Phytosterol Mixture Research Articles

Microbial transformation of phytosterols mixture from rice bran oil unsaponifiable matter by selected bacteria

Rice bran sample (12 Kg) was extracted and rice bran oil (RBO ≅ 76.8 g) was saponified. The resulted unsaponifiable matter of RBO (RBO unsap) was qualitatively and quantitatively estimated using different chromatographic analyses. RBO, produced 9.65% unsaponifiable matter with the following contents, cholesterol, 6.75%; stigmasterol, 3.4%; β. sitosterol, 10.23% and campesterol, 4.2%, in addition to unknown phytosterols, hydrocarbons and waxes. Microbial transformation process started by screening of 35 bacterial strains, locally isolated from rice bran, air and soil, using RBO unsap as a carbon and an energy source to produce some pharmaceutically useful C18 and C19 steroids. Moraxella ovis was the most potent isolate for its highest capability to utilize RBO unsap and selectively degrade the phytosterols side-chain producing androst-4-ene-3,17-dione (AD), androsta-1,4-diene-3,17-dione (ADD), testosterone (T) and estrone (E). The RBO unsap was the best carbon and energy source. Maximum production of the desired products was observed in 36 h, pH 7 and at 30°C by M. ovis.

Long-term phytosterol treatment alters gene expression in the liver of apo E-deficient mice

Dietary phytosterols significantly reduce plasma cholesterol concentrations and atherosclerosis in apolipoprotein E-knockout (apo E-KO) mice. We investigated the long-term effects of phytosterol treatment on gene expression in the liver of these mice. Male apo E-KO mice were fed an atherogenic diet supplemented with ( n=6) or without ( n=6) 2% (wt/wt) phytosterol mixtures for 14 weeks. Liver specimens were collected and stored in RNAlater immediately. mRNA was extracted and subjected to microarray analyses and real-time quantitative reverse transcription–polymerase chain reaction (RT-PCR) assay for confirmation. Oligonuleotide microarray analysis of pooled samples ( n=3) revealed that the expression of 132 genes/transcripts was significantly altered in treated animals, considering the false discovery rate (FDR) of 0.23. Real-time RT-PCR techniques confirmed these alterations in the expression of several of these genes, including Hmgcr (2.16-fold; P=.0002), Hmgcs1 (1.79-fold; P=.001), Hsd17b7 (2.11-fold; P=.028), Sqle (2.03-fold; P=.01), Cyp51 (1.8-fold; P=.001), Fads1 (1.55-fold; P=.031), Fads2 (2.17-fold; P=.047), Lpin1 (3.67-fold; P=.001), Ppargc1b (PGC-1β; a coactivator of sterol-regulatory element-binding proteins; 1.66-fold; P=.007) and Cyp7B1 (1.81-fold; P=.025). In summary, our data suggest that long-term dietary phytosterols can alter the expression of a number of hepatic genes that regulate sterol metabolism in apo E-KO mice. It is possible that these changes are due to inhibition of cholesterol absorption, but are not a direct effect of plant sterols. Further multivariate correlation or association analysis is needed to establish the relations between changes in the expression of these genes and prevention of atherosclerosis by phytosterols.

Endocrine modulating actions of a phytosterol mixture and its oxidation products in zebrafish ( Danio rerio)

In this study, a phytosterol preparation (“ultrasitosterol”; 80% β-sitosterol) and an oxidized ultrasitosterol preparation were evaluated for reproductive effects in zebrafish. Adults were exposed in a continuous flow to 10 μg/L and 100 μg/L ultrasitosterol and oxidized ultrasitosterol, and to 0.27 μg/L 17β-estradiol and 0.28 μg/L testosterone for 3 weeks. Biomarkers analysed included plasma vitellogenin, testosterone, 11-ketotestosterone, 17β-estradiol, and gonadal histopathology. Ovarian steroid production of testosterone and 17β-estradiol was examined in isolated zebrafish follicles exposed in vitro to the compounds at the same concentrations as in vivo. Vtg was induced in males exposed to ultrasitosterol, and in males and females exposed to 17β-estradiol. Males exposed to oxidized phytosterols showed increased levels of testosterone and 11-ketotestosterone, and accelerated spermatogenesis. Increased follicular atresia was observed in females exposed to oxidized phytosterols and 17β-estradiol. Correlation analyses between biomarkers revealed more intercorrelated values for females than for males, and the strongest associations were found in females exposed to oxidized phytosterols. Testosterone production was significantly increased in follicles exposed to the oxidized phytosterol preparations. These findings indicate that the phytosterol mixture is weakly estrogenic in male fish, and that the oxidized phytosterol mixture contains substances that may interfere with spermatogenesis, oogenesis and gonadal steroidogenesis in zebrafish.

