Β-sitosterol Oxidation Research Articles

β-Sitosterol Oxides From the Dried Stem of Salix gilgiana Inhibit the Proliferation of HL-60 Leukemic Cells

Background: Salix gilgiana is a deciduous tree that grows in northern Japan, the Korean peninsula, eastern Russia along the Ussuri River, and northeast China. The stem of this tree is dried and consumed orally as a folk medicine. Our intensive screening of various plant materials found that the MeOH extract of its dried stem exhibited significant antiproliferative activity against HL-60 leukemic cells with an IC50 of 16 ppm. We systematically investigated the biologically active compounds of the MeOH extract of the dried stem of S. gilgiana. Methods: The MeOH extract of S. gilgiana dried stem was fractionated by a repeated chromatography monitored by antiproliferative activity against HL-60 leukemic cells. Five active compounds were isolated and the structures were elucidated by MS, 1H- and 13C-NMR spectroscopy, and X-ray analysis. Results: The active compounds were identified as 7-ketositosterol (I), 7β-hydroxysitosterol (II), 7α- hydroxysitosterol (III), (4-hydroxyphenyl)ethanol (IV), and (4-hydroxyphenyl)propan-1-ol (V). The strongest activity was found for 7α-hydroxysitosterol (III), with an IC50 of 8.4 µM. This is the first report of the isolation of these compounds from S. gilgiana. Conclusion: Five compounds were isolated by a repeated chromatography under the guidance of antiproliferative bioassay using HL-60. The structures were identified as three β-sitosterol oxides and two phenolic compounds. Since Salix species, namely, willow trees, have beneficial characteristics including rapid growth, easy cloning, and resistance to high humidity and dryness, they may be utilized as a relatively inexpensive tool for the efficient production of useful bioactive materials.

Phytosterols and phytosterol oxides in Bronte’s Pistachio (Pistacia vera L.) and in processed pistachio products

This study provides data on phytosterols (campesterol, stigmasterol, β-sitosterol, Δ5-avenasterol) content in raw Bronte’s pistachios, four industrially processed pistachio products and roasted pistachios. The study also contributes to greater insight into the effect of processing on the formation of β-sitosterol oxidation products. The total sterol content in raw Bronte’s pistachio (279 mg/100 g oil) was higher than in processed pistachios where it varied with processing. β-sitosterol was the dominant sterol in all the samples studied. The total β-sitosterol oxides content ranged from 4.40 µg/g oil found in the raw sample up to 6.30 µg/g oil detected in the roasted samples at 150°. The most abundant β-sitosterol oxide was the 6-keto-sitostanol. The reported phytosterol oxides profile let us hypothesize that their synthesis is initiated via the epoxide pathway. Therefore, 6-keto-sitostanol could be used as a molecular marker of thermal stress in pistachio products.

Phytosterols as precursors for the synthesis of aromatase inhibitors: Hemisynthesis of testololactone and testolactone

Using β-sitosterol and stigmasterol as precursor materials, a concise and efficient hemisynthesis of aromatase inhibitors: testololactone and testolactone was accomplished in a well-established reaction scheme. It involves highly effective Oppaneur oxidation of both β-sitosterol as well as stigmasterol to generate the required enone moiety in ring ‘A’ of the desired steroid system. The Oppaneur oxidation products of both β-sitosterol and stigmasterol were then subjected to oxidative cleavage of the side chain to produce 4-androstene-3,17-dione. Baeyer–Villiger oxidation of 4-androstene-3,17-dione using m-CPBA yielded testololactone. Dehydrogenation of 4-androstene-3,17-dione using phenylselenyl chloride in ethyl acetate followed by selenoxide elimination with H2O2 in dichloromethane furnished androstenedienone. Baeyer–Villiger oxidation of the resulting androstenedienone yielded the desired testolactone (overall yield 33%). This expeditious reaction scheme may be exploited for the bulk production of aromatase inhibitors (especially testolactone marketed under the brand name Teslac) from the most abundant and naturally occurring phytosterols like β-sitosterol.

Oxidation of β-sitosterol and campesterol in sunflower oil upon deep- and pan-frying of French fries.

