Characterization of oxyphytosterols generated by β-sitosterol ozonization Igor Rodrigues Martins,

ABSTRACT

Your work provides valuable insight into both the benefits and potential risks associated with β-sitosterol (βSito), a prominent phytosterol in various plant-based foods. While βSito is recognized for its positive effects, such as lowering LDL cholesterol and reducing cardiovascular risks, the focus on the generation of oxyphytosterols through oxidative processes adds a critical dimension to understanding its overall impact on food quality and health.

The proposed mechanism of forming oxyphytosterols like βSec, βLac, and βCOOH under ozone exposure is fascinating. The susceptibility of βSito to oxidation at its unsaturated C5-C6 bond mirrors the behavior seen with cholesterol, emphasizing the influence of reactive oxygen species like ozone. These oxidation products, which reduced HepG2 cell viability in cytotoxicity assays, underscore the need for further investigation into their biological and health implications.

The broader discussion about ozone as a potent oxidizing agent highlights its dual role. While it has industrial and environmental applications, its presence at high concentrations in urban areas like Mexico City and São Paulo raises concerns about human and crop health. The interplay between ozone pollution and the formation of oxidized compounds in food systems has significant ramifications, particularly in megacities where environmental factors exacerbate these challenges.

Phytosterols like βSito are celebrated for their diverse benefits, such as tumor growth inhibition and improved immune responses. Yet, as highlighted, the molecular biological mechanisms of their actions remain incompletely understood, and the potential adverse effects of oxyphytosterols warrant careful consideration. Your study bridges a critical gap by synthesizing and characterizing βSito oxidation products, providing a foundation for future research on their impacts and mitigation strategies. It also raises awareness of the need to balance the health benefits of phytosterols with the risks posed by their oxidative derivatives, especially in ozone-polluted environments.

Your research delves into the complex role of oxyphytosterols, which, while potentially harmful in some contexts, exhibit a spectrum of biological effects, both adverse and beneficial. The detection of oxyphytosterols in various biological and clinical samples, such as plasma, serum, and aortic tissues, underscores their prevalence and relevance in metabolic and cardiovascular health.

The findings linking oxyphytosterols to atherosclerotic lesions in LDL receptor-deficient mice and their cytotoxicity toward cultured macrophage-derived cell lines point to their pro-atherogenic and potentially toxic effects. Additionally, their ability to attenuate vasorelaxation in rat aorta, driven by reactive oxygen species production and downstream signaling activation, further highlights their role in vascular dysfunction.

On the other hand, the anti-inflammatory, anti-diabetic, anti-viral, and even anti-carcinogenic properties of oxyphytosterols present an intriguing paradox. Notably, the observed antiproliferative and pro-apoptotic effects of oxyphytosterols against colon cancer cell lines HCT-116 and HT-29, as reported by Zhu et al., suggest their therapeutic potential. This duality highlights the need for a deeper understanding of specific oxyphytosterol species, their formation under different conditions, and their diverse biological impacts.

Your characterization of the three major oxyphytosterol species—βSec, βLac, and βCOOH—generated by βSito ozonization represents a significant contribution to the field. The use of advanced techniques like 1D and 2D NMR spectroscopy and high-resolution mass spectrometry (HR-MS) to analyze these species strengthens the reliability of your findings. The cytotoxic effects of βSito and βSec on HepG2 cell viability further reinforce the importance of understanding their mechanisms of action.

This study not only underscores the complexity surrounding oxyphytosterols but also provides a foundation for evaluating their biological roles in greater detail. Balancing their health risks and therapeutic potential, especially in the context of exposure to reactive oxidants like ozone, remains a critical area of investigation for advancing nutritional and medical science.

Materials and Methods

Various reagents and materials were utilized in the experimental procedures. β-Sitosterol, silica gel 60 (230-400 mesh), deuterium chloroform (CDCl3), anhydrous sodium sulfate, powdered zinc, dimethyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) were all procured from Sigma Aldrich, headquartered in Steinheim, Germany. High-purity oxygen (>99%) was supplied by Air Liquide, located in São Paulo, Brazil.