β-Sitosterol and 17β-estradiol alter gonadal steroidogenic acute regulatory protein (StAR) expression in goldfish, Carassius auratus

Fish exposed to the phytosterol β-sitosterol (β-sit) have decreased circulating hormone and cholesterol concentrations, and decreased gonadal intra-mitochondrial cholesterol pools. The current study examined the potential for β-sit to alter abundance of the key cholesterol transport protein, steroidogenic acute regulatory (StAR) protein, which delivers cholesterol to the first and rate-limiting steroidogenic enzyme P450 side chain cleavage (P450 scc) inside the mitochondria. Plasma testosterone (T) and lipids (cholesterol, lipoproteins and triglycerides) were also measured. Goldfish were exposed to 200 μg/g β-sit (97% pure or as a 72.6% pure phytosterol mixture) and 10 μg/g 17β-estradiol (E 2; estrogenic control) by intra-peritoneal Silastic ® implants for 21-days or for five-months. Plasma T was significantly decreased in male fish exposed to the phytosterol mixture following the long-term exposure ( p < 0.001). There were no differences in total cholesterol concentrations among treatments in the short- or long-term exposure, but male fish in the long-term exposure had significantly lower HDL as compared to control fish ( p < 0.025) with a corresponding increase in LDL. StAR transcript levels were unchanged following the short-term exposure, but were reduced after five months in male β-sit fish ( p = 0.05) and E 2-treated female fish ( p = 0.05). This reduction in StAR transcript abundance in conjunction with decreased plasma T and altered plasma lipoprotein fractions demonstrates a non-estrogenic effect of β-sit. This is the first study to show that β-sit has the capacity to alter gonadal StAR transcript abundance, offering a mechanism by which β-sit disrupts reproductive endocrine endpoints.

Long term phytosterol treatment alters global gene expression in the liver of apo E deficient mice

Background: We have consistently observed that dietary phytosterols significantly reduce plasma cholesterol concentrations and atherosclerosis in apo E-KO mice. We investigated long-term effects of phytosterol treatment on gene expression in the liver of these mice. Methods: Male apo E-KO mice were fed an atherogenic diet supplemented with (n=8) or without (n=7) 2% (w/w) phytosterol mixtures for 14 weeks. Liver specimens were collected and frozen immediately. mRNA was extracted from these samples, and subjected to microarray analyses. Results: The expression of 137 genes was significantly changed in the phytosterol-treated group, as compared to controls. Various database searches indicated that these genes are involved in a number of metabolic pathways including lipid metabolism, inflammation, glucose metabolism, protein metabolism, heme biosynthesis and others. These changes were associated with significant reductions in plasma cholesterol levels and the severity of atherosclerosis. Conclusions: Anti-atherogenic and cholesterol-lowering effects of plant sterols in apo E-KO mice may be mediated through beneficial alterations in the expression of genes in various pathways involved in pathogenesis of atherosclerosis. Further studies warrant mechanisms by which dietary phytosterols regulate gene expression. Acknowledgements: Supported by NSERC, CIHR, and Heart and Stroke Foundation.

Separation of Δ5- and Δ7-Phytosterols by Adsorption Chromatography and Semipreparative Reversed Phase High-Performance Liquid Chromatography for Quantitative Analysis of Phytosterols in Foods

A method for the separation, isolation, and identification of phytosterols was developed. A commercial phytosterols mixture, Generol 95S, was fractionated first by adsorption silica gel column chromatography and then separated by means of a semipreparative reverse phase high-performance liquid chromatography fitted with a Polaris C8-A column (250 mm x 10 mm i.d., 5 microm) using isocratic acetonitrile:2-propanol:water (2:1:1, v/v/v) as the mobile phase. Milligram scales of six individual phytosterols, including citrostadienol, campesterol, beta-sitosterol, Delta7-avenasterol, Delta7-campesterol, and Delta7-sitosterol, were obtained. Purities of these isolated sterols were 85-98%. Relative response factors (RRF) of these phytosterols were calculated against cholestanol as an authentic commercial standard. These RRF values were used to quantify by gas chromatography-mass spectrometry (GC-MS) the phytosterols content in a reference material, oils, and chocolates.

