Effect of a Solanesol-Enriched Extract from Nicotiana tabacum L. on Intracellular Oxidative Stress in Endothelial Cells

Effect of a Solanesol-Enriched Extract from Nicotiana tabacum L. on Intracellular Oxidative Stress in Endothelial Cells | 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 Effect of a Solanesol-Enriched Extract from Nicotiana tabacum L. on Intracellular Oxidative Stress in Endothelial Cells Yudeyki Almuiña Guerra, Oscar Galarce-Bustos, Claudio Aguayo Tapia, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9235979/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Oxidative stress and chronic inflammation play a key role in the development and progression of various cellular pathologies, mainly caused by the overproduction of reactive oxygen species. In this context, natural antioxidant compounds have attracted growing attention for their potential to prevent and reduce oxidative damage. In this study, we obtained a solanesol fraction with high-purity from Nicotiana tabacum L., Solanaceae, and evaluated its antioxidant capacity in vitro to determine its potential to mitigate oxidative stress. The extract was obtained from N. tabacum leaves by ultrasound-assisted extraction and further fractionated by column chromatography. The presence and concentration of solanesol were confirmed by high-performance liquid chromatography and mass spectrometry. The in vitro assays were performed to determine antioxidant activity, cytotoxicity in endothelial cells, and the generation of intracellular reactive oxygen species. The purified fractions showed strong antioxidant activity, with hydroxyl radical inhibition exceeding 38.81 ± 0.001%, low cytotoxicity, and a stable inhibition of reactive oxygen species (34.6 ± 0.006%) over the 5 min observation period. These results support the potential of the solanesol-enriched extract as a bioactive ingredient, highlighting its possible applicability in preventive or therapeutic strategies targeting oxidative stress associated to cellular disorders. Ultrasound-assisted extraction sonalensol-enriched extract EA.hy926 cell line intracellular redox balance vascular dysfunction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Solanesol ( 1 ) is a linear terpenoid alcohol composed of nine isoprene units (Yan et al. 2015 ). In recent years, it has gained increasing attention due to its role as a key precursor in the synthesis of high-value therapeutic compounds such as coenzyme Q10 and vitamin K2 (Qin et al. 2017 ). In addition to its industrial relevance, solanesol exhibits antioxidant and anti-inflammatory properties, mainly attributed to its multiple non-conjugated double bonds, which enable efficient free radical scavenging and modulation of oxidative stress-related pathways (Yao et al. 2015 , Yan et al. 2019 ). Oxidative stress is a major contributor to cellular damage and is closely associated with the development of chronic diseases, including cardiovascular and neurodegenerative disorders (Chen et al. 2021 , Pisoschi et al. 2021 ). In this context, natural antioxidants have attracted considerable interest as potential agents to prevent or mitigate oxidative damage at both the molecular and cellular levels. Solanesol is distributed in several species of the Solanaceae family, including Solanum tuberosum (potato), Solanum lycopersicum (tomato), Solanum melongena (eggplant), and Capsicum annuum (pepper), where it occurs in relatively low amounts in the leaves (0.09–0.35%, 0.04–0.4%, 0.2–0.4%, and 0.09–0.35% of dry weight, respectively). In contrast, significantly higher concentrations has been reported in Nicotiana tabacum L., where levels can reach up to 3.6%, making it one of the richest natural sources of this compound (Yan et al. 2015 , Campbell et al. 2016 ). Importantly, the tobacco industry generates large amounts of residual biomass (e.g., leaves, dust, and stems), which constitute an abundant, low-cost, and underutilized source of solanesol (Tita et al. 2021 ). According to the World Health Organization (WHO), the tobacco industry generates approximately 25 million tons of waste annually worldwide (World Health Organization (WHO) 2022 ). These residues represent a valuable and sustainable source of solanesol, particularly because the chemical synthesis of this compound is highly complex. Furthermore, the curing process has been shown to increase the proportion of free solanesol, facilitating its recovery from these residues (Machado et al. 2010 , Bravo Cardenas et al. 2021 ). In this context, solanesol concentrations of up to 598.9 µg/ml have been reported in various tobacco industry waste products, including leaf remnants, dust, and midribs (Banožić et al. 2019 ). Therefore, the aim of this study was to obtain a solanesol-enriched extract from Nicotiana tabacum waste using an efficient extraction and purification approach, and to evaluate its antioxidant capacity both in vitro and in endothelial cells, with special emphasis on its ability to modulate intracellular reactive oxygen species. Materials and methods Chemicals and Reagents Solanesol standard (≥ 90%) was obtained from Cayman Chemical (Michigan, USA), ethanol, hexane, ethyl acetate, formic acid, HPLC grade acetonitrile, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H -tetrazolium bromide (MTT), 2',7'-dichlorofluorescin diacetate (DCFH-DA), phosphate-buffered saline (PBS), and DMSO were purchased from Merck (Darmstadt, Germany). DPPH, Trolox (≥ 97%), gallic acid (98%), Folin–Ciocalteu (FC), ferrous chloride (FeCl 3 , ≥ 99%), sodium carbonate, hydrogen peroxide (H 2 O 2 ), ascorbic acid, deoxyribose solution, trichloroacetic acid (TCA), thiobarbituric acid (TBA), and silica gel for column chromatography (60 Å pore size, 70–230 mesh) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sample Processed tobacco strands of the Burley type ( N. tabacum L., Solanaceae) were obtained from a commercial source (brand: Tabaco Bristol Orgánico; Tabacalera S.A., Chile). The material originated from crops located in San Clemente, Maule Region, Chile (35°32′16.3″S, 71°27′31.1″W). Samples were dried at 45.0°C for 4 h, ground, and sieved to obtain a particle size < 45 µm prior to extraction. A representative sample was deposited in the laboratory collection of Pharmacognosy of the Faculty of Pharmacy, Universidad de Concepción, Chile. Ultrasound-Assisted Extraction of Solanesol Solanesol was extracted from 50 g of tobacco powder using an ultrasonic bath (Elma, Singen, Germany; 320 W) at 30.0°C for 30 min with 500 ml of ethanol. The extract was filtered and concentrated under reduced pressure at 50.0°C to obtain a crude extract (CE). To remove alkaloid-type compounds, the extract was partitioned with hexane and washed twice with 1% HCl, followed by distilled water. The organic phase was dried over anhydrous Na 2 SO 4 , filtered, and concentrated. The resulting extract (1.6 g) was re-dissolved in hexane:ethyl acetate (4:1, v/v) for further purification. Column Chromatography Fractionation The CE was subjected to open-column chromatography (2 cm × 16.5 cm) packed with silica gel (18 g). Elution was performed using hexane:ethyl acetate (4:1, v/v), and eighteen fractions (5 ml) were collected. Fractions were monitored by thin-layer chromatography (TLC) using vanillin reagent and pooled according to similar chromatographic profiles. Determination of Solanesol by UHPLC–DAD Identification of solanesol in the fractions was performed using an ultra-high performance liquid chromatography system coupled with diode array detection and tandem mass spectrometry (UHPLC–DAD–MS/MS) (Shimadzu LCMS-8030, Shimadzu, Kyoto, Japan). Chromatographic separation was performed on a Kinetex® Phenyl-Hexyl column (100 × 2.1 mm, 1.7 µm; Phenomenex, USA) using an isocratic mobile phase of acetonitrile:0.1% formic acid (80:20, v/v) at 0.4 ml/min and 35.0°C. Detection was carried out by ESI-MS in positive mode ( m/z 50–1000). Solanesol