whereby the higher lipophilicity and esterified nature of Bixin may facilitate its diffusion and interaction with intracellular targets (e.g., mitochondria), potentially contributing to increased apoptotic signaling compared to the more polar Norbixin [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Consequently, Bixin may serve as a promising scaffold for exploring pro-apoptotic activity in glioblastoma cells in vitro, highlighting the influence of molecular structure on cellular uptake and redox activity in a controlled experimental model.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eGlioblastoma multiforme (GBM) remains one of the most therapeutically challenging malignancies. Despite the current standard-of-care treatments, GBM is characterized by high rates of recurrence and poor overall survival [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The limited long-term efficacy of conventional therapies is largely attributed to factors such as intrinsic multidrug resistance (MDR), often mediated by ATP-binding cassette (ABC) transporters [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and significant treatment-related neurotoxicity. Consequently, the investigation of adjuvant or complementary therapeutic strategies, particularly those derived from natural products with minimal systemic toxicity, represents a promising avenue for exploratory investigation.\u003c/p\u003e \u003cp\u003eOur study aimed to address a significant gap in the literature by providing the first comparative evaluation of the cytotoxic and pro-apoptotic potential of Bixin and Norbixin in the human U87-MG glioblastoma cell line. Bixin has previously demonstrated broad antineoplastic effects across various \u003cem\u003ein vitro\u003c/em\u003e models, including melanoma and lung cancer, often involving mechanisms linked to cell cycle arrest and apoptosis [\u003cspan additionalcitationids=\"CR32 CR33 CR34 CR35 CR36\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The present findings extend these observations to a glioblastoma cell model under controlled in vitro conditions.\u003c/p\u003e \u003cp\u003eThe observed biological activity finds a strong rationale in the compounds' electronic structure. The UV\u0026ndash;Vis analysis revealed a smaller HOMO\u0026ndash;LUMO energy gap (E\u003csub\u003eg\u003c/sub\u003e) for Norbixin (2.36 eV) compared to Bixin (2.56 eV) (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This narrow band gap significantly enhances the apocarotenoids' propensity to participate in electron transfer processes, a fundamental requirement for the generation and scavenging of ROS [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. From a physicochemical perspective, this dual capability may be relevant for modulating redox balance in cancer cell models, where molecules may act as pro-oxidants under specific conditions. The high conjugation and physicochemical differences, corroborated by FTIR and NMR analyses (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), thus provide a molecular basis for hypothesizing selective redox modulation in U87-MG cells.\u003c/p\u003e \u003cp\u003eOur dose\u0026ndash;response evaluation established a clear structure\u0026ndash;activity relationship (SAR) driven by the differential polarity of the apocarotenoids. Bixin exhibited significantly higher cytotoxic potency (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;231.9 \u0026micro;M or 91.5 g/mL) compared to norbixin (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;532.9 \u0026micro;M or 202.79 g/mL) (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Based on established criteria for natural product activity [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], Bixin is classified as moderately active, while Norbixin falls into the weakly active range against U87-MG cells. These classifications apply specifically to in vitro screening contexts.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe superior efficacy of Bixin is primarily attributed to its esterified and highly lipophilic nature. This increased lipophilicity may facilitate passive diffusion across the lipid bilayer of the plasma and mitochondrial membranes, leading to faster intracellular accumulation and potentially more direct interaction with apoptotic regulators compared to the more polar, dicarboxylated Norbixin. Furthermore, Bixin exhibited a steep Hill slope (14.7\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;7.3), indicative of a highly cooperative or rapid, all-or-nothing cytotoxic mechanism once the threshold concentration is reached. Conversely, Norbixin's shallower Hill slope (5.94\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.85) suggests a more gradual reduction in cell viability under the experimental conditions employed.\u003c/p\u003e \u003cp\u003eA critical finding in the Bixin dose-response curve was the transient increase in cell viability in the concentration range of 100 \u0026micro;M to 175 \u0026micro;M, peaking at 145.5% viability, before the onset of cytotoxicity at 200 \u0026micro;M and higher. This biphasic pattern is consistent with a hormetic effect, as defined in vitro [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], where a low dose of an otherwise toxic or stressful agent elicits a beneficial or stimulatory response, while high doses are inhibitory. This hormetic behavior was observed exclusively in vitro and should be interpreted within this experimental framework.