Gram-scale chromatographic purification of β-sitosterol: Synthesis and characterization of β-sitosterol oxides

An effective purification method for β-sitosterol was developed starting from a commercial source of a phytosterol mixture using preparative adsorption column chromatography. β-Sitosterol (≥95% purity) was obtained on a gram-scale. Thus, the synthesis of six β-sitosterol oxides, including 7α-hydroxy, 7β-hydroxy, 5,6α-epoxy, 5,6β-epoxy, 7-keto, and 5α,6β-dihydroxysitosterol, were successfully carried out. The spectral characteristics of all the synthetic intermediates and target compounds (∼95% purity) were well-documented.

Bioconversion of phytosterols to androstanes by mycobacteria growing on sugar cane mud

Direct sterol conversion of sugar cane mud (residue) by Mycobacterium sp. was demonstrated to be possible technologically, thus avoiding sugar cane oil extraction and further processes of extraction and purification of phytosterols from this oil. Indeed, mycobacterial cells were able to convert phytosterols from sugar cane mud into 4-androstene-dione (AD) and 1,4 androsta-diene-3,17-dione (ADD). For the various concentrations assayed, concomitant higher yields for both androstanes were achieved at 20% (w/w) sugar cane mud in media. Furthermore, conversions were similar to those from other substrates, such as a mixture of phytosterols. The results suggest that the mycobacterial cell is able to easily access and bioconvert sugar cane mud phytosterols.

Toxicity of a Phytosterol Mixture to Grayling (Thymallus thymallus) during Early Developmental Stages

The study concerns the toxicity of a phytosterol mixture, ultrasitosterol, consisting mainly of beta-sitosterol 75.7% and beta-sitostanol 13.0%, to grayling (Thymallus thymallus) embryos. Eyed eggs were exposed to three concentrations (1 microg/l, 10 microg/l, and 50 microg/l) of ultrasitosterol for 4 weeks. Embryos and later on hatched fry were taken for triiodothyronine (T3), thyroxine (T4), and histopathological analyses after 7, 14, 21, and 28 days of exposure. Most of the eggs (>95%) hatched during the first week of exposure, and ultrasitosterol treatment shortened hatching time significantly (p < or = 0.0001) at all exposure concentrations in comparison to the control. Ultrasitosterol did not have any significant effect on T3 or T4 levels in the embryo extracts. However, an interesting observation was that T3 levels increased in all treatments and in the control near the time of hatching. In conclusion, ultrasitosterol showed potential to affect the development of grayling embryos and fry, but further long-term exposure experiments are needed to verify these changes in more detail.

Separation and purification of stigmasterol and β-sitosterol from phytosterol mixtures by solvent crystallization method

The separation and purification of stigmasterol and β-sitosterol from phytosterol mixtures by solvent crystallization method was studied based on the difference of the solubility of stigmasterol and β-sitosterol or the difference of solubility with temperature using n-pentanol or cyclohexanone as a solvent. The experimental results showed that the content of β-sitosterol was over 85% after three stages of recrystallization operation in cyclohexanone and the content of stigmasterol was more than 90% after five stages of fractional crystallization operation in n-pentanol or cyclohexanone under the experimental operating conditions.

Multigenerational exposure to phytosterols in the mouse

Due to beneficial health effects phytosterols (PS) are increasingly added to functional foods. The aim of the present study was to investigate the chronic effects of a dietary PS mixture (5 mg/kg/day), containing mainly β-sitosterol, on the reproduction of the mouse. General reproductive parameters, postnatal development, growth and survival of pups, weight of sex organs, the concentrations of plasma sex steroids and testicular testosterone were monitored across five generations (F 0–F 4). PS exposure increased the plasma levels of testosterone and decreased the relative uterine weights in the pups of F 2 and F 4 generations. Furthermore, PS exposure increased the concentrations of plasma estradiol in the female pups of F 3 generation. PS supplement also increased the testicular levels of testosterone in the male pups of F 2 generation. In spite of these transitory changes, PS exposure caused no permanent deleterious effects on the reproduction of the mouse.