quantification was performed by UHPLC–DAD using the same column under isocratic conditions (acetonitrile:water, 80:20, v/v) at 0.5 ml/min and 50.0°C. The injection volume was 5 µl. Calibration was carried out over a range of 10–100 µg/ml, and samples were filtered (0.45 µm) prior to analysis. Antioxidant Activity DPPH Radical Scavenging Assay DPPH radical scavenging activity was determined according to Galarce-Bustos et al. ( 2023 ) with minor modifications. Briefly, 20 µl of sample was mixed with 180 µl of 0.15 mM DPPH solution. After incubation for 30 min at room temperature in the dark, the absorbance was measured at 517 nm. Hydroxyl Radical Scavenging Assay Hydroxyl radical scavenging activity was determined according to Halliwell et al. ( 1987 ) with modifications. Briefly, 50 µl of FeCl 3 (20 mM), 50 µl of H 2 O 2 (42 mM), and 10 µl of ascorbic acid (50 mM) were mixed. Then, 10 µl of sample or control solution (gallic acid, 10 mM) and 100 µl of deoxyribose (2.8 mM) were added. The mixture was incubated at 37.0°C for 1 h. Subsequently, 1 ml of 2.8% TCA and 1 ml of TBA were added, followed by heating at 100°C for 15 min. After cooling to room temperature, absorbance was measured at 532 nm. Cellular Viability and Oxidative Stress Assays Cytotoxicity Assay on Endothelial Cells EA.hy926 endothelial cells were seeded in 96-well plates (1 × 10 4 cells/well) and incubated for 24 h at 37.0°C under 5.0% CO 2 . Cells were treated in quadruplicate ( n = 4) with samples (0.01–10 mg/ml) for 24 h. Cell viability was assessed using a modified MTT assay (Carrasco et al. 2024 ). After treatment, cells were washed with PBS and incubated with 10 µl of MTT solution (14.5 mM) for 4 h at 37.0°C. Formazan crystals were dissolved in 50 µl of DMSO, and absorbance was measured at 570 nm. Results were expressed as percentage cell viability: Total Reactive Oxygen Species Detection in Cells Intracellular ROS levels were determined according to (Huang et al. 2020 ) with modifications. EA.hy926 cells were seeded in 96-well plates (1 × 10 4 cells/well) and incubated for 24 h at 37.0°C under 5.0% CO 2 . Cells were treated in triplicate ( n = 3) with samples (0.01–10 mg/ml) for 24 h. After washing with PBS, oxidative stress was induced using 5 µl of H 2 O 2 (2% in PBS) for 30 min. ROS production was measured using DCFH-DA (0.5 µM) after 30 min incubation at 37°C. Fluorescence was recorded at 485/520 nm using a microplate reader, and results were expressed relative to control conditions. Results and Discussion Ultrasound-Assisted Extraction and Column-Based Enrichment of Solanesol Residual biomass generated during industrial tobacco processing (veins, dust, cured leaves, and stems) contains solanesol at levels ranging from 0.05% to 0.33% (w/w), corresponding to approximately 30% of the content found in fresh leaves. Notably, the curing process increases the proportion of free solanesol (Machado et al. 2010 , Banožić et al. 2019 ). Despite these lower concentrations, the large volumes of tobacco waste make these residues a practical, sustainable, and economically attractive source for solanesol recovery. In this study, solanesol was extracted using ultrasound-assisted extraction (UAE), a rapid and energy-efficient technique with low solvent consumption (Shen et al. 2023 ). Under the applied conditions, a yield of 3.2 ± 0.002% (w/w) relative to the dried material was obtained, exceeding previously reported UAE yields for tobacco residues (0.8–2.3% w/w) (Banožić et al. 2019 ). This yield is also competitive with other extraction techniques, such as microwave-assisted extraction (MAE), which reaches up to 0.91% (w/w) (Zhou and Liu 2006 ), and sequential microwave–ultrasound-assisted extraction (MUAE), which achieves approximately 3.0% (w/w) but requires multiple processing steps (Safitra et al. 2024 ). Although supercritical CO 2 extraction (scCO 2 ) can reach yields of up to 3.74% (w/w), it involves high-pressure equipment and increased operational costs (Huang et al. 2008 ). In this context, the UAE approach used here provides a competitive and simpler alternative. Following extraction, column chromatography proved to be an effective method for solanesol purification and enrichment. Separation was rapidly optimized, identifying the extraction solvent as a key factor influencing fractionation efficiency. Hexane extracts showed greater matrix complexity, leading to poorer resolution, whereas ethanol extracts yielded cleaner profiles, facilitating the removal of interfering compounds and improving solanesol enrichment. These results highlight the importance of solvent selection for both extraction and downstream purification. Qualitative and Quantitative Analysis of Solanesol Fractions The obtained fractions were initially analyzed by TLC, where solanesol was predominantly detected in fractions 7 to 14 with an R F value of 0.29 ± 0.001, consistent with the solanesol standard. The presence of the analyte was further confirmed by mass spectrometry, showing matching molecular ions at m/z 669.5, 653.6, and 613.6 [M + H]⁺ ( Fig. S1 ), corresponding to characteristic fragment ions of solanesol (Zhao et al. 2007 ). Based on TLC profiles, fractions 11–14, which exhibited lower band intensity, were combined into a single UF. Fractions 7–10 and UF were selected for further quantification and bioactivity evaluation. Solanesol content was determined by UHPLC–DAD, with a retention time ( t R ) of 12.93 ± 0.14 min (Fig. 1 ). As shown in Table 1 , the overall solanesol yield was 1.52 ± 0.004% (w/w) relative to the dried material. Table 1 Solanesol concentration in the analyzed fractions determined by UHPLC–DAD at 215 nm. Samples Concentration (mg/ml) Solanesol content (%) F7 1.43 ± 0,001 62.17± 0.021 F8 1.82 ± 0,001 96.79 ± 0.003 F9 1.96 ± 0,004 98.00 ± 0.001 F10 0.88 ± 0,001 52.77 ± 0.009 UF 1.71 ± 0,017 90.00 ± 0.002 CE 15.22 ± 0,052 F: fractions 7, 8, 9, 10; UF: unified fraction; CE: crude extract Column chromatography enabled effective enrichment of solanesol, achieving high relative purity in fractions F8, F9, and UF, ranging from 90.00 ± 0.017% to 98.00 ± 0.004%, as determined by UHPLC–DAD. These values are comparable to those reported in the literature, where silica gel chromatography yields purities of 83–85% (Tang et al. 2007 , Safitra et al. 2024 ), and normal-phase chromatography on alumina reaches up to 95% (Ingle and Lali 2015 ). Although higher purities (~ 99%) have been achieved through multi-step processes combining scCO 2 extraction and advanced chromatographic techniques (Dębczak et al. 2025 ), the results obtained here were achieved using a simpler purification strategy, highlighting the efficiency of the proposed method. Evaluation of In Vitro Antioxidant Activity DPPH Radical Scavenging Assay The DPPH assay evaluates antioxidant activity through a mixed mechanism involving electron transfer and hydrogen atom transfer, partially reflecting antioxidant behavior in biological systems (Xie and Schaich 2014 , Apak et al. 2016 ). In this context, radical scavenging capacity serves as a key indicator of antioxidant potential. DPPH radical scavenging activity was evaluated at equivalent concentrations to allow comparison among samples. Overall, a good antioxidant capacity was observed, with the highest inhibition for the CE (88.25 ± 0.05%), followed by the UF (70.57 ± 0.05%), showing values comparable to the reference antioxidant Trolox (67.94 ± 0.01%). The remaining fractions exhibited lower activity, particularly those with lower purity. These results indicate that antioxidant activity is associated with solanesol-enriched fractions, while also reflecting the contribution of additional co-extracted compounds present in the crude extract. Detailed results are provided in Supplementary Table S1 and Fig. S2 . The antioxidant activity observed was higher than that reported in previous studies. For example, tobacco extracts obtained by heat-reflux extraction