Phytosterol compositions and use thereof in foods, beverages, pharmaceuticals, nutraceuticals and the like

The present invention provides a esterified and subsequently hydrogenated phytosterol composition for use alone or for incorporation into foods, beverages, pharmaceuticals, nutraceuticals, and the like. The composition has the advantage of enhanced solubility/dispersability, increased molar potency and enhanced stability over naturally isolated phytosterol compositions. Methods for the esterification and subsequent hydrogenation of the phytosterols are also provided.

FIELD OF THE INVENTION 
This present invention relates to the field of phytosterol-based 
compositions suitable for incorporation into foods, pharmaceuticals, 
nutraceuticals and the like and to methods of making the same. 
BACKGROUND OF THE INVENTION 
While recent advances in science and technology are helping to improve 
quality and add years to human life, the prevention of atherosclerosis, 
the underlying cause of cardiovascular disease ("CVD") has not been 
sufficiently addressed. Research to date suggest that cholesterol may play 
a role in atherosclerosis by forming atherosclerotic plaques in blood 
vessels, ultimately cutting off blood supply to the heart muscle or 
alternatively to the brain or limbs, depending on the location of the 
plaque in the arterial tree (1,2). Overviews have indicated that a 1% 
reduction in a person's total serum cholesterol yields a 2% reduction in 
risk of a coronary artery event (3). Statistically, a 10% decrease in 
average serum cholesterol (e.g. from 6.0 mmol/L to 5.3 mmol/L) may result 
in the prevention of 100,000 deaths in the United States annually (4). 
Sterols are naturally occurring triterpenoids that perform many critical 
cellular functions. Phytosterols such as campesterol, stigmasterol and 
beta-sitosterol in plants, ergosterol in fungi and cholesterol in animals 
are each primary components of cellular and sub-cellular membranes in 
their respective cell types. The dietary source of phytosterols in humans 
comes from plant materials i.e. vegetables and plant oils. The estimated 
daily phytosterol content in the conventional western-type diet is 
approximately 60-80 milligrams in contrast to a vegetarian diet winch 
would provide about 500 milligrams per day. 
Phytosterols have received a great deal of attention due to their ability 
to decrease serum cholesterol levels when fed to a number of mammalian 
species, including humans. While the precise mechanism of action remains 
largely unknown, the relationship between cholesterol and phytosterols is 
apparently due in part to the similarities between the respective chemical 
structures (the differences occurring in the side chains of the 
molecules). It is assumed that phytosterols displace cholesterol from the 
micellar phase and thereby reduce its absorption. 
Over forty years ago, Eli Lilly marketed a sterol preparation from tall oil 
and later from soybean oil called Cytellin.TM. which was found to lower 
serum cholesterol by about 9% according to one report (5). Various 
subsequent researchers have explored the effects of sitosterol 
preparations on plasma lipid and lipoprotein concentrations (6) and the 
effects of sitosterol and campesterol from soybean and tall oil sources on 
serum cholesterols (7). A composition of phytosterols which has been found 
to be highly effective in lowering serum cholesterol is disclosed in 
PCT/CA95/00555 and comprises no more than 70% by weight beta-sitosterol, 
at least 10% by weight campesterol and stigmastanol. It is hypothesized in 
this patent application (which has already issued to patent in some 
countries) that there may be some form of synergy between the constituent 
phytosterols. 
Given that phytosterols in various combinations have been proven to have 
wide clinical and dietary applications in lowering total and low density 
lipoprotein cholesterol, the key problem now facing researchers in this 
field is the adaptation of the phytosterol delivery system. Studies have 
investigated how the form (for example, crystalline, suspension, granular) 
in which the phytosterols are dosed impacts on their ability to lower 
serum cholesterol levels. Phytosterols are highly hydrophobic, do not 
dissolve to any significant degree in the micellar phase in the digestive 
tract and therefore are not capable of efficiently blocking cholesterol 
absorption. Oils and fats are capable to a limited but not satisfactory 
degree of dissolving free phytosterols. Since only solubilized 
phytosterols inhibit the absorption of cholesterol, this "delivery" 
problem must be adequately addressed. 