Postmetamorphic Xenopus laevis shows decreased plasma triiodothyronine concentrations and phosphorylase activity due to subacute phytosterol exposure

Postmetamorphic South African clawed frogs ( Xenopus laevis) were exposed to a phytosterol mixture (ca. 80% β-sitosterol and less sitostanol, campesterol, and campestanol) for 14 days at 30 μg l −1 in a flow-through system. The effects of phytosterols (PS) on the plasma thyroid hormone (T 3 and T 4), testosterone, leptin-immunoreactive peptide and tissue glycogen concentrations were determined. The following enzyme activities were also analyzed from the liver and muscle: glycogen phosphorylase and lipase, and from the liver only: glucose-6-phosphatase. The plasma T 3 concentration was lower in the PS-exposed female frogs. Both muscle lipase and glycogen phosphorylase activities were also lower in the PS-exposed animals. These results could indicate that the basal metabolic rate and locomotion activity of the frogs were decreased. The effects could not be attributed to the possible estrogenicity of the PS mixture. Further studies will be needed to evaluate the possible significance of these effects.

Oral absorption of phytosterols and emulsified phytosterols by Sprague-Dawley rats

Clinical studies have demonstrated that consumption of phytosterol esters in lipid-based foods decreases serum concentrations of total and LDL cholesterol. These substances represent minimal potential for adverse effects when consumed orally because of their low bioavailability. However, some studies have reported estrogenic and other effects in laboratory animals treated parenterally with phytosterols, demonstrating that these substances may have the potential to cause adverse effects if absorbed. Water-soluble phytosterols have been prepared by formulation with emulsifiers to expand delivery options to include non–lipid-based foods. However, emulsifiers are used as excipients in the formulation of lipophilic pharmaceuticals to increase solubility, thereby increasing their absorption. Therefore, oral consumption of emulsified water-soluble phytosterols could potentially increase their absorption. In the current study, absorption of phytosterols prepared as water-soluble emulsified micelles with two different food-grade emulsifiers was evaluated in Sprague-Dawley rats and compared with absorption of non-micellar free phytosterols and esterified phytosterol mixtures dissolved in a lipophilic vehicle (soybean oil). Rats were dosed via gavage with 42 mg/kg of formulated phytosterol preparations. Blood was collected at 8, 16, 24, and 32 hours, extracted with hexane, derivatized with benzoyl chloride, and analyzed by high-performance liquid chromatography to determine concentrations of β-sitosterol, and campesterol. Plasma concentrations and AUC 0–32hours [μg/mL/h] of β-sitosterol and campesterol were lower in plasma obtained from rats treated with emulsified phytosterol preparations than in animals treated with free phytosterols dissolved in soybean oil. Because the pharmacokinetic profile of water-soluble phytosterols is similar to that of phytosterols administered in a lipid vehicle, the safety profile is likely to be the same as that of phytosterols and phytosterol esters in currently used applications.

Reproduction of the tundra vole ( Microtus oeconomus) with dietary phytosterol supplement

Phytosterols (PS) are plant-derived compounds with estrogenic activity in vitro and beneficial effects on the serum lipid profile in vivo. In nature, PS exposure can derive from pulp mill effluents. The effects of a pulp-mill derived PS mixture on the reproduction, endocrine variables and enzyme activities of the tundra vole ( Microtus oeconomus) were investigated in a two-generation study. The cumulative food intake of PS-treated females was higher than in the control group supporting previous results on the effects of PS on food consumption in rodents. 85% of the PS treated pairs reproduced, but the figure was only 60% for the control pairs. The plasma and testicular testosterone concentrations were lower in the adult PS males, but the PS-treated male offspring had higher testicular testosterone concentrations than their controls. In the female offspring, the liver lipase activity was higher in the PS-treated group, which could be a result of decreased cholesterol absorption in the gut. Chronic PS treatment increased the reproduction probability of the species and had a potential effect on the sex steroid hormones of maturing offspring, which could have applications in environmental monitoring.

Assimilation of grape phytosterols by Saccharomyces cerevisiae and their impact on enological fermentations.