showed approximately 19% inhibition, whereas scCO 2 extracts reached 58% (Huang et al. 2008 ). Similarly, Banožić et al. ( 2019 ) reported a maximum inhibition of 37.24% for tobacco waste extracts. In this context, the results obtained here are comparatively higher despite the use of residual biomass, suggesting that the proposed extraction and purification strategy enhances antioxidant performance. Hydroxyl Radical ( • OH) Scavenging Activity The hydroxyl radical (•OH) is one of the most reactive and damaging reactive oxygen species (ROS) in biological systems, contributing to oxidative damage in DNA, lipids, and proteins. Therefore, evaluating the inhibition of •OH formation provides a relevant indicator of a compound’s protective potential against severe oxidative stress (Shahidi and Samarasinghe 2025 ). Figure 2 shows hydroxyl radical scavenging activity assessed by the Fenton reaction. Gallic acid was used as a positive control, exhibiting 72.00 ± 0.005% inhibition. Among the samples, the UF showed the lowest activity (38.81 ± 0.001%) at the same concentration as the control (1.71 ± 0.05 mg/ml), whereas the CE exhibited higher inhibition (55.29 ± 0.003%) at a slightly lower concentration (1.52 ± 0.02 mg/ml). Although comparable data for tobacco-derived extracts are limited, the results are consistent with those reported for other species. For example, Solanum muricatum showed 46.46% inhibition at 0.2 mg/ml, increasing to 89.63% at 1 mg/ml (Sudha et al. 2011 ). Similarly, •OH inhibition between 28.10% and 74.2% has been reported for Flammulina velutipes at comparable concentrations (Zhang et al. 2013 ). Cellular Evaluation of Cytotoxicity and Antioxidant Effects in Endothelial Cells Cytotoxicity Assessment The cell viability results shown in Fig. 3 confirm the low cytotoxic profile of the evaluated samples. As expected, the positive control significantly reduced cell viability, validating the sensitivity of the assay. In contrast, the negative control and the solanesol standard maintained high viability, indicating that solanesol itself does not exert cytotoxic effects under the tested conditions. The CE maintained high cell viability across all tested concentrations, with values close to or above 100%. In contrast, the UF showed a more pronounced decrease at specific concentrations, particularly at 0.1 and 10 mg/ml, where viability decreased to approximately 75% and 70%, respectively. However, even under these conditions, viability remained significantly higher than that of the positive control, indicating moderate effects. No clear dose–response relationship was observed, suggesting that the observed effects are not solely concentration-dependent but may be influenced by the composition of each fraction. Overall, both the CE and solanesol-enriched fractions exhibited low cytotoxicity in endothelial cells, supporting their suitability for non-cytotoxic applications. Previous studies further support this behavior. In human placental BeWo cells, solanesol at concentrations ranging from 0.001 to 0.2 mg/ml for 24 h did not reduce cell viability, as determined by the MTT assay (Lateef et al. 2023 ). Similarly, in L-02 hepatocytes, solanesol showed no significant cytotoxic effects compared to the control group at concentrations below 160 µM (0.10 mg/ml) (Liu et al. 2023 ). These findings are consistent with the low cytotoxicity observed in the present study. Taken together, these data confirm that solanesol-enriched extracts exhibit a favorable cytocompatibility profile, supporting their use in further studies aimed at evaluating their antioxidant effects at the cellular level. Intracellular ROS Scavenging Assay EA.hy926 endothelial cells are a suitable in vitro model for evaluating antioxidant capacity, as they exhibit functional and biochemical characteristics of human endothelium, including the expression of antioxidant enzymes and responsiveness to oxidative stimuli relevant to vascular pathophysiology (Huang et al. 2004 , Wang et al. 2019 ). In this context, intracellular ROS levels were assessed using the DCFH-DA probe, showing clear differences among treatments (Fig. 4 ). As expected, the positive control exhibited a marked increase in fluorescence compared to the negative control, confirming effective oxidative stress induction. Treatment with the CE and the unified fraction UF reduced intracellular ROS levels relative to the positive control, indicating a protective effect. This effect was more evident at intermediate concentrations (0.01–0.1 mg/ml), where both samples showed a noticeable decrease in fluorescence. However, the response was not strictly dose-dependent, as higher concentrations did not consistently enhance ROS reduction. In contrast, the solanesol standard showed a clearer concentration-dependent trend, with maximal ROS inhibition at 10 mg/ml. The more variable behavior of CE and UF likely reflects the influence of co-extracted compounds that may modulate antioxidant activity at the cellular level. These findings are consistent with previous studies in EA.hy926 cells, where compounds such as α-lipoic acid and phenolics (e.g., dihydrocaffeic acid) reduced intracellular ROS and enhanced antioxidant defenses (Jones et al. 2002 , Huang et al. 2004 ). Similarly, phenolic extracts have been shown to attenuate ROS production under inflammatory conditions in this model (Wang et al. 2019 ). Despite structural differences, these studies and the present results support a common outcome: modulation of intracellular oxidative stress. Overall, the CE and solanesol-enriched fractions effectively modulate intracellular ROS levels in endothelial cells. These findings highlight the complexity of cellular antioxidant responses and suggest potential applications in vascular protection and in conditions associated with oxidative stress, particularly those involving endothelial dysfunction (Youdim et al. 2000 , Wang et al. 2019 ). From a therapeutic perspective, modulation of redox balance in endothelial cells may contribute to preserving vascular function, preventing cellular senescence, and enhancing cell survival under stress conditions, which is relevant in cardiovascular, metabolic, and neurodegenerative diseases (Kamaruddin et al. 2022 ). Conclusion This study demonstrates that tobacco-derived residues are a viable and sustainable source of solanesol that can be efficiently recovered through a simple extraction and purification strategy. The low cytotoxicity observed in endothelial cells, together with the ability to modulate intracellular oxidative stress, supports a biocompatible antioxidant profile rather than a purely radical-scavenging effect. These findings highlight the importance of evaluating antioxidant activity at the cellular level, where compound interactions and biological context are critical. Declarations Acknowledgments: This work is part of Yudeyki Almuiña thesis to obtain the degree of Magister in Pharmaceutical Sciences from the University of Concepcion. National Fund for Scientific and Technological Development (FONDECYT) project N° 11220752, and the University of Concepcion financially supported this work. Authors' contributions: Study conception and design: YAG, MAL, OGB, CAT. Data collection: YAG, COF, DGH. Data analysis and interpretation: YAG, COF, OGB, CAT, MAL. Statistical analysis: YAG, DGH. Manuscript writing: YAG, MAL, OGB. Critical review of the manuscript: COF, MAL, OGB, CAT. 