Early research focused on grinding or milling the phytosterols in order to 
enhance their solubility (U.S. Pat. Nos.: 3,881,005 and 4,195,084 both to 
Eli Lilly). In addition, researchers have looked to the esterification of 
phytosterols in order to enhance their solubility in delivery systems. 
German Patent 2035069/Jan. 28, 1971 (analogous to U.S. Pat. No. 3,751,569) 
describes the addition of phytosterol fatty acid esters to cooking oil. 
The esterification is carried out between a free sterol and a fatty acid 
anhydride, with perchloric acid as the catalyst. The significant drawback 
to this process, along with others, is the use of non-food grade catalysts 
and reagents. 
U.S. Pat. No. 4,588,717 to David E. Mitchell Medical Research Institute 
describes a vitamin supplement which comprises a fatty acid ester of a 
phytosterol, wherein the fatty acid forming the ester has from about 18 to 
20 carbon atoms in the main carbon chain. 
U.S. Pat. No. 5,270,041 to Marigen S. A. teaches the use of small amounts 
of sterols, their fatty acid esters and glucosides for the treatment of 
tumours. The method of preparation of these compositions involving the use 
of hazardous chemical reagents effectively precludes their use in foods or 
as dietary additives. 
Other research has demonstrated that phytostanols, the 5 alpha saturated 
derivatives of phytosterols, are more effective as therapeutic agents in 
lowering serum cholesterol on a molecular weight basis than phytosterols 
(8). Similarly, in a further comparison, sitosterols infused into the GI 
tract resulted in a 50% reduction in serum cholesterol as opposed to an 
85% reduction when sitostanols were infused (9). The advantages of stanols 
over sterols with respect to inhibition of cholesterol absorption from the 
GI tract are two-fold. Firstly, stanols are more chemically stable than 
their unsaturated counterparts in heat and air due to the absence of 
carbon-carbon bonds in the former. Secondly, stanols are more effective at 
lowering serum cholesterol on a molecular weight basis than their 
unsaturated counterparts. 
U.S. Pat. No. 5,502,045 to Raision Tehtaat Oy AB (hereinafter the "Raision 
Patent") describes the preparation of a beta-sitostanol fatty acid ester 
mixture prepared by interesterifying beta-sitostanol with a fatty acid 
ester containing from 2 to 22 carbon atoms in the presence of an 
interesterification catalyst. This process renders the sitostanol 
appreciably more soluble in fats and oils. 
South African Patent Application 967616 also to Raision Tehtaat Oy AB 
(hereinafter the "SA Raision Patent") describes a similar composition to 
that in the Raision Patent but which further contains at least 10% 
campestanol obtained by hydrogenation of the phytosterol mixture. 
U.S. Pat. No. 5,244,887 to Straub discloses a method of making a food 
additive composition which comprises dissolving a stanol (sitostanol; 
clionastanol; 22,23-dihydrobrassicastanol; campestanol and mixtures 
thereof) with an edible solubilizing agent, an anti-oxidant and a carrier 
or dispersant. 
Although the Raision Patent and the Raision SA Patent both attempt to 
produce a phytostanol delivery system which is stable and effective, there 
are significant problems with the long-term stability of these esterified 
products due to the ultimate oxidation of the unsaturated fatty acid 
moiety. 
It is an object of the present invention to obviate or mitigate the above 
disadvantages. 
SUMMARY OF THE INVENTION 
The present invention provides a composition suitable for use alone or for 
incorporation into foods, beverages, pharmaceuticals, nutraceuticals and 
the like which comprises one or more esterified and subsequently 
hydrogenated phytosterols. 