Fried foods, both deep-fried and pan-fried, are enjoyed by people worldwide. Frying is one of the main factors leading to formation of phytosterols (PS) oxidation products (POP) in vegetable oils. The aim of this study was to measure the oxidation of β-sitosterol (24α-ethyl-5-cholesten-3β-ol) and campesterol (24α-methyl-5-cholesten-3β-ol) in commercial sunflower oil (SFO) during deep- and pan-frying of French fries for different periods (30, 60, 120 and 240min). The total amount of PS in SFO was 4732μg/g, wherein the major PS were β-sitosterol and campesterol. The results of POP were confirmed by the GC-MS analysis that monitored the formation of oxides during frying. Upon frying, total PS content decreased whereas the highest decrease was measured after 240min of frying. The oxidative stability (OS) of different sitosterol and campesterol during both frying methods was evaluated. In general, pan frying resulted in more PS oxidation than deep frying. β-Sitosterol oxides predominated while campesterol oxides were formed to a lesser extent. 7-Ketositosterol, followed by 7β-hydroxysitosterol, 5,6-epoxy derivatives and 7α-hydroxysitosterol were the main POP induced during frying. The proportion of 7-keto derivatives decreased during frying while the proportion of 7β-hydroxy derivatives increased. The formation of POP might be a limiting factor for frying in SFO for long periods.

Solid phase selective oxidation of β-sitosterol and 4β-hydroxy cholesterol by N-bromosuccinimide – A green approach

Solid phase selective oxidation of β-sitosterol and 4β-hydroxy cholesterol by N-bromosuccinimide – A green approach

Plant sterol oxides in functional beverages: Influence of matrix and storage

Three plant sterol (PS)-enriched beverages, milk based fruit juice (MFJPS), fruit juice (FJPS) and milk beverage (MPS), were stored at 4, 24, or 37°C and analysed at regular time intervals of 2months until 6months. PS stability was analysed from the production of phytosterol oxidation products (POPs). The β-sitosterol oxides (7α/7β-hydroxy, β/α-epoxy, triol, and 7-keto) and campesterol oxides (β/α-epoxy, and 7-keto) were detected in all beverages and at all storage times and temperatures. Total POP contents followed the order MPS≫FJPS>MFJPS. In general, the beverages showed low PS oxidation levels (<0.17%). Predictive models of POP content versus storage time were established. These models explain total POP content by over 75% and individual POP content by over 50%. We propose 7-ketositosterol and 7-ketocampesterol as PS oxidation markers during storage of beverages of this kind.

Characterisation of non-polar dimers formed during thermo-oxidative degradation of β-sitosterol

Thermo-oxidative degradation of sterols at temperature typical for frying leads to the formation of oxidised derivatives, fragmented sterols and oligomers. Recent research on sterol oxidation focuses mainly on the oxysterol derivatives formation to the exclusion of compounds with high molecular mass. The aim of this work was to decipher the chemical structure of non-polar dimers formed during β-sitosterol oxidation at 180°C in the presence of oxygen. The dimer fraction was separated by size-exclusion chromatography (SEC) after pre-fractionation on silica gel. The chemical structure of the dimers was assessed by 1D and 2D NMR, IR, Raman and MS spectroscopies. NMR data confirmed that the predominant non-polar dimer formed during β-sitosterol oxidative degradation has a configuration of 3β,3β′-disitosteryl ether. Data from IR and Raman spectroscopies further proved it chemical structure. Applied analytical techniques also confirmed presence of dimers with different configuration than disteryl ethers.

β-Sitosterol oxidation products attenuate vasorelaxation by increasing reactive oxygen species and cyclooxygenase-2

