Stigmasterol Oxidation Research Articles

Thermo-oxidative stability of asymmetric distigmasterol-modified acylglycerols as novel derivatives of plant sterols

The study investigated the thermo-oxidative stability of distigmasterol-modified acylglycerols as a new structured acylglycerols. Samples were heated at 60 and 180 °C for 8 h. Their percentage degradation and products formed during heating were compared with free stigmasterol and stigmasteryl esters. The remaining of stigmasterol and fatty acid parts, the formation of stigmasterol oxidation products and the composition of polar and non-polar fractions were analysed using chromatographic methods. The cytotoxicity and genotoxicity were determined with the use of an MTT test and a comet assay, respectively. The highest stability during heating was observed for 2,3-distigmasterylsuccinoyl-1-oleoyl-sn-glycerol (dStigS-OA) and the lowest for 2,3-distigmasterylcarbonoyl-1-oleoyl-sn-glycerol (dStigC-OA). Data showed that the formation of thermo-oxidative degradation products is affected by the temperature and chemical structure of lipids present in the molecule. The dStigMAs bonded by a succinate linker and products formed during their thermo-oxidation showed no cytotoxic or genotoxic activity to normal human cells.

Cytotoxic activity of stigmasteryl esters and products of their thermo-oxidative degradation against drug sensitive and drug resistant human acute lymphoblastic leukemia cells.

Phytosterols are mainly known as a cholesterol-lowering factor, although they form oxidation products during food storage and processing. Moreover, phytosterol oxidation products (POP) can be ab- sorbed and found in human serum, so there is the need to investigate their impact on different kinds of cells. Esters of fatty acids (oleic, linoleic and linolenic) with stigmasterol were synthetized and heated at 180°C, for 1–12 hours. The cytotoxic effect on the leukemic cells of unheated stigmasteryl esters and the mixture of compounds after heating was determined using MTT assays. POP were identified using GC-MS. The total number of POP was analysed by SPE fractionation and GC-FID separation. Dimers, trimers and oligomers in non-polar fraction were determined by gel permeation chromatography with refractive index detection. After heating, stigmasterol oxidation products were formed (up to 1.1 mg/g ester). The heating increased the potency of the compounds to reduce cell population and form POPs and oligomers in a time-dependent manner. The cytotoxicity depends on the kind of ester, dose and time. The strongest cytotoxic effect was found after 72 hours of cell treatment. Among the three stigmasteryl esters tested the most cytotoxic effect was caused by stigmasteryl linoleate.

Synthesis and assessment of the relative toxicity of the oxidised derivatives of campesterol and dihydrobrassicasterol in U937 and HepG2 cells

The cytotoxic effects of the oxidised derivatives of the phytosterols, stigmasterol and β-sitosterol, have previously been shown to be similar but less potent than those of the equivalent cholesterol oxides in the U937 cell line. The objective of the present study was to compare the cytotoxic effects of the oxidised derivatives of synthetic mixtures of campesterol and dihydrobrassicasterol in both the U937 and HepG2 cell lines. The parent compounds consisted of a campesterol: dihydrobrassicasterol mix at a ratio of 2:1 (2CMP:1DHB) and a dihydrobrassicasterol:campesterol mix at a ratio of 3:1 (3DHB:1CMP). The 2CMP:1DBH oxides were more cytotoxic in the U937 cells than the 3DBH:1CMP oxides but the difference in cytotoxicity was less marked in the HepG2 cells. The order of toxicity of the individual oxidation products was found to be similar to that previously observed for cholesterol, β-sitosterol and stigmasterol oxidation products in the U937 cell line. There was an increase in apoptotic nuclei in U937 cells incubated with the 7-keto and 7β-OH derivatives of both 2CMP:1DHB and 3DHB:1CMP and also in the presence of 3DHB:1CMP-3β,5α,6β-triol and 2CMP:1DHB-5β,6β-epoxide. An additional oxidation product synthesised from 2CMP:1DHB, 5,6,22,23-diepoxycampestane, was cytotoxic but did not induce apoptosis. These results signify the importance of campesterol oxides in the overall paradigm of phytosterol oxide cytotoxicity.