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Phytochem Rev 14:403–417. https://doi.org/10.1007/s11101-015-9393-5 Yan N, Liu Y, Liu L, Du Y, Liu X, Zhang H, Zhang Z (2019) Bioactivities and Medicinal Value of Solanesol and Its Accumulation, Extraction Technology, and Determination Methods. Biomolecules 9:334. https://doi.org/10.3390/biom9080334 Yao X, Bai Q, Yan D, Li G, Lü C, Xu H (2015) Solanesol protects human hepatic L02 cells from ethanol-induced oxidative injury via upregulation of HO-1 and Hsp70. Toxicol Vitro 29:600–608. https://doi.org/10.1016/j.tiv.2015.01.009 Youdim KA, Martin A, Joseph JA (2000) Incorporation of the elderberry anthocyanins by endothelial cells increases protection against oxidative stress. Free Radic Biol Med 29:51–60. https://doi.org/10.1016/S0891-5849(00)00329-4 Zhang Z, Jin Q, Lv G, Fan L, Pan H, Fan L (2013) Comparative study on antioxidant activity of four varieties of Flammulina velutipes with different colour. Int J Food Sci Technol 48:1057–1064. https://doi.org/10.1111/ijfs.12062 Zhao C, Li C, Zu Y (2007) Rapid and quantitative determination of solanesol in Nicotiana tabacum by liquid chromatography–tandem mass spectrometry. J Pharm Biomed Anal 44:35–40. https://doi.org/10.1016/j.jpba.2007.01.021 Zhou H-Y, Liu C-Z (2006) Microwave-assisted extraction of solanesol from tobacco leaves. J Chromatogr A 1129:135–139. https://doi.org/10.1016/j.chroma.2006.07.083 Supplementary Files GraphicalAbstractRBF.jpeg SupplementarymaterialRBFA.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 23 Apr, 2026 Reviewers invited by journal 20 Apr, 2026 Editor invited by journal 08 Apr, 2026 Editor assigned by journal 07 Apr, 2026 First submitted to journal 07 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9235979","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626397942,"identity":"1396cade-88c4-4b4d-9144-68a3a18e493e","order_by":0,"name":"Yudeyki Almuiña Guerra","email":"","orcid":"","institution":"Universidad de Concepcion","correspondingAuthor":false,"prefix":"","firstName":"Yudeyki","middleName":"Almuiña","lastName":"Guerra","suffix":""},{"id":626397943,"identity":"821d700b-9b8b-437d-914e-61c9ea3f07d2","order_by":1,"name":"Oscar Galarce-Bustos","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYBADGTD5gYEZRDEeIEYLD1jpDIgWBuK1MPMQo4Wf//DDzxW/7Hj4Zx9++Ni2zVqOv4H5AV4tkjPSjCXP9iXzSJxLMzbObUs3ljjAZoBXi8ENHgbJxh6gk87wsEnnth1O3AB0JH4t588w/2zsqeeRP8PD/tuy7XA9YS0HctgkG34c5jEA2sLM2HY4wYCQFqBfzCwbG47zGJ5hM5bsOZduOOMwAb8AQ+zxzYY/1XJyZ5gffvhRZi3P39788AE+LWDA2IbMYyaoHgT+EKVqFIyCUTAKRioAAPudReiQCSJIAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4252-1133","institution":"Universidad de Concepcion","correspondingAuthor":true,"prefix":"","firstName":"Oscar","middleName":"","lastName":"Galarce-Bustos","suffix":""},{"id":626397944,"identity":"5bb2e7f3-add5-40ea-96fb-78ae622d75df","order_by":2,"name":"Claudio Aguayo Tapia","email":"","orcid":"","institution":"Universidad de Concepcion","correspondingAuthor":false,"prefix":"","firstName":"Claudio","middleName":"Aguayo","lastName":"Tapia","suffix":""},{"id":626397945,"identity":"67214a1a-5fa7-49cb-8f20-4b08f3bd029a","order_by":3,"name":"Camilo Obregón-Berg","email":"","orcid":"","institution":"Universidad de Concepcion","correspondingAuthor":false,"prefix":"","firstName":"Camilo","middleName":"","lastName":"Obregón-Berg","suffix":""},{"id":626397946,"identity":"748a4d1f-7494-4f32-be4a-6edc118f8310","order_by":4,"name":"Deliany González Hernández","email":"","orcid":"","institution":"Universidad de Concepcion","correspondingAuthor":false,"prefix":"","firstName":"Deliany","middleName":"González","lastName":"Hernández","suffix":""},{"id":626397947,"identity":"acb52f96-3201-4119-8bf7-7829f4dace10","order_by":5,"name":"Marcia Avello Lorca","email":"","orcid":"https://orcid.org/0000-0003-2004-5487","institution":"Universidad de Concepcion","correspondingAuthor":false,"prefix":"","firstName":"Marcia","middleName":"Avello","lastName":"Lorca","suffix":""}],"badges":[],"createdAt":"2026-03-26 15:28:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9235979/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9235979/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108087236,"identity":"da5b065d-28a0-4442-9087-3fbb4929db8b","added_by":"auto","created_at":"2026-04-29 08:47:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1165240,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of solanesol at 215 nm. (a)\u003cstrong\u003e \u003c/strong\u003esolanesol standard, (b) crude extract, and (c) fraction 7-14\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9235979/v1/d08b659b269bed56ff724128.png"},{"id":108087237,"identity":"734ce046-8c90-4d57-a521-43b860fe5f00","added_by":"auto","created_at":"2026-04-29 08:47:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":148825,"visible":true,"origin":"","legend":"\u003cp\u003eHydroxyl radical scavenging activity expressed as percentage inhibition. Data are presented as the mean ± standard error of triplicates. *Different letters indicate statistically significant differences between means, according to one-way ANOVA, p \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9235979/v1/88a8a7e9a4723fab2d8813bc.png"},{"id":108491562,"identity":"83d7dcbf-4bdd-45e5-8a5d-569f331a793b","added_by":"auto","created_at":"2026-05-05 09:54:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":252248,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of the evaluated samples on endothelial cell viability. Values are expressed as mean ± SD (n = 4).*Different letters indicate statistically significant differences between means, according to one-way ANOVA, p \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9235979/v1/206e79a612a9aedf89c2e82a.png"},{"id":108087239,"identity":"e89bb111-6b17-4f4c-bb29-8902f2c65cdf","added_by":"auto","created_at":"2026-04-29 08:47:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":318519,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of \u003cem\u003eNicotiana tabacum\u003c/em\u003e L. fractions on intracellular ROS levels in EA.hy926 endothelial cells under H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress (0.3%). Control+ (with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), Control− (without H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and C0.01–10 (sample concentrations). Data are expressed as mean ± SE (n=3). Different letters indicate significant differences (one-way ANOVA, p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9235979/v1/0cca56b47210acc83e9144e6.png"},{"id":108490895,"identity":"e4c9ea0b-69ef-4bcb-bf0a-3b066534b785","added_by":"auto","created_at":"2026-05-05 09:49:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":33350,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the Introduction section.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9235979/v1/d22f3436c578e05fadd8887b.png"},{"id":108804746,"identity":"9f98d89c-41fe-4782-bae2-6727bb74ac33","added_by":"auto","created_at":"2026-05-08 15:23:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2001887,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9235979/v1/155d48e4-64b5-482c-8cec-e7919fdb0466.pdf"},{"id":108181786,"identity":"6cfcb32e-6752-4e0e-8e67-fc329903b861","added_by":"auto","created_at":"2026-04-30 08:58:55","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3377251,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstractRBF.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9235979/v1/b6c6a9eff51c7eb2a39c7c4a.jpeg"},{"id":108087240,"identity":"0809d8a3-49df-4528-844d-36f3054b0bcc","added_by":"auto","created_at":"2026-04-29 08:47:59","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1899835,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialRBFA.docx","url":"https://assets-eu.researchsquare.com/files/rs-9235979/v1/3cc60b73b68836a3757f43c3.docx"}],"financialInterests":"","formattedTitle":"Effect of a Solanesol-Enriched Extract from Nicotiana tabacum L. on Intracellular Oxidative Stress in Endothelial Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSolanesol (\u003cb\u003e1\u003c/b\u003e) is a linear terpenoid alcohol composed of nine isoprene units (Yan et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In recent years, it has gained increasing attention due to its role as a key precursor in the synthesis of high-value therapeutic compounds such as coenzyme Q10 and vitamin K2 (Qin et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In addition to its industrial relevance, solanesol exhibits antioxidant and anti-inflammatory properties, mainly attributed to its multiple non-conjugated double bonds, which enable efficient free radical scavenging and modulation of oxidative stress-related pathways (Yao et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Yan et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOxidative stress is a major contributor to cellular damage and is closely associated with the development of chronic diseases, including cardiovascular and neurodegenerative disorders (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Pisoschi et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this context, natural antioxidants have attracted considerable interest as potential agents to prevent or mitigate oxidative damage at both the molecular and cellular levels.