β-Sitosterol has become a popular cholesterol-lowering functional food product worldwide. β-Sitosterol can be oxidized to β-sitosterol oxidation products (SOPs) during food processing. Little is known about the impact of SOPs and β-sitosterol on the functionality of arteries. This study investigated the effects of SOPs and β-sitosterol on vasorelaxation and the possible cellular mechanisms involved. By isometric tension measurement, SOPs but not β-sitosterol blunted relaxation induced by acetylcholine or Ca(2+) ionophore A23187 in endothelium-intact aortae. SOPs-impaired vasorelaxation was completely reversed by cyclooxygenase (COX)-2 inhibitor DuP-697, whereas the reversal by COX-1 inhibitor SC-560 was only partial. Western blotting and immunohistochemistry showed that SOPs increased the protein expression of COX-2 but not COX-1 in the endothelium. Using dihydroethidium staining and electron paramagnetic resonance spin trapping techniques, SOPs were found to elevate the level of reactive oxygen species in rat aortic endothelial cells, and the effects were reversed by antioxidants tempol, tiron, or diphenylene iodonium. Consistently, these antioxidants reversed SOPs-induced impairment of endothelium-dependent relaxation. Up-regulation of COX-2 expression by SOPs was also reversed by tempol. Moreover, SOPs attenuated nitric oxide donor sodium nitroprusside-induced relaxation in endothelium-intact, but not endothelium-denuded rings, confirming that SOPs act on the endothelium. Interestingly, a thromboxane-prostanoid (TP) receptor blocker S18886 reversed SOPs-impaired vasorelaxation, suggesting the involvement of TP receptor in mediating the downstream effect. SOPs decreased cGMP production, and the effect could be reversed by inhibiting COX-2 or TP receptor. This study provides novel experimental evidence showing the harmful effects of SOPs on endothelial function.

Identification and Quantitative Analysis of β-sitosterol Oxides in Oil of 2 Macrotermitinae Varieties from Congo

Identification and Quantitative Analysis of β-sitosterol Oxides in Oil of 2 Macrotermitinae Varieties from Congo

Sterol stability in functional fruit beverages enriched with different plant sterol sources

Two different plant sterol (PS) sources (free PS from tall oil and esterified PS from vegetable oils) were used for manufacturing two types of functional beverages (fruit and milk-based fruit beverages), and their PS and phytosterol oxidation product (POP) contents were determined. Gas chromatography–tandem mass spectrometry (GC–MS/MS) was used for identification and gas chromatography–flame ionization detection (GC–FID) for quantitation purposes. Brassicasterol, campesterol, campestanol, stigmasterol, β-sitosterol and sitostanol were the quantified PS, conforming a profile in order with current legislation. The relative percentages of PS differed according to the enrichment source involved, though the enrichment levels (g/100g beverage) were of the same order (1.77 from tall oil and 1.84 from vegetable oils). Only POPs from β-sitosterol (the prevalent PS in the analyzed beverages) were detected — the predominant representative being 7β-hydroxysitosterol (39–58.5% of total POP content). The following POPs were quantified: 7α-hydroxy, β-epoxy, α-epoxy, and 7-ketositosterol, yielding a total POP content ranging between 42.9 and 57.4mg/100g of PS. No statistically significant differences (p>0.05) in total and individual POP content according to the source of PS were found. The mean β-sitosterol oxidation percentage was <0.07%, which reflected a low PS oxidation extent, though manufacture was on a laboratory scale regardless of the PS source used in enrichment of the functional beverages. These functional drinks therefore can be regarded as healthy food products and as an adequate PS vehicle as well.

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.

Stability of Plant Sterols in Ingredients Used in Functional Foods

The content of plant sterol (PS) and their oxidation products (POPs) in eight ingredients used to enrich functional foods was studied. A gas chromatographic (GC) technique with mass-spectrometric detection was used for identification, while GC with a flame ionization detector (GC-FID) was used for quantification. β-Sitosterol was the most abundant phytosterol, and the main POPs found were derived from this compound (7α/β-hydroxysitosterol, 7-ketositosterol, and sitostanetriol). The total amount of POPs found in the ingredients ranged from 29.03 to 110.02 μg/100 g PS. The β-sitosterol oxidation rates ranged from 10 to 50 μg β-sitosterol oxides/100 g of β-sitosterol. In view of this low rate of oxidation in the ingredients tested, it can be concluded that the PS remain stable in these ingredients. Significant correlations (p < 0.01) were found between total oxysitosterols versus β-sitosterol contents (R(2) = 86.5%) and between total POPs and total PS (R(2) = 81.6%).

Interaction of fatty acids with oxidation of cholesterol and β-sitosterol

The present study studied the effect of stearic acid, oleic acid, linoleic acid and α-linolenic acid on oxidation of cholesterol and β-sitosterol. Both cholesterol and β-sitosterol were instable with 85% of cholesterol and β-sitosterol being degraded at 180 °C for 120 min. Cholesterol and β-sitosterol had a similar degradation pattern and produced 7-keto, 7α-hydroxy, 7β-hydroxy, 5,6α-epoxy and 5,6β-epoxy oxidative derivatives, indicating position 7 was the most susceptible site of oxidation. Four fatty acids accelerated the degradation of cholesterol and β-sitosterol for the first 60 min at 180 °C and thereafter their oxidation-promoting effect became weaker or diminished. When the oxidation progressed to 120 min, cholesterol and β-sitosterol in the presence of α-linolenic acid, was less oxidised when compared with the control, suggesting that it might inhibit the oxidation. It was concluded that the surrounding fatty acids affected sterol oxidation in a time-dependent manner and their effect was unlikely related to their degree of unsaturation.