Effect of phytosterols and their oxidation products on lipoprotein profiles and vascular function in hamster fed a high cholesterol diet

Human diets contain phytosterols and their oxidation products. We investigated effect of β-sitosterol (Si), stigmasterol (St), β-sitosterol oxidation products (SiOP) and stigmasterol oxidation products (StOP) on plasma total cholesterol and their interaction with the gene expression of enzymes, proteins and transporters involved in cholesterol absorption and metabolism. Sixty male hamsters were fed the control diet or one of four experimental diets containing 0.1% Si, 0.1% SiOP, 0.1% St and 0.1% StOP, respectively, for six weeks. SiOP and StOP groups had the relative liver weights greater than their corresponding non-oxidized forms, indicating they were possibly toxic. Results showed both Si and St groups reduced while SiOP and StOP hamsters lost the capacity of lowering plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL) and triacylglycerols (TG) compared with the control group. Si and St but not SiOP and StOP were anti-atherosclerotic. RT-PCR analysis demonstrated Si and St but not SiOP and StOP down-regulated mRNA levels of intestinal acyl CoA: cholesterol acyltransferase (ACAT2) and microsomal triglyceride protein (MTP). Aortas from Si and St hamsters relaxed better than those from the control and their corresponding SiOP and StOP-treated hamsters. It was concluded that Si and St not SiOP and StOP were beneficial in improving lipoprotein profile and aortic function.

Dimer formation during the thermo-oxidation of stigmasterol

The monomeric oxidation products formed during the thermo-oxidation of phytosterols do not explain the loss of unoxidized phytosterols, leaving a gap in the knowledge on the products that are being formed during thermo-oxidation. Previous research showed that the gap can be explained by the formation of dimers and polymers. The aim of this research was to investigate the dimers that are formed during the thermo-oxidation of phytosterols, using stigmasterol as a model compound. Following the oxidation of stigmasterol at 180 °C, several dimeric fractions were obtained by solid phase extraction and high-performance size-exclusion chromatography. Individual dimers were separated by reversed-phase high-performance liquid chromatography, and their molecular masses were determined by combining atmospheric pressure chemical ionization-mass spectrometry and coordination-ion spray-mass spectrometry. This study resulted, for the first time, in the assignment of several individual stigmasterol dimers. The most dominant dimer was found to have a molecular mass of 822.7 Da and is suggested to represent a C–C linked dimer of stigmasterol, in accordance with literature. Several other stigmasterol dimers were found to be formed during thermo-oxidation, which are suggested to differ in monomeric subunit composition, linkage type, and stereochemistry.

Synthesis and Characterization of Stigmasterol Oxidation Products

The synthesis and structural characterization of a series of oxides of stigmasterol is described providing a valuable series of reference standards for these oxides, analogous to the cholesterol oxidation products (COPs) which have been shown to have detrimental biological effects. Biological evaluation of the oxides of phytosterols is significant in the context of increased dietary use of phytosterols in the drive to reduce cholesterol absorption.

Sterol Oxidation in Ready-to-Eat Infant Foods During Storage

The effect of storage on sterol oxidation of ready-to-eat infant foods was evaluated. Two different liquid infant foods (honey or fruits flavors), prepared with milk and cereals, were stored for 0, 2, 4, 7 and 9 months at 25 degrees C. Sterol oxidation products (SOP) were isolated by cold saponification, purified by silica solid-phase extraction, and analyzed by gas chromatography (GC) and GC-mass spectrometry. beta-Sitosterol was the most representative sterol, followed by cholesterol and campesterol. No significant differences in the total and single SOP content (0.8-1 mg/kg of product) were observed with respect to storage time and type of sample; the main SOP found was 7-ketositosterol (<0.2 mg/kg of product). The extent of stigmasterol oxidation (2.9%) was higher than that of cholesterol (1.9%) and beta-sitosterol (1.4%). The type and quality of raw materials, as well as the processing conditions, seem to greatly influence SOP formation and accumulation in infant foods.

Determination of phytosterol oxides in some food products by using an optimized transesterification method

An increased intake of phytosterols (PS), as well as new products launched on the market enriched with PS and phytostanols, increases the demands for improved analytical techniques. In the present study, a method for the determination of phytosterol oxidation products (POP) was evaluated and optimized. The method included transesterification, enrichment with amino SPE cartridges and quantification by GC. The methodology was evaluated with authentic samples of sito-, campe- and stigma-sterol oxidation products and the linearity and recovery for some POP were >0.95 and 80–120%, respectively. The lowest spiking level determined was 0.5 μg and lower levels could be quantified. In addition, the formation of POP during thermal oxidation of vegetable oils at 180 °C for 0–2 h was studied and increased levels were observed. A commercially available PS ester-enriched margarine was also included in the investigation and the level of POP was 12 μg/g of spread.