\u003c/p\u003e \u003cp\u003eSolanesol is distributed in several species of the Solanaceae family, including \u003cem\u003eSolanum tuberosum\u003c/em\u003e (potato), \u003cem\u003eSolanum lycopersicum\u003c/em\u003e (tomato), \u003cem\u003eSolanum melongena\u003c/em\u003e (eggplant), and \u003cem\u003eCapsicum annuum\u003c/em\u003e (pepper), where it occurs in relatively low amounts in the leaves (0.09\u0026ndash;0.35%, 0.04\u0026ndash;0.4%, 0.2\u0026ndash;0.4%, and 0.09\u0026ndash;0.35% of dry weight, respectively). In contrast, significantly higher concentrations has been reported in \u003cem\u003eNicotiana tabacum\u003c/em\u003e L., where levels can reach up to 3.6%, making it one of the richest natural sources of this compound (Yan et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Campbell et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Importantly, the tobacco industry generates large amounts of residual biomass (e.g., leaves, dust, and stems), which constitute an abundant, low-cost, and underutilized source of solanesol (Tita et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to the World Health Organization (WHO), the tobacco industry generates approximately 25\u0026nbsp;million tons of waste annually worldwide (World Health Organization (WHO) \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These residues represent a valuable and sustainable source of solanesol, particularly because the chemical synthesis of this compound is highly complex. Furthermore, the curing process has been shown to increase the proportion of free solanesol, facilitating its recovery from these residues (Machado et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Bravo Cardenas et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this context, solanesol concentrations of up to 598.9 \u0026micro;g/ml have been reported in various tobacco industry waste products, including leaf remnants, dust, and midribs (Banožić et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTherefore, the aim of this study was to obtain a solanesol-enriched extract from \u003cem\u003eNicotiana tabacum\u003c/em\u003e waste using an efficient extraction and purification approach, and to evaluate its antioxidant capacity both \u003cem\u003ein vitro\u003c/em\u003e and in endothelial cells, with special emphasis on its ability to modulate intracellular reactive oxygen species.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and Reagents\u003c/h2\u003e \u003cp\u003eSolanesol standard (\u0026ge;\u0026thinsp;90%) was obtained from Cayman Chemical (Michigan, USA), ethanol, hexane, ethyl acetate, formic acid, HPLC grade acetonitrile, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2\u003cem\u003eH\u003c/em\u003e-tetrazolium bromide (MTT), 2',7'-dichlorofluorescin diacetate (DCFH-DA), phosphate-buffered saline (PBS), and DMSO were purchased from Merck (Darmstadt, Germany). DPPH, Trolox (\u0026ge;\u0026thinsp;97%), gallic acid (98%), Folin\u0026ndash;Ciocalteu (FC), ferrous chloride (FeCl\u003csub\u003e3\u003c/sub\u003e, \u0026ge;\u0026thinsp;99%), sodium carbonate, hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), ascorbic acid, deoxyribose solution, trichloroacetic acid (TCA), thiobarbituric acid (TBA), and silica gel for column chromatography (60 \u0026Aring; pore size, 70\u0026ndash;230 mesh) were purchased from Sigma-Aldrich (St. Louis, MO, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample\u003c/h3\u003e\n\u003cp\u003eProcessed tobacco strands of the Burley type (\u003cem\u003eN. tabacum\u003c/em\u003e L., Solanaceae) were obtained from a commercial source (brand: Tabaco Bristol Org\u0026aacute;nico; Tabacalera S.A., Chile). The material originated from crops located in San Clemente, Maule Region, Chile (35\u0026deg;32\u0026prime;16.3\u0026Prime;S, 71\u0026deg;27\u0026prime;31.1\u0026Prime;W). Samples were dried at 45.0\u0026deg;C for 4 h, ground, and sieved to obtain a particle size\u0026thinsp;\u0026lt;\u0026thinsp;45 \u0026micro;m prior to extraction. A representative sample was deposited in the laboratory collection of Pharmacognosy of the Faculty of Pharmacy, Universidad de Concepci\u0026oacute;n, Chile.\u003c/p\u003e\n\u003ch3\u003eUltrasound-Assisted Extraction of Solanesol\u003c/h3\u003e\n\u003cp\u003eSolanesol was extracted from 50 g of tobacco powder using an ultrasonic bath (Elma, Singen, Germany; 320 W) at 30.0\u0026deg;C for 30 min with 500 ml of ethanol. The extract was filtered and concentrated under reduced pressure at 50.0\u0026deg;C to obtain a crude extract (CE). To remove alkaloid-type compounds, the extract was partitioned with hexane and washed twice with 1% HCl, followed by distilled water. The organic phase was dried over anhydrous Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, filtered, and concentrated. The resulting extract (1.6 g) was re-dissolved in hexane:ethyl acetate (4:1, v/v) for further purification.\u003c/p\u003e\n\u003ch3\u003eColumn Chromatography Fractionation\u003c/h3\u003e\n\u003cp\u003eThe CE was subjected to open-column chromatography (2 cm \u0026times; 16.5 cm) packed with silica gel (18 g). Elution was performed using hexane:ethyl acetate (4:1, v/v), and eighteen fractions (5 ml) were collected. Fractions were monitored by thin-layer chromatography (TLC) using vanillin reagent and pooled according to similar chromatographic profiles.\u003c/p\u003e\n\u003ch3\u003eDetermination of Solanesol by UHPLC–DAD\u003c/h3\u003e\n\u003cp\u003eIdentification of solanesol in the fractions was performed using an ultra-high performance liquid chromatography system coupled with diode array detection and tandem mass spectrometry (UHPLC\u0026ndash;DAD\u0026ndash;MS/MS) (Shimadzu LCMS-8030, Shimadzu, Kyoto, Japan). Chromatographic separation was performed on a Kinetex\u0026reg; Phenyl-Hexyl column (100 \u0026times; 2.1 mm, 1.7 \u0026micro;m; Phenomenex, USA) using an isocratic mobile phase of acetonitrile:0.1% formic acid (80:20, v/v) at 0.4 ml/min and 35.0\u0026deg;C. Detection was carried out by ESI-MS in positive mode (\u003cem\u003em/z\u003c/em\u003e 50\u0026ndash;1000).\u003c/p\u003e \u003cp\u003eSolanesol quantification was performed by UHPLC\u0026ndash;DAD using the same column under isocratic conditions (acetonitrile:water, 80:20, v/v) at 0.5 ml/min and 50.0\u0026deg;C. The injection volume was 5 \u0026micro;l. Calibration was carried out over a range of 10\u0026ndash;100 \u0026micro;g/ml, and samples were filtered (0.45 \u0026micro;m) prior to analysis.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant Activity\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eDPPH Radical Scavenging Assay\u003c/h2\u003e \u003cp\u003eDPPH radical scavenging activity was determined according to Galarce-Bustos et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) with minor modifications. Briefly, 20 \u0026micro;l of sample was mixed with 180 \u0026micro;l of 0.15 mM DPPH solution. After incubation for 30 min at room temperature in the dark, the absorbance was measured at 517 nm.