Although yeasts are known to be able to incorporate a wide variety of exogenous sterols under strict anaerobiosis, no data are available on the assimilation of grapevine phytosterols under enological conditions and the eventual impact on fermentation kinetics. We used therefore a mixture of pure phytosterols, in a proportion representative of the different grape skins phytosterols, to supplement a synthetic fermentation medium simulating a grape must. Under anaerobiosis, normal biomass formation was achieved with 5 mg phytosterols l(-1). Similar results were obtained in comparison with the observed maximal fermentation rates. These results clearly indicated that grape phytosterols may efficiently act as a substitute for ergosterol in the yeast membrane for promoting yeast growth and initial fermentative activity. Analysis of total yeast sterols indicated that phytosterols are accumulated without further modification, mainly in their esterified form. However, all the fermentations performed with synthetic media supplemented with phytosterols led to stuck fermentations, linked to a correlative strong decrease in cell viability during the stationary phase. Therefore, grape phytosterols are easily incorporated by yeast cells under enological conditions for promoting initial growth and fermentative activity, but rapidly perturb the yeast membrane properties by being the predominant sterols.

Phytosterols and lecithin do not have an additive effect in lowering plasma and hepatic cholesterol levels in diet‐induced hypercholesterolemic rats

Both plant sterols and lecithin are used as dietary supplements for lowering blood cholesterol in Western countries. This study evaluated the possibility of an additive effect of these ingredients on the regulation of lipid concentrations and cholesterol metabolism. Male Sprague-Dawley rats were randomly divided into three groups, and fed one of the following diets for 5 weeks; high cholesterol diet (HCD), phytosterol mixture-supplemented diet (PD, HCD+0.25% phytosterols), or phytosterol mixture and lecithin-supplemented diet (PLD, PD+0.15% lecithin). Feeding the PD for 5 weeks resulted in a 34% and 41% decrease in plasma total- and VLDL+LDL-cholesterol levels, respectively, and a 23% decrease in hepatic cholesterol content compared to those for the HCD rats (p < 0.05). These cholesterol-lowering properties of the phytosterol mixture were also associated with the down-regulation of hepatic acyl CoA:cholesterol acytransferase (ACAT) activity (p < 0.05). Addition of lecithin plus phytosterol mixture to the hypercholesterolemic diet did not significantly affect blood and hepatic lipid concentrations (with the exception of 36% decrease in hepatic triglyceride level, p < 0.05) as well as hepatic ACAT activity compared to feeding the hypercholesterolemic diet supplemented with phytosterol alone. These results indicate that combining lecithin, at a 0.15% level, with a phytosterol mixture-supplemented diet does not exhibit an additive effect in regulating hepatic ACAT activity or lowering blood cholesterol in hypercholesterolemic rats.

In vivo implants of β-sitosterol cause reductions of reactive cholesterol pools in mitochondria isolated from gonads of male goldfish ( Carassius auratus)

β-Sitosterol, a phytosterol found in high concentrations in pulp mill effluents, has been proposed as one of the causative agents for steroid depressions observed in fish exposed to pulp mill effluents. Previous studies have suggested a cholesterol-mediated mechanism; however, it is unknown how β-sitosterol depresses gonadal steroidogenesis. In this study, adult male goldfish ( Carassius auratus) were exposed for 24–31 days to β-sitosterol (55% of a phytosterol mixture or 96% pure; 150 μg/g; Silastic implant) after which gonadal mitochondria were isolated. Pregnenolone production, an indicator of the size of the pool of reactive cholesterol, was then measured in the isolated mitochondria. Sterol exposure did not affect P450 side-chain cleavage enzyme (converts cholesterol to pregnenolone) activity but did decrease the size of the mitochondrial pool of reactive cholesterol, suggesting β-sitosterol is impeding cholesterol transfer across the mitochondrial membrane. This finding is supported by the observation that 25-hydroxycholesterol, which passes through mitochondrial membranes without need for a membrane transporter, restores β-sitosterol-induced reductions in pregnenolone production.