Additionally, silica gel 60 F254 plates were sourced from Macherey-Nagel in Düren, Germany, while acetic acid was obtained from Labsynth in São Paulo, Brazil. Water used in all experimental procedures was of ultrapure grade, prepared using MilliQ® Reference equipment, manufactured by Merck Millipore in Darmstadt, Germany. For solvent requirements, high-performance liquid chromatography (HPLC) grade solvents were acquired from J.T. Baker, Mexico. Essential cell culture reagents, including penicillin, streptomycin, fetal bovine serum (FBS), and DMEM, were purchased from Thermo Fisher Scientific, based in Massachusetts, USA. Furthermore, the HepG2 human liver cancer cell line (Code 0103) was obtained from the Rio de Janeiro Cell Bank (BCRJ), Brazil.

The ozonization of β-sitosterol was carried out under controlled conditions at a temperature of 4°C. The procedure employed an Aquazone Plus instrument, designed by Red Sea Fish Pharm Ltd. in Eilat, Israel, to produce ozone. A 24 mM β-sitosterol solution (300 mg) was dissolved in CH2Cl2 and subjected to an ozone flow rate of 90 mL/min (52 mM) for a duration of 1.5 hours. After completing the ozonization, the organic solvent was evaporated under reduced pressure.

This was followed by a reduction reaction using 150 mg of powdered zinc, combined with 20 mL of an acetic acid-water solution (20:1, v/v), which was stirred continuously for 2 hours. For the extraction process, 100 mL of dichloromethane was used, and the solution underwent triple washes with 100 mL of water. Residual water was removed using anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. Purification of the β-sitosterol oxidation products was achieved via flash column chromatography, using silica gel 60 (230-400 mesh).

The chromatography column was initially equilibrated with hexane, followed by the application of a gradient mixture comprising hexane and ethyl acetate or hexane and diethyl ether. Additional purification was performed using preparative thin-layer chromatography (TLC) on Merck Kieselgel 60 F254 plates (0.25 mm coating thickness). Elution was carried out with an ethyl acetate and hexane solution (1:1). The resultant compounds were subsequently characterized through spectroscopic techniques, including nuclear magnetic resonance (NMR), infrared (IR) analysis, and high-resolution mass spectrometry (HR-MS). For isolating oxyphytosterol βCOOH, high-performance liquid chromatography (HPLC) was employed, incorporating analytic and semi-preparative biphenyl columns (Phenomenex, USA) in an isocratic mode. The mobile phase consisted of 85% acetonitrile and 15% water, delivered at a flow rate of 4 mL/min.

The isolated oxyphytosterols underwent NMR analysis using a Bruker-Biospin Ascend 500 instrument from the Avance III series (Rheinstetten, Germany). Operating at 11.7 Tesla, the instrument was equipped with a 5-mm TXI probe with z-gradient capabilities and a 5-mm TCI prodigy cryoprobe. Temperature control was facilitated by a BCU I accessory. Chemical shifts were expressed in parts per million (ppm) relative to the deuterium solvent (CHCl3 in CDCl3: 7.25 ppm for 1H NMR, and 77.00 ppm for 13C NMR) or tetramethylsilane (TMS). Data acquisition and processing were performed using the TOPSPIN 3.2 software from Bruker-Biospin. The 1H NMR (500.13 MHz) and 13C NMR (125.77 MHz) spectra were recorded at 0°C using CDCl3 as the solvent.

For mass spectrometric characterization, oxyphytosterols were analyzed using a high-resolution microTOF-Q QTOF instrument from Bruker Daltonik Inc. (Massachusetts, USA). Positive electrospray ionization mode (ESI+) was employed for the analysis. A 100 µM solution of oxyphytosterols in a 9:1 methanol-water mixture was injected into the instrument at a flow rate of 180 µL/h. Parameters such as the drying gas flow rate (5.0 L/min), nebulizing gas pressure (2.0 bar), capillary voltage (4.5 kV), and gas temperature (180°C) were optimized for accurate data collection.

Infrared analysis was performed using a Prestige 21 IR spectrometer from Shimadzu (Tokyo, Japan). Samples were dissolved in chloroform (100 µL) and applied to a KCl disk. The solvent was evaporated prior to analysis.