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eHydroxyl Radical Scavenging Assay\u003c/h3\u003e\n\u003cp\u003eHydroxyl radical scavenging activity was determined according to Halliwell et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1987\u003c/span\u003e) with modifications. Briefly, 50 \u0026micro;l of FeCl\u003csub\u003e3\u003c/sub\u003e (20 mM), 50 \u0026micro;l of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (42 mM), and 10 \u0026micro;l of ascorbic acid (50 mM) were mixed. Then, 10 \u0026micro;l of sample or control solution (gallic acid, 10 mM) and 100 \u0026micro;l of deoxyribose (2.8 mM) were added. The mixture was incubated at 37.0\u0026deg;C for 1 h. Subsequently, 1 ml of 2.8% TCA and 1 ml of TBA were added, followed by heating at 100\u0026deg;C for 15 min. After cooling to room temperature, absorbance was measured at 532 nm.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCellular Viability and Oxidative Stress Assays\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eCytotoxicity Assay on Endothelial Cells\u003c/h2\u003e \u003cp\u003eEA.hy926 endothelial cells were seeded in 96-well plates (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) and incubated for 24 h at 37.0\u0026deg;C under 5.0% CO\u003csub\u003e2\u003c/sub\u003e. Cells were treated in quadruplicate (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4) with samples (0.01\u0026ndash;10 mg/ml) for 24 h. Cell viability was assessed using a modified MTT assay (Carrasco et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). After treatment, cells were washed with PBS and incubated with 10 \u0026micro;l of MTT solution (14.5 mM) for 4 h at 37.0\u0026deg;C. Formazan crystals were dissolved in 50 \u0026micro;l of DMSO, and absorbance was measured at 570 nm. Results were expressed as percentage cell viability:\u003c/p\u003e \u003cp\u003eTotal Reactive Oxygen Species Detection in Cells\u003c/p\u003e \u003cp\u003eIntracellular ROS levels were determined according to (Huang et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) with modifications. EA.hy926 cells were seeded in 96-well plates (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) and incubated for 24 h at 37.0\u0026deg;C under 5.0% CO\u003csub\u003e2\u003c/sub\u003e. Cells were treated in triplicate (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) with samples (0.01\u0026ndash;10 mg/ml) for 24 h. After washing with PBS, oxidative stress was induced using 5 \u0026micro;l of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (2% in PBS) for 30 min. ROS production was measured using DCFH-DA (0.5 \u0026micro;M) after 30 min incubation at 37\u0026deg;C. Fluorescence was recorded at 485/520 nm using a microplate reader, and results were expressed relative to control conditions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eUltrasound-Assisted Extraction and Column-Based Enrichment of Solanesol\u003c/h2\u003e \u003cp\u003eResidual biomass generated during industrial tobacco processing (veins, dust, cured leaves, and stems) contains solanesol at levels ranging from 0.05% to 0.33% (w/w), corresponding to approximately 30% of the content found in fresh leaves. Notably, the curing process increases the proportion of free solanesol (Machado et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, Banožić et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Despite these lower concentrations, the large volumes of tobacco waste make these residues a practical, sustainable, and economically attractive source for solanesol recovery.\u003c/p\u003e \u003cp\u003eIn this study, solanesol was extracted using ultrasound-assisted extraction (UAE), a rapid and energy-efficient technique with low solvent consumption (Shen et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Under the applied conditions, a yield of 3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002% (w/w) relative to the dried material was obtained, exceeding previously reported UAE yields for tobacco residues (0.8\u0026ndash;2.3% w/w) (Banožić et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This yield is also competitive with other extraction techniques, such as microwave-assisted extraction (MAE), which reaches up to 0.91% (w/w) (Zhou and Liu \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and sequential microwave\u0026ndash;ultrasound-assisted extraction (MUAE), which achieves approximately 3.0% (w/w) but requires multiple processing steps (Safitra et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Although supercritical CO\u003csub\u003e2\u003c/sub\u003e extraction (scCO\u003csub\u003e2\u003c/sub\u003e) can reach yields of up to 3.74% (w/w), it involves high-pressure equipment and increased operational costs (Huang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In this context, the UAE approach used here provides a competitive and simpler alternative.\u003c/p\u003e \u003cp\u003eFollowing extraction, column chromatography proved to be an effective method for solanesol purification and enrichment. Separation was rapidly optimized, identifying the extraction solvent as a key factor influencing fractionation efficiency. Hexane extracts showed greater matrix complexity, leading to poorer resolution, whereas ethanol extracts yielded cleaner profiles, facilitating the removal of interfering compounds and improving solanesol enrichment. These results highlight the importance of solvent selection for both extraction and downstream purification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eQualitative and Quantitative Analysis of Solanesol Fractions\u003c/h2\u003e \u003cp\u003eThe obtained fractions were initially analyzed by TLC, where solanesol was predominantly detected in fractions 7 to 14 with an \u003cem\u003eR\u003c/em\u003e\u003csub\u003e\u003cem\u003eF\u003c/em\u003e\u003c/sub\u003e value of 0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001, consistent with the solanesol standard. The presence of the analyte was further confirmed by mass spectrometry, showing matching molecular ions at \u003cem\u003em/z\u003c/em\u003e 669.5, 653.6, and 613.6 [M\u0026thinsp;+\u0026thinsp;H]⁺ (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), corresponding to characteristic fragment ions of solanesol (Zhao et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on TLC profiles, fractions 11\u0026ndash;14, which exhibited lower band intensity, were combined into a single UF. Fractions 7\u0026ndash;10 and UF were selected for further quantification and bioactivity evaluation. Solanesol content was determined by UHPLC\u0026ndash;DAD, with a retention time (\u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003eR\u003c/em\u003e\u003c/sub\u003e) of 12.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the overall solanesol yield was 1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004% (w/w) relative to the dried material.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSolanesol concentration in the analyzed fractions determined by UHPLC\u0026ndash;DAD at 215 nm.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003cp\u003e(mg/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSolanesol content\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.43 \u0026plusmn; 0,001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e62.17\u0026plusmn; 0.021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.82 \u0026plusmn; 0,001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e96.79 \u0026plusmn; 0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.96 \u0026plusmn; 0,004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e98.00 \u0026plusmn; 0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.88 \u0026plusmn; 0,001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e52.77 \u0026plusmn; 0.009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.71 \u0026plusmn; 0,017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e90.00 \u0026plusmn; 0.002\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.22 \u0026plusmn; 0,052\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003eF: fractions 7, 8, 9, 10; UF: unified fraction; CE: crude extract\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eColumn chromatography enabled effective enrichment of solanesol, achieving high relative purity in fractions F8, F9, and UF, ranging from 90.