Effects of sterol structure, temperature, and lipid medium on phytosterol oxidation.

Factors contributing to the oxidative stability of phytosterols were studied. Unsaturated stigmasterol and saturated sitostanol were used as model compounds and were heated at different temperatures in different lipid matrices for various periods of time. Accumulations of the major secondary oxidation products were used as a marker of the stability of heated compounds, and the products were analyzed by gas chromatography-mass spectrometry. The results showed that both temperature and heating time, as well as sterol structure and lipid matrix composition, affected phytosterol oxidation. In particular, the interactions between different lipid matrices and temperatures had drastic effects on the total contents of the phytosterol oxides formed and also on the reaction pathways of oxidation. During heating at high temperatures for prolonged periods, >20% of stigmasterol was oxidized. At moderate temperatures the oxidation of stigmasterol was rather slow. Sitostanol oxide contents were low under all heating conditions studied.

Inhibition of Stigmasterol Oxidation by Antioxidants in Purified Sunflower Oil

A study was conducted to analyze the effect of the antioxidants butylated hydroxytoluene, alpha-tocopherol, ethanolic extracts of rosemary, and green tea on stigmasterol resistance against degradation and formation of its oxidation products in purified triacylglycerols (TAG) from sunflower oil. The content of stigmasterol and its oxidation products 7alpha- and 7beta-hydroxy, alpha- and beta-epoxy, triol, and 7-ketostigmasterol were determined during incubation at 60 degrees C for 3, 6, and 9 days. In addition, peroxide value and fatty acid composition were also determined in the samples. Correlation between the levels of the accumulated stigmasterol oxides and peroxide value of the TAG with antioxidants during incubation was significant only for rosemary extract (R = 0.6799, p < 0.05). The lack of correlation precludes the use of peroxide values to determine the level of sterol oxidation products in the used model system. Correlation between stigmasterol content and the level of stigmasterol oxides was significant for all samples (R = 0.8874, p < 0.05). The total increase of the stigmasterol oxidation products was the lowest in samples with alpha-tocopherol, but the content of stigmasterol-triol increased the most in this sample. In all the analyzed samples, alpha-epoxy-stigmasterol was formed in the highest amounts among the analyzed stigmasterol oxidation products.

Oxidation of stigmasterol in heated triacylglycerols

A triacylglycerol mixture containing 5% stigmasterol by weight and devoid of other unsaponifiables was heated at 180 ± 5°C for several hours. Oxidation products from stigmasterol were separated and isolated by column chromatography and preparative thin-layer chromatography. Characterization of individual compounds was based on chromatographic mobilities, specific colour reactions, UV and IR spectroscopy and fragmentation patterns from the mass spectra of the purified oxidation products. A hydrocarbon (stigmasta-3,5,22-triene), a triunsaturated ketosteroid (stigmasta-3,5,22-trien-7-one), a diunsaturated ketosteroid (stigmasta-4,22-dien-3-one) and a pregnane derivative ( Δ 5-pregnen-3 β-ol-2-one) were identified. There is also evidence for the presence of more polar compounds such as hydroxy and epoxy derivatives.

Micro method for the quantitative determination of Δ22-sterols

A rapid and efficient method for the quantitative determination of sterols containing a Δ 22-unsaturation has been developed. The technique consists of a controlled periodate-permanganate oxidation of the C 27, C 28, or C 29 sterol, extraction of the acidic fragments, and analysis by gas-liquid chromatography of the C 5, C 6, or C 7 carboxylic acid cleavage product using a 10% diethylene glycol succinate + 2% phosphoric acid liquid phase. Oxidation of 22-dehydrocholesteryl acetate and 7,22-bisdehydrocholesteryl acetate produced isovaleric acid, and oxidation of ergosterol and stigmasterol gave α-methylisovaleric acid and α-ethylisovaleric acid, respectively. Except for the stigmasterol oxidation, the yields were high. As expected, no short-chain acidic products could be detected from cholesterol, 7-dehydrocholesterol, or lathosterol.

Sterols. XXXII. Oxidation of Stigmasterol by Selenium Oxide

Sterols. XXXII. Oxidation of Stigmasterol by Selenium Oxide