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

doi:10.1016/j.jnutbio.2003.08.013

The Journal of Nutritional Biochemistry

Volume 15, Issue 5, May 2004, Pages 289-295

https://doi.org/10.1016/j.jnutbio.2003.08.013 Get rights and content

Abstract

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.

Introduction

Phytosterols are naturally occurring substances found in plants and that are structurally similar to cholesterol [1]. Although numerous unique phytosterols have been identified, β-sitosterol, campesterol, and stigmasterol account for the largest proportion in most sources [2]. While similar in structure to cholesterol, these phytosterols possess substitutions at position C-24 that are responsible for their poor absorption [3], [4], [5].

Phytosterols inhibit cholesterol absorption from the gastrointestinal system. Many clinical studies have demonstrated significant decreases in serum cholesterol concentrations after consumption of foods into which phytosterols were incorporated [6], [7], [8]. In those studies, phytosterols were administered as fatty acid esters to increase solubility and to facilitate incorporation into lipid-based foods. However, the cholesterol-lowering activity is attributable to the free phytosterols as they are hydrolyzed to free sterols and fatty acids in the gut [9], [10].

Laboratory animals administered phytosterols parenterally have demonstrated adverse effects including evidence of estrogenicity [11], [12], [13]. In contrast, oral exposure to phytosterols does not appear to cause adverse effects. Laboratory rats consuming dietary mixtures of phytosterol or pure β-sitosterol demonstrated no evidence of estrogenic activity even in assays sensitive enough to respond to weakly estrogenic phytochemicals such as coumesterol [14], [15]. From these studies, it appears that the adverse effects of phytosterols are dependent on bioavailability.

Water-soluble phytostanols have been formulated as lecithin emulsified micelles with potential applications in non–lipid-based foods [16]. However, micellar preparations of lipophilic pharmaceuticals are formulated specifically to increase their solubility and hence also their absorption from the gut [17], [18], [19]. Phytosterols formulated with emulsifiers into water-soluble micelles may be more soluble within the gut and have the potential for increased absorption when consumed orally. The present studies were conducted to compare the absorption of emulsified phytosterol micelles with absorption of phytosterols and phytosterol esters in laboratory rats.

Section snippets

Materials

Reference compounds campesterol (C₂₈H₄₈O; MW 400.7), stigmasterol (C₂₄H₄₈O; MW 412.7), β-sitosterol (C₂₉H₅₀O; MW 414.7), and other chemicals including KOH, ethanol, benzoyl chloride, 1,2-dichloroethane, and pyridine were obtained from Sigma Chemical (St. Louis, MO).

Phytosterols and phytosterol esters

The free phytosterols (lot #4763-36-6) used in this work were obtained from soybean oil distillates through a multi-step purification process (Cargill NutriProducts, Eddyville, IA). The product used was a mixture of phytosterols. The

Analytical validation

To validate the ability to detect phytosterols in rat plasma, pure campesterol, stigmasterol, and β-sitosterol were added to plasma from untreated rats. After extraction and derivatization, the samples were analyzed via HPLC as described above (see “Methods and materials” section). Significant concentrations of cholesterol were detected owing to the similarity in structure between cholesterol and the phytosterols. Retention times for cholesterol, campesterol, stigmasterol, and β-sitosterol were

Discussion

Phytosterols are structurally similar to cholesterol [1] except that they possess substitutions at C-24. This substitution appears to be responsible for inhibiting their absorption from the gastrointestinal system [3], [4], [5]. Although Mellanen et al. demonstrated estrogenic activity of phytosterols when evaluated in vitro [21], these compounds exhibit relatively little potential for toxicity because they are poorly absorbed.. Long-term studies on the oral consumption of phytosterols in

Acknowledgements

This study was sponsored by Cargill, Inc., Health and Food Technologies, Wayzata, MN.