Oxidation of Cholesterol and β-Sitosterol and Prevention by Natural Antioxidants

Consumption of cholesterol oxidation products (COPs) is a growing health concern, but little is known about the intake of beta-sitosterol oxidation products (SOPs). The present study was performed (i) to compare the oxidative stability of cholesterol with that of beta-sitosterol; (ii) to investigate the oxidative pattern of cholesterol and beta-sitosterol in lard, corn oil, and olive oil; and (iii) to examine the effectiveness of green tea catechins (GTC), alpha-tocopherol, and quercetin in prevention of cholesterol and beta-sitosterol oxidation compared with butylated hydroxytoluene (BHT). Results showed both cholesterol and beta-sitosterol were thermally unstable with 75% of cholesterol and beta-sitosterol being oxidized at 180 degrees C for 2 h. The oxidation behavior of beta-sitosterol was similar to that of cholesterol in terms of oxidative rate and oxidation products. The major COPs produced were 7-ketocholesterol, 7alpha-hydroxycholesterol, 7beta-hydroxycholesterol 5,6alpha-epoxycholesterol, and 5,6beta-epoxycholesterol, whereas the major SOPs were 7-ketositosterol, 7alpha-hydroxysitosterol, 7beta-hydroxysitosterol, 5,6alpha-epoxysitosterol, and 5,6beta-epoxysitosterol. Under the same experimental conditions, both cholesterol and beta-sitosterol were oxidized more slowly in corn oil, lard, and olive oil, attributable to the unsaponified antioxidants present in these fat and oils. GTC, alpha-tocopherol, and quercetin were more effective than BHT in preventing the oxidation of cholesterol and beta-sitosterol.

The biocatalytic obtainment of physiologically active compounds based on vegetative β-sitosterol

This paper determines the optimal conditions for the directed biocatalytic oxidation of β-sitosterol by actinobacilli of the genus Rhodococcus to compounds claimed in the pharmaceutical practice: stigmast-4-en-3-one, androst-4-ene-3.17-dione, and androsta-1.4-diene-3.17-dione. Rhodococci were ascertained to convert approximately 98.0% of β-sitosterol into stigmast-4-en-3-one in the presence of n-hexadecane. The cells of rhodococci preliminarily adapted to β-sitosterol catalyze the reaction of the removal of the aliphatic lateral β-sitosterol chain with the formation of 31.5% of androstan derivatives in the presence of glucose and 2.2′-dipyridyl that is the inhibitor of 9α-hydroxylase. It was discovered for the first time that cells of R. ruber catalyzed the reaction of sterol acylation with the formation of β-sitosterol acetate along with oxidative processes.

Separation and isolation of β-sitosterol oxides and their non-mutagenic potential in the Salmonella microsome assay

The recent increase in phytosterol-enriched functional food provokes questions concerning the safety of their oxidation products. However, most of the existing toxicity studies have been performed with mixtures instead of single compounds, a consequence of the lack of pure phytosterol oxidation product (POP)-standards. The objectives of this study were to take in use a method for the isolation of β-sitosterol oxidation products and to assess their mutagenic and pro-oxidative potential. Oxides were prepared by thermo-oxidation, purified by column chromatography and separated by a NP–HPLC–UV system. 7-ketositosterol, 7β-OH-sitosterol, 7α-OH-sitosterol, a mixture of 6β-OH-3-ketositosterol/6α-OH-3-ketositosterol (ratio 4:3) and a mixture of polar oxides were fractionated. Yields in the range of several milligrams per fraction were achieved within one HPLC-run. Identification and quantification was done by GC–MS and GC–FID, respectively. In the Ames test the collected fractions failed to show a mutagenic activity towards Salmonella typhimurium strains TA98, TA100 and TA102.