The present invention further comprises foods, beverages, pharmaceuticals, 
nutraceuticals and the like which comprise one or more esterified and 
subsequently hydrogenated phytosterols. These "formulations" include, but 
are not limited to, the composition incorporated into edible oils and 
fat-based foods (such as margarines, butter, mayonnaise, dressing, 
shortenings, and cheeses), and formed into suspensions, emulsions, 
microemulsions, liposomes, niosomes and general hydrated lipid phases. The 
composition additionally may be incorporated into numerous pharmaceutical 
dosage forms as described in detail below. 
The present invention further comprises the use of a composition which 
comprises one or more esterified and subsequently hydrogenated 
phytosterols to lower serum cholesterol in animals, including humans. 
The present invention further comprises methods of making a composition 
suitable for incorporation into foods, beverages, pharmaceuticals, 
nutraceuticals and the like which comprises condensing a suitable 
aliphatic acid with a phytosterol to form a phytosterol ester and 
subsequently hydrogenating the phytosterol ester to form a hydrogenated 
phytosterol ester. 
The composition of the present inventions which comprises one or more 
esterified and subsequently hydrogenated phytosterols has marked 
advantages over the phytosterol compositions previously known and 
described, particularly those compositions taught in the Raision Patent 
and the SA Raision Patent. The composition of the present invention not 
only enhances the solubility and dispersability of phytosterols in lipid 
or fat-based systems and aqueous systems and increases the molar potency 
of phytosterols as agents to lower serum cholesterol but also greatly 
improves and extends the stability and shelf-life of the composition, 
alone and in association with other forms of conveyance or administration. 
These advantages and their commercial implications are described in more 
detail below. 
PREFERRED EMBODIMENTS OF THE INVENTION 
According to one aspect of the present invention, there is provided a 
composition suitable for use alone or for incorporation into foods, 
beverages, pharmaceuticals, nutraceuticals and the like which comprises 
one or more esterified and subsequently hydrogenated phytosterols. 
The key feature of this invention, which affords the advantages of enhanced 
solubility/dispersability, increased molar potency and particularly 
enhanced stability, hinges on the esterification of the phytosterols prior 
to hydrogenation (i.e. saturation). In this way, all unsaturated bonds not 
only in the phytosterol ring of the ester but in the aliphatic acid moiety 
are hydrogenated and thereby significantly protected from the effects of 
oxidation. The resultant composition is also protected from microbial 
oxidation and/or degradation which is critical when the composition is 
incorporated into foods such as cheeses and yogurt. In addition, the 
composition of the present invention is more heat stable and therefore 
amenable to many food, beverage, pharmaceutical, and nutraceutical 
processing techniques. The saturation of both the aliphatic acid moiety 
and phytosterol ring also enhances the solubility of the composition, even 
without further treatments. Furthermore, this enhanced degree of 
saturation overcomes the tendency in phytosterols to develop bitterness in 
aqueous systems which is of importance in the field of preparing 
comestibles. None of the prior researchers have explored or appreciated 
the advantages of an esterified and subsequently hydrogenated phytosterol 
composition. 
As used herein, the term "phytosterol" includes all phytosterols without 
limitation, for example: sitosterol, campesterol, stigmasterol, 
brassicasterol, desmosterol, chalinosterol, poriferasterol, clionasterol 
and all natural or synthesized forms and derivatives thereof, including 
isomers. It is to be understood that modifications to the phytosterols 
i.e. to include side chains also falls within the purview of this 
invention. It is also to be understood that this invention is not limited 
to any particular combination of phytosterols forming a composition. In 
other words, any phytosterol alone or in combination with other 
phytosterols in varying ratios as required depending on the nature of the 
ultimate formulation may be subject to the esterification and subsequent 
hydrogenation method of the present invention. For example, the 
composition described in PCT/CA95/00555 which comprises no more than 70% 
by weight beta-sitosterol, at least 10% by weight campesterol and 
stigmastanol may be esterified and hydrogenated to yield a stable and 
favourably soluble product for incorporation into foods. 