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For over 50 years PS have been shown to reduce serum LDL cholesterol by up to 10% when consumed in adequate quantities (Jones et al., 2018; Ostlund, 2002). Over 250 types of PS have been identified, with beta-sitosterol, campesterol, and stigmasterol being the most abundant (Delaney et al., 2004; Marangoni & Poli, 2010). Some evidence suggests these different PS types can differentially affect cholesterol absorption or inflammation (Sabeva et al., 2011), but the mechanism of action is still unclear.
There is great interest in reaching a consensus method for in vitro digestion of foods. The COST INFOGEST network of researchers has developed a standardized protocol, which is appropriate for basic digestibility studies, but may be insufficient for more complex studies, such as micronutrient bioaccessibility. In the present study, the bioaccessibility of phytosterols (PS) was evaluated using the standardized and modified versions of the in vitro digestion protocol. The INFOGEST protocol was used without modification for 2% solutions of either free phytosterols (FPS) or esterified phytosterols (EPS) in soybean oil. The effect of two variables which have yet to be explored using the INFOGEST protocol was subsequently evaluated. Those variables are: (1) changing the mode of agitation from orbital shaking to head over heels (HOH) rotation and (2) adding pancreatic cholesterol esterase (CE) during the intestinal phase. Bioaccessibility of FPS was greater than that of EPS, and HOH rotation caused a further increase in bioaccessibility of FPS but not EPS. Addition of CE caused a slight, but significant improvement in bioaccessibility of EPS, though hydrolysis was incomplete. Our findings highlight a need for further evaluation of the standardized in vitro digestion protocol, especially for complex studies such as lipid bioaccessibility.
Phytosterols of marine algae: Insights into the potential health benefits and molecular pharmacology
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Marine algae are rich in some unique biologically active secondary metabolites having diverse pharmacological benefits. Of these, sterols comprise a group of functional lipid compounds that have attracted much attention to natural product scientists.
This review was aimed to update information on the health effects of algae-derived phytosterols and their molecular interactions in various aspects of human health and diseases and to address some future perspectives that may open up a new dimension of pharmacological potentials of algal sterols.
A literature-based search was carried out to retrieve published research information on the potential health effects of algal phytosterols with their pharmacological mechanisms from accessible online databases, such as Pubmed, Google Scholar, Web of Science, and Scopus, using the key search terms of ‘marine algae sterol’ and ‘health potentials such as antioxidant or anti-inflammatory or anti-Alzheimer's or anti-obesity or cholesterol homeostasis or hepatoprotective, antiproliferative, etc.’
Phytosterols of marine algae, particularly fucosterol, have been investigated for a plethora of health benefits, including anti-diabetes, anti-obesity, anti-Alzheimer's, antiaging, anticancer, and hepatoprotection, among many others, which are attributed to their antioxidant, anti-inflammatory, immunomodulatory and cholesterol-lowering properties, indicating their potentiality as therapeutic leads. These sterols interact with enzymes and various other proteins that are actively participating in different cellular pathways, including antioxidant defense system, apoptosis and cell survival, metabolism, and homeostasis.
In this review, we briefly overview the chemistry, pharmacokinetics, and distribution of algal sterols, and provide critical insights into their potential health effects and the underlying pharmacological mechanisms, beyond the well-known cholesterol-lowering paradigm.
Microencapsulation of Phytosterols by Spray Drying
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Moreover, a typical western diet includes 80 mg/day, whereas 500 mg/day is consumed in vegetarian diets [35,39]. Although the recommended doses are not clearly established, high intakes of phytosterols do not exert side effects, subchronic toxicity, or mutagenic activity [1,7,8,26]. Actually, the Food and Drugs Administration includes the phytosterols, phytostanols, and their esters in the Generally Recognized as Secure (GRAS) list [54].
Phytosterols (PS) are vegetable sterols with a similar structure to cholesterol. Although PS are poorly absorbed into the blood stream, they are widely recognized as lowering absorption of cholesterol and their serum levels. It has been found that PS exert their hypocholesterolemic effect if they are dispersed. Indeed, the PS must be administered finely divided in order to facilitate their exposure to the bile salts, preferably in particles smaller than 25 μm to reduce the sandy mouth feel and favor their incorporation into the micellar phase in the intestine. Several authors focused on particle-size reduction to improve the PS dispersibility by several techniques. However, all of them are complex and time- and energy consuming because they require more than one step (homogenization or milling, including cooling or heating) to obtain the desired particle size. Furthermore, for solid PS, several abrasive effects of the homogenizer valves or parts of the milling equipment have been found.