\u003c/p\u003e \u003cp\u003eAt these non-cytotoxic concentrations (100\u0026ndash;175 \u0026micro;M), we propose that Bixin, a known antioxidant carotenoid, functions as a mild stressor. This subtle perturbation likely triggers an adaptive stress response in U87-MG cells, often mediated through the Nrf2/Keap1 signaling pathway. Nrf2 activation results in the transcriptional upregulation of cytoprotective genes, including Phase II detoxification enzymes and antioxidant proteins (e.g., HO-1 and NQO1). This adaptive supercompensation mechanism will enhances the cell's endogenous defense capacity, leading to an apparent increase in cell viability or proliferation. Such mechanisms are proposed based on existing literature and were not directly measured in this study.\u003c/p\u003e \u003cp\u003eThe transition from the hormetic (stimulatory) phase to the cytotoxic (inhibitory) phase above 200 \u0026micro;M suggests a saturation of the cellular adaptive capacity. At these higher concentrations, the inherent pro-oxidant activity of Bixin, particularly when accumulated in the mitochondrial membrane due to its lipophilicity, may become dominant. The resulting mitochondrial dysfunction, increased reactive oxygen species (ROS) generation, and collapse of the membrane potential are plausible contributors, to the commitment of cell apoptosis via the intrinsic pathway. The ability of Bixin to traverse cell membranes effectively is, therefore, hypothesized to be relevant for both its hormetic effects at low concentrations and its potent cytotoxic action at high concentrations.\u003c/p\u003e \u003cp\u003eIn stark contrast, the Norbixin dose-response curve did not show a significant increase in cell viability at low concentrations, failing to elicit the characteristic hormetic effect. Norbixin's lack of a hormetic phase is most likely attributable to its hydrophilic nature. Being highly water-soluble, Norbixin may have reduced capacity to cross the lipophilic cell and mitochondrial membranes to reach and trigger intracellular sensors (like Keap1/Nrf2) or induce the subtle mitochondrial stress required to activate the adaptive response. Consequently, Norbixin's action is predominantly inhibitory, and occurred at higher concentrations, further supporting the role of lipophilicity in modulating cellular responses in this model system.\u003c/p\u003e \u003cp\u003eAlthough cytotoxic potency is modest compared to conventional chemotherapeutics, the IC\u003csub\u003e50\u003c/sub\u003e values suggest that these apocarotenoids may serve as exploratory scaffolds for selective, mechanism-based cellular effects in vitro. Such a role remains speculative and requires validation in additional experimental models. The identification of a hormetic window for Bixin is nonetheless relevant for experimental design, highlighting the importance of dose selection in studies involving redox-active compounds.\u003c/p\u003e \u003cp\u003eTo further elucidate the MOA, we conducted morphological and quantitative analyses of apoptosis (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Both compounds induced classic hallmark apoptotic features (cell shrinkage, membrane blebbing, etc.), consistent with programmed cell death [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Crucially, the comparative assay revealed that for Bixin, the IC\u003csub\u003e50\u003c/sub\u003e for apoptotic induction (\u0026asymp; 130.1 \u0026micro;M) was substantially lower than the IC\u003csub\u003e50\u003c/sub\u003e for metabolic viability reduction (231.9 \u0026micro;M). This AIIC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;MTSIC\u003csub\u003e50\u003c/sub\u003e relationship confirms that apoptosis is the early and predominant event in the cytotoxic cascade of Bixin, preceding the loss of general metabolic function. Conversely, Norbixin required higher concentrations for both effects, suggesting a slower and less efficient activation of apoptotic pathways.\u003c/p\u003e \u003cp\u003eMechanistically, this pro-apoptotic effect may involve mitochondrial perturbation and ROS generation, as suggested in the literature [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Carotenoids, particularly those with higher lipophilicity, are known to integrate into cellular membranes and modulate Bcl-2 family proteins, thereby enhancing caspase-dependent apoptosis [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. While consistent with existing literature, these mechanisms were not directly assessed in the present study. The pronounced difference between Bixin and Norbixin underscores how subtle structural modifications, such as the ester hydrolysis, can profoundly influence the bioavailability, subcellular distribution, and resulting cytotoxic and pro-apoptotic efficacy.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides, to the best of our knowledge, the first evidence that the natural apocarotenoids Bixin and Norbixin exert differential cytotoxic and pro-apoptotic effects in U87-MG human glioblastoma cells. Bixin exhibited significantly greater potency, displaying a characteristic biphasic dose-response profile indicative of a hormetic effect at low concentrations, followed by robust cytotoxicity at high concentrations. The IC\u003csub\u003e50\u003c/sub\u003e for apoptosis was markedly lower than that for overall metabolic viability, indicating that programmed cell death is an early and prominent event associated with its cytotoxicity. In contrast, Norbixin showed weaker and slower apoptotic induction, consistent with its higher polarity and reduced lipophilicity. These findings underscore the critical role of molecular structure and lipophilicity in influencing cellular uptake and apoptotic efficiency within this experimental model.