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017% to 98.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004%, as determined by UHPLC\u0026ndash;DAD. These values are comparable to those reported in the literature, where silica gel chromatography yields purities of 83\u0026ndash;85% (Tang et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Safitra et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and normal-phase chromatography on alumina reaches up to 95% (Ingle and Lali \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Although higher purities (~\u0026thinsp;99%) have been achieved through multi-step processes combining scCO\u003csub\u003e2\u003c/sub\u003e extraction and advanced chromatographic techniques (Dębczak et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), the results obtained here were achieved using a simpler purification strategy, highlighting the efficiency of the proposed method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of In Vitro Antioxidant Activity\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003eDPPH Radical Scavenging Assay\u003c/h2\u003e \u003cp\u003eThe DPPH assay evaluates antioxidant activity through a mixed mechanism involving electron transfer and hydrogen atom transfer, partially reflecting antioxidant behavior in biological systems (Xie and Schaich \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Apak et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this context, radical scavenging capacity serves as a key indicator of antioxidant potential. DPPH radical scavenging activity was evaluated at equivalent concentrations to allow comparison among samples. Overall, a good antioxidant capacity was observed, with the highest inhibition for the CE (88.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05%), followed by the UF (70.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05%), showing values comparable to the reference antioxidant Trolox (67.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01%). The remaining fractions exhibited lower activity, particularly those with lower purity. These results indicate that antioxidant activity is associated with solanesol-enriched fractions, while also reflecting the contribution of additional co-extracted compounds present in the crude extract. Detailed results are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and \u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe antioxidant activity observed was higher than that reported in previous studies. For example, tobacco extracts obtained by heat-reflux extraction showed approximately 19% inhibition, whereas scCO\u003csub\u003e2\u003c/sub\u003e extracts reached 58% (Huang et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Similarly, Banožić et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported a maximum inhibition of 37.24% for tobacco waste extracts. In this context, the results obtained here are comparatively higher despite the use of residual biomass, suggesting that the proposed extraction and purification strategy enhances antioxidant performance.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHydroxyl Radical (\u003c/b\u003e\u0026bull;\u003cb\u003eOH) Scavenging Activity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe hydroxyl radical (\u0026bull;OH) is one of the most reactive and damaging reactive oxygen species (ROS) in biological systems, contributing to oxidative damage in DNA, lipids, and proteins. Therefore, evaluating the inhibition of \u0026bull;OH formation provides a relevant indicator of a compound\u0026rsquo;s protective potential against severe oxidative stress (Shahidi and Samarasinghe \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows hydroxyl radical scavenging activity assessed by the Fenton reaction. Gallic acid was used as a positive control, exhibiting 72.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005% inhibition. Among the samples, the UF showed the lowest activity (38.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001%) at the same concentration as the control (1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mg/ml), whereas the CE exhibited higher inhibition (55.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003%) at a slightly lower concentration (1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mg/ml).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough comparable data for tobacco-derived extracts are limited, the results are consistent with those reported for other species. For example, \u003cem\u003eSolanum muricatum\u003c/em\u003e showed 46.46% inhibition at 0.2 mg/ml, increasing to 89.63% at 1 mg/ml (Sudha et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Similarly, \u0026bull;OH inhibition between 28.10% and 74.2% has been reported for \u003cem\u003eFlammulina velutipes\u003c/em\u003e at comparable concentrations (Zhang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCellular Evaluation of Cytotoxicity and Antioxidant Effects in Endothelial Cells\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003eCytotoxicity Assessment\u003c/h2\u003e \u003cp\u003eThe cell viability results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e confirm the low cytotoxic profile of the evaluated samples. As expected, the positive control significantly reduced cell viability, validating the sensitivity of the assay. In contrast, the negative control and the solanesol standard maintained high viability, indicating that solanesol itself does not exert cytotoxic effects under the tested conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe CE maintained high cell viability across all tested concentrations, with values close to or above 100%. In contrast, the UF showed a more pronounced decrease at specific concentrations, particularly at 0.1 and 10 mg/ml, where viability decreased to approximately 75% and 70%, respectively. However, even under these conditions, viability remained significantly higher than that of the positive control, indicating moderate effects. No clear dose\u0026ndash;response relationship was observed, suggesting that the observed effects are not solely concentration-dependent but may be influenced by the composition of each fraction. Overall, both the CE and solanesol-enriched fractions exhibited low cytotoxicity in endothelial cells, supporting their suitability for non-cytotoxic applications.\u003c/p\u003e \u003cp\u003ePrevious studies further support this behavior. In human placental BeWo cells, solanesol at concentrations ranging from 0.001 to 0.2 mg/ml for 24 h did not reduce cell viability, as determined by the MTT assay (Lateef et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Similarly, in L-02 hepatocytes, solanesol showed no significant cytotoxic effects compared to the control group at concentrations below 160 \u0026micro;M (0.10 mg/ml) (Liu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These findings are consistent with the low cytotoxicity observed in the present study.\u003c/p\u003e \u003cp\u003eTaken together, these data confirm that solanesol-enriched extracts exhibit a favorable cytocompatibility profile, supporting their use in further studies aimed at evaluating their antioxidant effects at the cellular level.