PS and their derivatives (stanols, esters, and stanol esters) have been included in fat- or oil-based food products, which are clearly restricted in diets for hypercholesterolemia. Therefore, the incorporation of PS in aqueous-based formulations (like beverages, soups, and others) is an attractive field of application. The hydrophobic and water-insoluble nature of PS, which make them poor candidates for stable dispersions, hinder their applicability on intermediate or final aqueous-based products. In this context, the microencapsulation by spray drying appears as a good choice to provide a physical barrier between the PS and the aqueous medium.
In this chapter, the microencapsulation of PS by spray drying was studied to obtain particles (microcapsules) with small sizes (lower than 25 μm) in order to keep the suspension stable. The feed suspensions to be spray dried were analyzed in details (e.g., composition, treatment conditions, rheological behavior, and interfacial properties). The influence of these factors on the microcapsule structure and on the particle size of PS in suspension was particularly investigated. The main operating variables were also rationally studied in order to identify the optimum set in terms of low particles size and high process yield, encapsulation efficiency, and PS retention in the powder of microcapsules. Several parameters involved in the microcapsules formation were affected by the spray-drying conditions. Finally, the microcapsules of PS were incorporated in aqueous matrices and the stability of the product suspension was analyzed.
The microencapsulation of PS by spray drying using a mixture of Arabic gum and maltodextrin was successfully achieved. Moreover, by including a surface active material in low concentrations (especially sodium lauryl sulfate at 2%, m/v), better results were obtained (low mean particle size and relatively high encapsulation efficiency and PS retention). Infrared spectroscopy, powder X-ray diffraction, and differential scanning calorimetry of the raw materials and microcapsules indicated that no changes in chemical structure nor strong interaction between microcapsules components took place.
Encapsulation of β-sitosterol plus γ-oryzanol in O/W emulsions: Formulation characteristics and stability evaluation with microchannel emulsification
2017, Food and Bioproducts Processing
β-Sitosterol and γ-oryzanol have reduced solubility in aqueous based formulations. In this study β-sitosterol (β-st) and γ-oryzanol (γ-oz) were encapsulated at relatively high concentrations in different food-grade oil-in-water (O/W) emulsions using straight-through microchannel emulsification. The innovative aspect of this study was the production of monodisperse droplets with high encapsulation efficiency and stability of β-sitosterol and γ-oryzanol. Milli-Q water containing 1% (w/w) Tween 20 or decaglycerol monolaurate (ML-750) was used as the continuous phase and the dispersed phase contained 0.5–4% (w/w) each of β-st and γ-oz in medium chain triglycerides. Successful droplet generation was conducted with different concentrations of β-st and γ-oz. The Sauter mean diameter of 1% (w/w) β-st and γ-oz loaded O/W emulsions ranged between 26 and 28 μm with relative span factor width below 0.21. These emulsions were stable at 4 and 25 °C during evaluated storage period. The emulsions stabilized with Tween 20 have encapsulation efficiencies of β-st and γ-oz (EE_β-st and EE_γ-oz) above 80% at 4 and 25 °C; those stabilized with ML-750 have EE_β-st over 80% and EE_γ-oz above 50% at 4 and 25 °C.
The use of Arabic gum, maltodextrin and surfactants in the microencapsulation of phytosterols by spray drying
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Emulsification implies the use of high temperatures (above the PS melting point, i.e., ≈ 136 °C) [9,18] or esterified PS (which have lower melting points) [19–21]. However, high temperatures could negatively affect the PS oxidative stability while esterified PS need to be hydrolyzed in order to inhibit cholesterol absorption [22]. On the other hand, the stabilization of aqueous suspensions can be achieved by adding surfactants [23].
The addition of phytosterols in aqueous-based food matrices is challenging because of their poor physicochemical properties (non-water soluble and hydrophobic powder). By using spray drying, phytosterols microparticles were formulated and developed in this work. Arabic gum, maltodextrin and one of two different surfactants were thoroughly studied as wall materials. Increasing concentrations of Tween 20 (T20) or sodium lauryl sulfate (SDS), from 0.1 to 2.65% w/v, were evaluated. The feed suspension characteristics (viscosity, interfacial properties and particle size distribution), process yield (PY), encapsulation efficiency (EE), phytosterols retention (R) and size of the microparticles were analyzed. The presence of surfactants in the suspension to be spray dried has significant effects on the studied responses. T20 led to process yields around 65% (2% w/v surfactant concentration). On the other hand, the microparticles obtained using 2% w/v of SDS were the best in terms of EE (about 50%), R (close to 40%) and particle size (5.89 μm), being the PY acceptable (almost 55%). According to the open literature, which indicates that average particle sizes lower than 25 μm favor the phytosterols bioavailability, the microparticles obtained in this work are promising for phytosterols delivery.
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Oral absorption of phytosterols and emulsified phytosterols by Sprague-Dawley rats

Abstract

Introduction

Section snippets

Materials

Phytosterols and phytosterol esters

Analytical validation

Discussion

Acknowledgements

J Am Diet Assoc

Atherosclerosis

J Lipid Res

J Nutr

Am J Clin Nutr

J Ethnopharmacol

Food Chem Toxicol

Food Chem Toxicol

Am J Clin Nutr

J Chromatgr

Toxicol Appl Pharmacol

Pharmacol Ther

J Nutr Biochem

J Nutr