\u003c/p\u003e \u003cp\u003eOverall, Bixin emerges as a promising molecular scaffold for further in vitro investigation, rather than a therapeutic agent, in the rational design of carotenoid-derived compounds targeting aggressive glioma cells. Additional mechanistic studies and in vivo validation are required to further explore its mode of action and potential relevance in neuro-oncological research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCredit authorship contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlvaro Antonio Alencar de Queiroz:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Software, Investigation, Visualization, Formal analysis, Conceptualization. \u003cstrong\u003eCarlos Roberto Jorge Soares:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; review, Investigation, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Formal analysis, Data curation, Conceptualization. \u003cstrong\u003eNicoli Dolores Gon\u0026ccedil;alves Correa:\u0026nbsp;\u003c/strong\u003eInvestigation,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMethodology, Data curation, Formal analysis, Validation.\u0026nbsp;\u003cstrong\u003eSirlene da Silva Rodrigues\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eInvestigation, Methodology, Validation, Resources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was partially supported by National Council for Scientific and Technological Development\u003cstrong\u003e-\u003c/strong\u003eCNPq (PQ 307047/2023-7/ DT 302970/2025-8) and by Coordination for the Improvement of Higher Education Personnel-CAPES.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest that could have influenced the work reported in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the manuscript preparation process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no generative artificial intelligence (AI) or AI-assisted technologies were used in the preparation of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSipos D et al (2025) Glioblastoma: clinical presentation, multidisciplinary management, and long-term outcomes. 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Antioxid (Basel) 13(9):1060\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"23681b14-e3d7-4c51-b89c-0dc5fb6b0945","identifier":"10.13039/501100003593","name":"Conselho Nacional de Desenvolvimento Científico e Tecnológico","awardNumber":"PQ 307047/2023-7/ DT 302970/2025-8","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Instituto de Pesquisas Energéticas e Nucleares - IPEN","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":"Human glioblastoma, Bixin, Norbixin, Cytotoxicity, Apoptosis, Hormesis, Redox modulation, U87MG cells","lastPublishedDoi":"10.21203/rs.3.rs-8620168/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8620168/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlioblastoma multiforme (GBM) is an aggressive, treatment-resistant brain tumor with limited therapeutic success. Natural apocarotenoids, such as Bixin (lipophilic) and Norbixin (hydrophilic), derived from annatto (\u003cem\u003eBixa orellana\u003c/em\u003e), have demonstrated anticancer potential, but their effects on glioblastoma remain largely unexplored. This study evaluated, under in vitro conditions and for the first time, the cytotoxic and pro-apoptotic activities of Bixin and Norbixin against the human glioblastoma cell line U87-MG. Molecular characterization was performed using FTIR and \u003csup\u003e1\u003c/sup\u003eH-NMR spectroscopy, and reactivity was estimated via Frontier Molecular Orbitals (FMO) analysis. Cell viability was quantified using the tetrazolium-based colorimetric assay (MTS), and apoptosis was monitored via phase-contrast microscopy to determine IC\u003csub\u003e50\u003c/sub\u003e values and evaluate morphological changes. Bixin exhibited significantly higher potency, displaying a distinctive biphasic dose-response pattern consistent with a hormetic effect at low concentrations. Bixin yielded IC\u003csub\u003e50\u003c/sub\u003e values of 231.9 \u0026micro;M (viability) and 130.1 \u0026micro;M (apoptosis), indicating that apoptotic signaling precedes metabolic viability loss (AIIC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;MTSIC\u003csub\u003e50\u003c/sub\u003e). Norbixin showed IC\u003csub\u003e50\u003c/sub\u003e of 532.9 \u0026micro;M for viability; 400.1 \u0026micro;M for apoptosis and a slower apoptotic induction profile. Morphological analyses corroborated these findings, confirming classic apoptotic features (e.g., cell shrinkage and membrane blebbing), particularly under cytotoxic concentrations, and were more pronounced in Bixin-treated cells. These results reveal distinct cytotoxic and apoptotic profiles between the two apocarotenoids, driven by their structural and physicochemical differences (lipophilicity vs. hydrophilicity). Collectively, the findings support Bixin and Norbixin as promising molecular scaffolds for further investigation, rather than therapeutic agents per se, in the context of carotenoid-based strategies targeting malignant glioma cells, warranting further mechanistic and in vivo validation.\u003c/p\u003e","manuscriptTitle":"Hormetic and Differential Apoptotic Effects of Bixin and Norbixin in Human Glioblastoma Cells (U87-MG)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-19 06:07:59","doi":"10.21203/rs.3.rs-8620168/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":"eb85a465-542d-4eab-94ac-8da344299d91","owner":[],"postedDate":"January 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61269560,"name":"Cancer Biology"},{"id":61269561,"name":"Biological Chemistry"},{"id":61269562,"name":"Natural Product Chemistry"}],"tags":[],"updatedAt":"2026-01-19T06:07:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-19 06:07:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8620168","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8620168","identity":"rs-8620168","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}