The phytosterols for use in this invention may be procured from a variety 
of natural sources. For example, they may be obtained from the processing 
of plant oils (including aquatic plants) such as corn oil and other 
vegetable oils), wheat germ oil, soy extract, rice extract, rice bran, 
rapeseed oil, sesame oil and fish oil. Without limiting the generality of 
the foregoing, it is to be understood that there are other sources of 
phytosterols such as marine animals from which the composition of the 
present invention may be prepared. U.S. patent Ser. No. 4,420,427 teaches 
the preparation of sterols from vegetable oil sludge using solvents such 
as methanol. Alternatively, phytosterols may be obtained from tall oil 
pitch or soap, by-products of the forestry practise as described in 
PCT/CA95/00555, incorporated herein by reference. 
The order of the steps in the method of the present invention is of 
critical importance. Esterification of the phytosterol must occur before 
the hydrogenation step. This way, the entire ester is saturated during 
hydrogenation and not just the phytosterol component, thereby removing all 
unstable double or pi bonds from the molecule. 
To form the phytosterol esters, one or more suitable aliphatic acids or 
their esters with low boiling alcohols are condensed with the 
phytosterols. A wide variety of aliphatic acids or their esters may be 
used successfully within the scope of the present invention and include 
all aliphatic acids consisting of one or more alkyl chains with one or 
more terminal carboxyl groups. These aliphatic acids may be natural or 
synthetic and are represented by the following chemical formulae: 
a) R1--COOH (monocarboxylic acid) wherein: 
R1 is an unbranched saturated alky group, represented by CH3--, CH3CH2-- or 
CH3(CH2)nCH2-- WHERE n=3-25; or 
R1 is a branched saturated alkyl group represented by CnH2n+1-- where 
n=1-25 is the number of carbon atoms contained in the group R1; the 
branching typically refers, but is not limited to one or more methyl group 
side chains (branches); or 
R1 is an unbranched or branched unsaturated alkyl group, represented by the 
formula CnH2n--2m+1, where n=1-25 is the number of carbon atoms in R1 and 
m=degree of unsaturation; or 
b) HOOC--R2--COOH is a dicarboxylic acid wherein: 
R2 is an unbranched saturated alkly group, represented by --CH2--, or 
--CH2CH2--, or --CH2(CH2)nCH2 where n=3-25; or 
R2 is a branched saturated alkyl group represented by --CnH2n-- where 
n=1-25 is the number of carbon atoms contained in the group R2; the 
branching typically refers, but is not limited to, one or more methyl 
group side chains (branches); or 
CnH2n--2m, where n=1-25 is the number of carbon atoms in R2 and m=degree of 
unsaturation; or 
c) a tricarboxylic acid represented by the formula: 
##STR1## 
wherein, in this formula: R3 is a branched saturated alkyl group 
represented by --CnH2n--1-- where n=1-25 is the number of carbon atoms 
contained in the group R3; the branching typically refers, but is not 
limited to, one or more methyl group side chains (branches); or 
R3 is a branched unsaturated alkyl group, represented by CnH2n--2m--1-- 
wherein n=1-25 is the number of carbon atoms in R3 and m=the degree of 
unsaturation; or 
d) a mono-, di-, or tricarboxylic acid as defined above, which may contain 
one, two or three hydroxyl groups in the molecule. 
In a preferred form, the aliphatic acid is either a straight-chain or 
branched unsaturated or saturated fatty acid selected, inter alia, from 
the following list: valeric acid, isovaleric acid, sorbic acid, isocaproic 
acid, lauric acid, myrestic acid, palmitic acid, stearic acid, caproic 
acid, ascorbic acid, arachidic acid, behenic acid, hexacosanoic acid, 
octacosanoic acid, pentadecanoic acid, erucic acid, linoleic acid, 
linolenic acid, arachidonic acid, acetic acid, citric acid, tartaric acid, 
palmitoleic acid and oleic acid. The most preferable fatty acids within 
the scope of the present invention are linoleic acid, linolenic acid and 
arachidonic acid which may be obtained from natural sources such as 
safflower oil, sunflower oil, olive oil and corn oil (linoleic acid), 
safflower oil, sunflower oil, olive oil and jojoba oil (linolenic acid and 
arachidonic acid) and rapeseed oil (erucic acid). 