For the cell viability assay, HepG2 cells were cultured in DMEM supplemented with 10% FBS and antibiotics (100 U/mL penicillin and 0.1 µg/mL streptomycin) at 37°C in a humidified environment containing 5% CO2. Cells (1.5 x 104) were seeded into 96-well plates containing 100 µL of DMEM without FBS and treated with either β-sitosterol (β-Sito) or oxysecophytosterol (βSec) at concentrations of 10 µM or 100 µM (prepared in 1% ethanol) for 24, 48, and 72 hours. Post-treatment, the medium was replaced with 100 µL of DMEM without phenol red and 20 µL of MTT solution (5 mg/mL), followed by incubation at 37°C for 4 hours. Formazan crystals formed during the assay were dissolved in 100 µL of DMSO, and absorbance readings were taken at 570 nm and 630 nm. Results were expressed as the mean ± standard error of the mean (SEM) relative to the control group. Data were obtained from three independent experiments, with six replicates per sample. Statistical analysis was conducted using the Dunnett ANOVA test for multiple comparisons, with the significance levels denoted as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Results and Discussion

The primary product, βSec (Scheme 1, compound 5), is characterized by the absence of olefin hydrogen in its 1H NMR spectrum, replaced by a distinctive broad peak at 9.62 ppm (Figure 1B). This peak is indicative of an aldehyde group, which is notably absent in the βSito spectrum (Figure 1A). This identification is further substantiated by HSQC analysis, revealing a direct correlation between the aldehyde's hydrogen and carbon at 203.2 ppm (Supplementary Material, Figures S1 and S6). Protons associated with methyl groups exhibit distinct signals in the 1H NMR spectrum. For instance, C18 (δ 11.47) and C19 (δ 17.55) manifest as singlets, while C21 (δ 18.54), C26 (δ 18.83), C27 (δ 19.84), and C29 (δ 11.89) appear as doublets. These signals display strong correlations with their respective carbons as confirmed by the HSQC spectrum.

HMBC analysis, which combines information from both 1H and HSQC spectra, elucidates correlations between hydrogen and carbon atoms separated by two or three bonds (Figure S8). For the aldehyde hydrogen H6 (δ 9.62), HMBC reveals 2J and 3J correlations with carbons C7 (δ 44.05) and C8 (δ 34.71), respectively. HSQC further assigns hydrogen H7α and H7β (δ 2.30 and δ 2.42) and H8β (δ 1.82). A carbonyl group at C5 corresponds to a peak at 218.0 ppm in the spectrum. Correlations were observed between H4α and H4β (δ 3.13 and δ 2.30), H3α (δ 4.51), H1α (δ 1.73), and H19 (δ 1.02) in the HMBC analysis, with carbon assignments verified using the HSQC spectrum. The compound was obtained as a colorless oil with a yield of 26.7%. The spectra and chemical shifts further confirm the structural composition: 1H NMR (500 MHz, CDCl3) δ 0.68 (s, 3H), 0.80 (m, 3H), 0.82 (m, 6H), 0.83 (m, 3H), among others.

The shielding effects observed under chemical shifts for hydrogens 3 and 4 on βSec, βLac, and βCOOH (Figures 1B, 1C, and 1D) when compared to βSito (Figure 1A) are noteworthy. The olefin hydrogen present in βSito at δ 5.32 ppm is absent in the 1H NMR spectra of oxyphytosterols, indicative of oxidation occurring at βSito's double bond upon exposure to ozone. Aldehyde-specific peaks around δ 9.3 ppm, observed in spectra B and C (Figure 1), confirm the presence of aldehyde groups in compounds βSec and βLac. These peaks are absent in the βCOOH spectrum, highlighting structural differences. Figure 2 provides comparative 13C NMR spectra for βSito (A), βSec (B), βLac (C), and βCOOH (D), highlighting variations in carbonyl chemical shifts and carbon assignments across compounds. Detailed 1H and 13C chemical shifts for oxyphytosterols are comprehensively assigned using 1D and 2D NMR data, as documented in the supplementary material.