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular ROS Scavenging Assay\u003c/h2\u003e \u003cp\u003eEA.hy926 endothelial cells are a suitable \u003cem\u003ein vitro\u003c/em\u003e model for evaluating antioxidant capacity, as they exhibit functional and biochemical characteristics of human endothelium, including the expression of antioxidant enzymes and responsiveness to oxidative stimuli relevant to vascular pathophysiology (Huang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Wang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this context, intracellular ROS levels were assessed using the DCFH-DA probe, showing clear differences among treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). As expected, the positive control exhibited a marked increase in fluorescence compared to the negative control, confirming effective oxidative stress induction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTreatment with the CE and the unified fraction UF reduced intracellular ROS levels relative to the positive control, indicating a protective effect. This effect was more evident at intermediate concentrations (0.01\u0026ndash;0.1 mg/ml), where both samples showed a noticeable decrease in fluorescence. However, the response was not strictly dose-dependent, as higher concentrations did not consistently enhance ROS reduction. In contrast, the solanesol standard showed a clearer concentration-dependent trend, with maximal ROS inhibition at 10 mg/ml. The more variable behavior of CE and UF likely reflects the influence of co-extracted compounds that may modulate antioxidant activity at the cellular level.\u003c/p\u003e \u003cp\u003eThese findings are consistent with previous studies in EA.hy926 cells, where compounds such as α-lipoic acid and phenolics (e.g., dihydrocaffeic acid) reduced intracellular ROS and enhanced antioxidant defenses (Jones et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Huang et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Similarly, phenolic extracts have been shown to attenuate ROS production under inflammatory conditions in this model (Wang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Despite structural differences, these studies and the present results support a common outcome: modulation of intracellular oxidative stress.\u003c/p\u003e \u003cp\u003eOverall, the CE and solanesol-enriched fractions effectively modulate intracellular ROS levels in endothelial cells. These findings highlight the complexity of cellular antioxidant responses and suggest potential applications in vascular protection and in conditions associated with oxidative stress, particularly those involving endothelial dysfunction (Youdim et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, Wang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). From a therapeutic perspective, modulation of redox balance in endothelial cells may contribute to preserving vascular function, preventing cellular senescence, and enhancing cell survival under stress conditions, which is relevant in cardiovascular, metabolic, and neurodegenerative diseases (Kamaruddin et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that tobacco-derived residues are a viable and sustainable source of solanesol that can be efficiently recovered through a simple extraction and purification strategy. The low cytotoxicity observed in endothelial cells, together with the ability to modulate intracellular oxidative stress, supports a biocompatible antioxidant profile rather than a purely radical-scavenging effect. These findings highlight the importance of evaluating antioxidant activity at the cellular level, where compound interactions and biological context are critical.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e This work is part of Yudeyki Almui\u0026ntilde;a thesis to obtain the degree of Magister in Pharmaceutical Sciences from the University of Concepcion. National Fund for Scientific and Technological Development (FONDECYT) project N\u0026deg; 11220752, and the University of Concepcion financially supported this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e Study conception and design: YAG, MAL, OGB, CAT. Data collection: YAG, COF, DGH. Data analysis and interpretation: YAG, COF, OGB, CAT, MAL. Statistical analysis: YAG, DGH. Manuscript writing: YAG, MAL, OGB. Critical review of the manuscript: COF, MAL, OGB, CAT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: National Fund for Scientific and Technological Development (FONDECYT) project N\u0026deg; 11220752.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e: not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eApak R, \u0026Ouml;zy\u0026uuml;rek M, G\u0026uuml;\u0026ccedil;l\u0026uuml; K, \u0026Ccedil;apanoğlu E (2016) Antioxidant Activity/Capacity Measurement. 2. Hydrogen Atom Transfer (HAT)-Based, Mixed-Mode (Electron Transfer (ET)/HAT), and Lipid Peroxidation Assays. 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J Chromatogr A 1129:135\u0026ndash;139. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.chroma.2006.07.083\u003c/span\u003e\u003cspan address=\"10.1016/j.chroma.2006.07.083\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ultrasound-assisted extraction, sonalensol-enriched extract, EA.hy926 cell line, intracellular redox balance, vascular dysfunction","lastPublishedDoi":"10.21203/rs.3.rs-9235979/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9235979/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOxidative stress and chronic inflammation play a key role in the development and progression of various cellular pathologies, mainly caused by the overproduction of reactive oxygen species. In this context, natural antioxidant compounds have attracted growing attention for their potential to prevent and reduce oxidative damage. In this study, we obtained a solanesol fraction with high-purity from \u003cem\u003eNicotiana tabacum\u003c/em\u003e L., Solanaceae, and evaluated its antioxidant capacity \u003cem\u003ein vitro\u003c/em\u003e to determine its potential to mitigate oxidative stress. The extract was obtained from \u003cem\u003eN. tabacum\u003c/em\u003e leaves by ultrasound-assisted extraction and further fractionated by column chromatography. The presence and concentration of solanesol were confirmed by high-performance liquid chromatography and mass spectrometry. The \u003cem\u003ein vitro\u003c/em\u003e assays were performed to determine antioxidant activity, cytotoxicity in endothelial cells, and the generation of intracellular reactive oxygen species. The purified fractions showed strong antioxidant activity, with hydroxyl radical inhibition exceeding 38.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001%, low cytotoxicity, and a stable inhibition of reactive oxygen species (34.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006%) over the 5 min observation period. These results support the potential of the solanesol-enriched extract as a bioactive ingredient, highlighting its possible applicability in preventive or therapeutic strategies targeting oxidative stress associated to cellular disorders.\u003c/p\u003e","manuscriptTitle":"Effect of a Solanesol-Enriched Extract from Nicotiana tabacum L. on Intracellular Oxidative Stress in Endothelial Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-29 08:47:54","doi":"10.21203/rs.3.rs-9235979/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-04-23T17:24:17+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-20T13:59:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Revista Brasileira de Farmacognosia","date":"2026-04-08T12:09:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-07T14:02:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Revista Brasileira de Farmacognosia","date":"2026-04-07T10:02:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"af91a421-b566-4720-ad82-4a19a8c33f79","owner":[],"postedDate":"April 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T08:47:54+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-29 08:47:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9235979","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9235979","identity":"rs-9235979","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}