A particular advantage in using fatty acids to form esterifed and 
subsequently hydrogenated phytosterols, i.e. saturated fats, in accordance 
with the present invention lies in the fact that saturated fats increase 
lipoprotein lipase activity. The activity of this latter enzyme reduces 
visceral fat formation. 
To form a phytosterol ester in accordance with the present invention, the 
selected phytosterol and aliphatic acid or its ester with volatile alcohol 
are mixed together under reaction conditions to permit condensation of the 
phytosterol with the aliphatic acid to produce an ester. A most preferred 
method of preparing these esters which is widely used in the edible fat 
and oil industry is described in U.S. Pat. No. 5,502,045 (which is 
incorporated herein by reference). As no substances other than the free 
phytosterol, a fatty acid ester or mixture thereof and an 
interesterification catalyst like sodium ethylate are used, the technique 
is highly suitable for preparing products ultimately for human 
consumption. In overview, this preferred method, adapted for use within 
the present invention, comprises heating the phytosterol(s) with a 
vegetable oil fatty acid ester (preferably a methyl ester) at a 
temperature from 90-120.degree. C. and subsequently adding a suitable 
catalyst such as sodium ethylate. The catalyst is then removed/destroyed 
by any one of the techniques known in the art e.g. adding water and/or 
filtration/centrifugation. 
Another method which may be used in accordance with the present invention 
is described in U.S. Pat. No. 4,588,717, which is also incorporated herein 
by reference. A preferred method is to mix the phytosterol and the fatty 
acid together bringing the mixture to a temperature of from about 
15.degree. C. to about 45.degree. C. at about atmospheric pressure for 
approximately one to three hours. 
Once the phytosterol ester is formed in accordance with the present 
invention it must then be hydrogenated. The conversion of the phytosterol 
ester to its saturated form may be achieved by one of many known 
hydrogenation techniques (10, incorporated herein by reference) based on 
the use of Pd/C catalyst in organic solvents. Other suitable catalysts 
include platinum and Raney nickel. When this step is carried out under 
optimal conditions, only very small amounts of unsaturated sterol esters 
remain unconverted. 
Within the scope of the present invention, it is possible to produce two 
classes of esterified and subsequently hydrogenated phytosterol 
compositions. The first class of composition, hereinafter referred to as 
the oil-based composition derives from a method in which the 
esterification step proceeds in oil, for example, a vegetable oil. This is 
the esterification process generally described in U.S. Pat. No. 5,502,045. 
The second class of composition, hereinafter referred to as the 
solvent-derived composition is generated by a method in which the 
esterification step proceeds in a suitable solvent, including an aqueous 
solution and not oil. The end product of these two classes, although both 
esterified and subsequently hydrogenated phytosterols, are each suitable 
for use in various delivery systems as disclosed further below. 
Oil-Based Composition 
The preferred method of preparing the oil-based composition comprises 
selecting one or more phytosterols and esterifying these phytosterols in a 
suitable oil. Commonly known interesterification techniques are provided 
in references 11, 12 and 13. The resultant esterified phytosterol 
composition is then hydrogenated. Although the oil-based composition of 
the present invention may be used alone or in various delivery systems, 
greatest efficacy is achieved when the esterified and subsequently 
hydrogenated phytosterols are further treated so as to ensure even 
distribution throughout the food, beverage, pharmaceutical or 
nutraceutical to which they are added. This is most readily accomplished 
by first enhancing the solubility and/or dispersability of the composition 
in a delivery system. Such enhancement may be achieved by a number of 
suitable means such as, for example, solubilizing or dispersing the 
composition to form emulsions, solutions and dispersions and 
self-emulsifying systems and the like as described further below. 