The βLac compound (Scheme 1, compound 9) represents a structural derivative of βSec, featuring a lactone group formation within the A ring. This structural modification is validated through 1D and 2D NMR, HR-MS (Figure S26), and infrared analyses (Figure S29). In the 1H NMR spectrum, the aldehyde hydrogen is observed at δ 9.70, accompanied by the aldehyde carbon signal at δ 206.56 in the 13C NMR spectrum (Figures 1C, 2C, S9, and S10). Protons from methyl groups within the ring exhibit distinct singlet peaks at δ 0.68 and δ 1.37, while overlapping signals are observed for other methyl groups at δ 0.82–0.85. The HSQC spectrum corroborates the carbon associations. The lactone's carbonyl group is characterized by a peak at δ 171.92 in the 13C NMR spectrum and a strong infrared absorption at 1735 cm–1, underscoring its structural presence. HMBC analysis confirms strong correlations of the carbonyl group with a methylene group directly bonded to oxygen (δ 2.98). The compound was obtained as a colorless oil with a yield of 20%.

The βCOOH compound (Scheme 1, compound 6) emerges as an autoxidation product of βSec. This transformation results in the presence of carboxylic acid and ketone groups, replacing the aldehyde and lactone functionalities. Carbonyl signals at δ 177.91 (carboxylic acid, C6) and δ 219.50 (ketone, C5) are prominent in the 13C NMR spectrum (Figure 2D, S18). The assignments for other 1H and 13C signals mirror those observed for βSec and βLac. This translucent jelly-like solid had a yield of 2%. The HR-MS analysis and infrared spectra (Figures S27 and S29) further validate the structure, with detailed 1D and 2D NMR assignments provided in the supplementary material.

The formation mechanism of βSec aligns closely with the Criegee pathway described for the ozonization of cholesterol. Olefin compounds exposed to ozone generate unstable ozonides, leading to cleavage and the formation of aldehyde, ketone, and peroxide derivatives. The Criegee mechanism involves bond cleavage within βSito (Scheme 1, compound 1) by ozone, producing an unstable primary ozonide (compound 2) that transitions into a carbonyl oxide intermediate (compound 3). This intermediate undergoes rearrangement, leading to the formation of an unstable secondary ozonide (compound 4), which is subsequently reduced in zinc acid media to yield βSec (compound 5) as the primary product. Autoxidation further converts βSec into the carboxylic acid product βCOOH (compound 6). This mechanism parallels processes described for cholesterol ozonization in different solvent systems, as previously explored in extensive studies.

The study and observations surrounding the mechanism of formation and cytotoxic effects of oxyphytosterols such as βSec (5), βCOOH (6), and βLac (9) provide substantial insights into their chemical pathways and biological impacts. The ozonization of β-sitosterol (βSito, 1) involves a fascinating sequence of reactions. Initially, ozone reacts with βSito to form an unstable primary ozonide intermediate (2). This intermediate then undergoes cleavage, yielding the zwitterionic carbonyl oxide known as the Criegee intermediate (3). The Criegee intermediate can proceed through multiple reaction pathways, influenced by the surrounding solvent environment.

In aprotic solvents, the Criegee intermediate engages in intramolecular reactions, leading to the formation of a secondary ozonide (4). This compound is subsequently reduced to produce βSec (5), a secoaldehyde product. Further oxidation of βSec results in the formation of βCOOH (6), the carboxylic acid derivative. Alternatively, in protic solvents, water molecules interact with the Criegee intermediate, generating intermediates (7 and that eventually rearrange to form the lactone derivative βLac (9).

Apart from βSec and βCOOH, βLac, an additional carbonyl compound, was identified. The formation mechanism of βLac involves the tertiary carbonyl oxide (compound 3) undergoing intramolecular attack by the carbonyl group at C5, forming a dipolar intermediate (compound 7). This intermediate, in the presence of water, rearranges to yield βLac (compound 9). These findings underscore the multifaceted nature of ozonization and its potential to yield structurally diverse oxyphytosterols.

Building on the insights from Tomono et al. (2013), which demonstrated the cytotoxicity of various secosterols, the study evaluated the cytotoxic effects of βSec on HepG2 cells using the MTT assay. The experiments revealed that both βSito and βSec reduced the viability of HepG2 cells over time. Treatment with 10 µM of βSito led to a decrease in cell viability to 61.1 ± 3.3% at 6 hours, 47.2 ± 4.9% at 24 hours, and 40.3 ± 7.6% at 48 hours. At a higher concentration of 100 µM, the reductions were 62.1 ± 1.9%, 47.9 ± 3.2%, and 44.5 ± 8%, respectively. On the other hand, βSec exhibited no significant cytotoxicity at 10 µM across all time points. However, at 100 µM, βSec induced a sharp decline in cell viability to 46.3 ± 8.2% at 24 hours and 21.2 ± 3.1% at 48 hours, highlighting its potent cytotoxic effect at elevated concentrations.