Effects of a phytosterol mixture on male fish plasma lipoprotein fractions and testis P450scc activity

Plant sterols (phytosterols) have been identified as one potential source of reproductive effects in fish living downstream of pulp mills. β-Sitosterol, the predominant plant sterol in pulp mill effluent, has previously been shown to decrease plasma sex steroid and cholesterol levels and in vitro gonadal steroid production in fish. In this study, male brook trout ( Salvelinus fontinalis) and goldfish ( Carassius auratus) were exposed to a phytosterol mixture (72% β-sitosterol) via Silastic intraperitoneal implants to help elucidate the mechanisms of action of phytosterols on steroid depression. As cholesterol is exogenously supplied for gonadal steroidogenesis, changes in plasma cholesterol fractions were examined. In male brook trout, low-density lipoprotein cholesterol and triglyceride levels decreased significantly, 43 and 50%, respectively, in phytosterol-treated fish. It is improbable, however, that these decreases are linked to depressed gonadal steroidogenesis in fish. The activity of P450scc, which converts cholesterol to pregnenolone (the first step in the steroidogenic pathway), was not affected in testis mitochondria isolated from brook trout or goldfish. Further investigation of the mechanisms of action of phytosterols is required.

A single daily dose of soybean phytosterols in ground beef decreases serum total cholesterol and LDL cholesterol in young, mildly hypercholesterolemic men , , ,

Consumption of phytosterol-supplemented margarine lowers total plasma cholesterol (TC) and LDL-cholesterol concentrations in older middle-aged hypercholesterolemic individuals. The effects of incorporating phytosterols into lower-fat foods on the plasma lipids of young men at increased risk of developing cardiovascular disease have not been studied. We tested the hypothesis that a single daily dose of soybean phytosterols added to ground beef will lower plasma TC and LDL-cholesterol concentrations in mildly hypercholesterolemic young men. In a triple-blind, 4-wk study, 34 male college students with elevated plasma TC (5.85 +/- 0.70 mmol/L), LDL cholesterol (4.02 +/- 0.60 mmol/L), and TC:HDL cholesterol (5.5 +/- 1.2) were randomly assigned to the control (ground beef alone) or treatment (ground beef with 2.7 g of phytosterols) group. The phytosterol mixture was two-thirds esterified and one-third nonesterified and consisted of beta-sitosterol (48%), campesterol (27%), and stigmasterol (21%). Consumption of phytosterol-supplemented ground beef lowered plasma TC and LDL-cholesterol concentrations and TC:HDL cholesterol from baseline by 9.3%, 14.6%, and 9.1%, respectively (P < 0.001). The LDL particle size did not change, suggesting that the decrease was primarily of particle number. The decreases were similar in subjects with (n = 8) and without (n = 9) a family history of premature cardiovascular disease. No significant changes were found in the control group. Phytosterol-supplemented ground beef effectively lowers plasma TC and LDL cholesterol and has the potential to become a functional food to help reduce the risk of cardiovascular disease.

In vitro and in vivo (SCID mice) effects of phytosterols on the growth and dissemination of human prostate cancer PC-3 cells.

The dietary effect of phytosterols (PS) versus cholesterol on the growth and metastasis of the PC-3 human prostate cancer cells in SCID mice was studied. Also, their direct effect on the growth and migration of these cells in vitro was analysed. In the in vivo experiment, SCID mice were fed a diet containing 2% of either PS mixture or cholesterol plus 0.2% cholic acid and implanted with 2 x 10(6) tumour cells per mouse. Tumour growth was monitored for 8 weeks post inoculation. Animals fed the PS diet had tumours 40-43% smaller than those fed the cholesterol diet. Furthermore, the number of mice with lymph node and lung metastasis was almost one-half that of the cholesterol-fed group. In the in vitro studies, both beta-sitosterol and campesterol inhibited the growth of PC-3 cells by 70% and 14%, respectively, while cholesterol supplementation increased the growth by 18% when compared with controls. PS inhibited the invasion of PC-3 cells into Matrigel-coated membranes by 78% while cholesterol increased it by 43% as compared with the cells in the control media. Migration of tumour cells through 8 microm pore membranes was reduced by 60-93% when the PC-3 cells were in PS media, as compared with a 67% increase after cholesterol supplementation. PS supplementation reduced the binding of PC-3 cells to laminin by 15-38% and fibronectin by 23% while cholesterol increased binding to type IV collagen by 36%. It was concluded that PS indirectly (in vivo as a dietary supplement) and directly (in tissue culture media) inhibited the growth and metastasis of PC-3 cells. beta-Sitosterol was more effective than campesterol in offering this protection in most of the parameters studied.