β-Sitosterol oxidation products failed to show mutagenic potential in the Ames test

β-Sitosterol oxidation products failed to show mutagenic potential in the Ames test

Quantitative Analysis of β-Sitosterol Oxides Induced in Vegetable Oils by Natural Sunlight, Artificially Generated Light, and Irradiation

UV radiation is able to induce lipid peroxidation. Photooxidation-induced beta-sitosterol oxides were monitored in four vegetable oils exposed to sunlight for 10, 20, and 30 days during May 2005 (northeastern France), exposed to artificial light generated by a high-pressure Hg lamp for 21, 42, and 63 h at room temperature, and exposed to a 10 MeV electron beam at 0.93, 2.69, and 9.30 kGy at 8 degrees C. Quantification was performed by capillary gas chromatography-mass spectrometry according to the total ion current mode and using a reconstructed ion trace chromatogram with specific ion fragments. Sunlight induced the formation of higher amounts of oxides than UV light, while no significant oxidizing effect was observed with electron beam irradiation. However, data suggested that the amount of the main oxides formed was strongly dependent on the dose rate (length of exposure). Accordingly, shorter but more intense treatments had lower oxidizing effects.

Qualitative and quantitative comparison of the cytotoxic and apoptotic potential of phytosterol oxidation products with their corresponding cholesterol oxidation products

Phytosterols contain an unsaturated ring structure and therefore are susceptible to oxidation under certain conditions. Whilst the cytotoxicity of the analogous cholesterol oxidation products (COP) has been well documented, the biological effects of phytosterol oxidation products (POP) have not yet been fully ascertained. The objective of the present study was to examine the cytotoxicity of beta-sitosterol oxides and their corresponding COP in a human monocytic cell line (U937), a colonic adenocarcinoma cell line (CaCo-2) and a hepatoma liver cell line (HepG2). 7beta-Hydroxysitosterol, 7-ketositosterol, sitosterol-3beta,5alpha,6beta-triol and a sitosterol-5alpha,6alpha-epoxide-sitosterol-5beta,6beta-epoxide (6:1) mixture were found to be cytotoxic to all three cell lines employed; the mode of cell death was by apoptosis in the U937 cell line and necrosis in the CaCo-2 and HepG2 cells. 7beta-Hydroxysitosterol was the only beta-sitosterol oxide to cause depletion in glutathione, indicating that POP-induced apoptosis may not be dependent on the generation of an oxidative stress. A further objective of this study was to assess the ability of the antioxidants alpha-tocopherol, gamma-tocopherol and beta-carotene to modulate POP-induced cytotoxicity in U937 cells. Whilst alpha/gamma-tocopherol protected against 7beta-hydroxycholesterol-induced apoptosis, they did not confer protection against 7beta-hydroxysitosterol- or 7-ketositosterol-induced toxicity, indicating that perhaps COP provoke different apoptotic pathways than POP. beta-Carotene did not protect against COP- or POP-induced toxicity. In general, results indicate that POP have qualitatively similar toxic effects to COP. However, higher concentrations of POP are required to elicit comparable levels of toxicity.

Identification and quantitative analysis of β-sitosterol oxides in vegetable oils by capillary gas chromatography–mass spectrometry

As vegetable oils and phytosterol-enriched spreads are marketed for frying food or cooking purposes, temperature is one of the most important factors leading to the formation of phytosterol oxides in food matrix. A methodology based on saponification, organic solvent extraction, solid-phase extraction (SPE), followed by mass spectrometric identification and quantitation of β-sitosterol oxides using capillary gas chromatography–mass spectrometry (GC–MS) in selected ion monitoring (SIM) mode was developed and characterized. Relative response factors of six β-sitosterol oxides, including 7α-hydroxy, 7β-hydroxy, 5,6α-epoxy, 5,6β-epoxy, 7-keto, and 5α,6β-dihydroxysitosterol, were calculated against authentic standards of 19-hydroxycholesterol or cholestanol. Linear calibration data, limit of detection, and sample recoveries during analytical process. Recoveries of these oxidation compounds in spiked samples ranged from 88 to 115%, while relative standard derivation (R.S.D.) values were below 10% in most cases. The analytical method was applied to quantify β-sitosterol oxides formed in thermal-oxidized vegetable oils which were heated at different temperatures and for varying time periods. Sitosterol oxidation is strikingly higher in sunflower oil relative to olive oil under all conditions of temperature and heating time.