These findings align with prior studies that reported reduced cell viability upon exposure to various oxidized sitosterol derivatives. For instance, treatments with compounds like 7βOH-sitosterol, 7-ketositosterol, and polar oxide mixtures demonstrated cytotoxic effects on HepG2 cells. Interestingly, the βSec structure bears similarity to cholesterol-derived oxysterol CSec, which has been linked to pro-proliferative effects at certain concentrations. The study also highlighted that βSito exhibited greater toxicity than βSec. For example, at 10 µM for 6 hours, βSito reduced cell viability to 61%, whereas 100 µM for 48 hours maintained viability at 44%. In contrast, 100 µM of βSec reduced viability to 21% at 48 hours, emphasizing the distinct toxicity profiles of these compounds.

The variability in cytotoxicity among phytosterols and oxyphytosterols can be attributed to differences in cell line characteristics, uptake and transport mechanisms, membrane incorporation, molecular structures, and their effects on cellular metabolism. These disparities necessitate further dose-response studies to ascertain IC50 values and compare compound efficacies and potencies. The IC50 of oxyphytosterols tends to be higher than that of their phytosterol counterparts, suggesting variations in their bioactivity.

While cytotoxicity and oxidative stress induction are documented, the underlying mechanisms remain a topic of ongoing investigation. For example, studies on stigmasterol oxides demonstrated increased caspase-3 activity in U937 cells, suggesting apoptosis as a potential pathway. However, findings on U937 and HepG2 cells indicate that oxidative stress markers such as catalase content, lipoperoxidation, and DNA damage are not consistently elevated, highlighting the complexity of the cytotoxic mechanisms. These initial observations pave the way for detailed investigations into the biological effects and mechanisms of βSec and other oxyphytosterols. Future studies aim to unravel the pathways contributing to their cytotoxicity, with implications for their potential applications in therapeutic settings.

Conclusion

The effects of oxyphytosterols on biological systems remain largely underestimated, and this is primarily due to the complexity involved in synthesizing and isolating these pure compounds. The present study stands as a groundbreaking effort to not only synthesize and characterize three novel oxyphytosterols, which were derived through the ozonization of β-sitosterol (βSito), but also to elucidate their mechanisms of formation and evaluate the biological effects of the parent compound βSito and its major product, βSec, on the viability of HepG2 cells. This comprehensive approach sheds new light on the interaction between phytosterols and their oxidation products within biological environments.

Given the rising trend in phytosterol consumption and the potential biological impacts of their oxidized derivatives, the necessity for continued research becomes evident. Future investigations should focus on detecting these compounds in vivo and further delving into their molecular mechanisms of action within biological systems. By enhancing the understanding of the pathways and effects related to these compounds, the field can advance toward identifying their implications for health and disease.

The following abbreviations have been utilized throughout the study:
- βSito: β-Sitosterol.
- βSec: 2-[(7aR)-5-[(1R,4S)-4-hydroxy-1-methyl-2-oxocyclohexyl]-1,7a-dimethyl-1,2,3,3a,4,5,6,7-octahydroinden-4-yl]acetaldehyde.
- βLac: 2-[(7aR)-5-[(2R,5S)-5-hydroxy-2-methyl-7-oxo-oxepan-2-yl]-1,7a-dimethyl-1,2,3,3a,4,5,6,7-octahydroinden-4-yl]acetaldehyde.
- βCOOH: 2-((7aR)-5-((1R,4S)-4-hydroxy-1-methyl-2-oxocyclohexyl)-1,7a-dimethyloctahydro-1Hinden-4-yl)acetic acid.

This study marks a significant milestone in addressing the challenges associated with understanding oxyphytosterols, paving the way for meaningful advancements in their characterization and exploration of their biological impacts. If there’s any particular aspect of the study you’d like to further elaborate on, just let me know!