Emulsions 
Emulsions are finely divided or colloidal dispersions comprising two 
immiscible phases, e.g. oil and water, one of which (the internal or 
discontinuous phase) is dispersed as droplets within the other (external 
or discontinuous phase). Thus an oil-in-water emulsion consists of oil as 
the internal phase, dispersed water as the external phase, the 
water-in-oil emulsion being the opposite. 
A wide variety of emulsified systems may be formed which comprise the 
composition of the present invention including standard emulsions, 
microemulsions and those which are self-emulsifying (emulsify on exposure 
to agitated aqueous fluids such as gastric or intestinal fluids). 
Generally, emulsions may include oil and water phases, emulsifiers, 
emulsion stabilizers and optionally preservatives, flavouring agents, pH 
adjusters and buffers, chelating agents, antifoam agents, tonicity 
adjusters and anti-oxidants. Suitable emulsifiers (wherein bracketed 
numerals refer to the preferred HLB values) include: anionic surfactants 
such as alcohol ether sulfates, alkyl sulfates (30-40), soaps (12-20) and 
sulfosuccinates; cationic surfactants such as quaternary ammonium 
compounds; zwtterionic surfactants such as alkyl betaine derivatives; 
amphoteric surfactants such as fatty amine sulfates, difatty alkyl 
triethanolamine derivatives (16-17); and nonionic surfactants such as the 
polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, 
saturated fatty acids and alkyphenols, water-soluble polyethyleneoxy 
adducts onto polypropylene glycol and alkyl polypropylene glycol, 
nonylphenol polyethoxyethanols, castor oil polyglycol ethers, 
polypropylene/polyethylene oxide adducts, 
tributylphenoxy-polyethoxyethanol, polyethylene glycol, 
octylphenoxy-polyethoxethanol, lanolin alcohols, polyoxyethylated (POE) 
alkyl phenols, POE fatty amides, POE fatty alcohol ethers, POE fatty 
amines, POE fatty esters, poloxamers (7-19). POE glycol monoethers 
(13-16), polysorbates and sorbitan esters. This list is not intended to be 
exhaustive as other emulsifiers are equally suitable. 
Appropriate emulsion stabilizers include, but are not limited to, lyophilic 
colloids such as polysaccharides (e.g. acacia, agar, alginic acid, 
carrageenin, guar gum, karaya gum, tragacanth xanthan gum), amphoterics 
(e.g. gelatin) and synthetic or semi-synthetic polymers (e.g. carbomer 
resins, cellulose ethers, carboxymethyl chitin, polyethylene glycol-n 
(ethylene oxide polymer H(OCH2CH2)nOH); finely divided solids including 
clays (e.g. attapulgite, bentonite, hectorite, kaolin, magnesium aluminum 
silicate and montmorillonite), microcrystalline cellulose oxides and 
hydroxides (e.g. aluminum hydroxide, magnesium hydroxide and silica); and 
cybotactic promoters/gellants including amino acids, peptides, proteins 
lecithin and other phospholipids and poloxamers. 
Suitable anti-oxidants for use in the formation of emulsions include: 
chelating agents such as citric acid, EDTA, phenylalanine, phosphoric 
acid, tartaric acid and tryptophane; preferentially oxidized compounds 
such as ascorbic acid, sodium bisulfite and sodium sulfite; water soluble 
chain terminators such as thiols and lipid soluble chain terminators such 
as alkly gallates, ascorbyl palmitate, t-butyl hydroquinone, butylated 
hydroxyanisole, butylated hydroxyrtoluene, hydroquinone, 
nordihydroguaiaretic acid and alpha-tocopherol. Suitable preservatives, pH 
adjustment agents, and buffers, chelating agents, osmotic agents, colours 
and flavouring agents are discussed hereinbelow under "Supensions", but 
are equally applicable with respect to the formation of emulsions. 
The general preparation of emulsions is as follows: the two phases (oil and 
water) are separately heated to an appropriate temperature (the same in 
both cases, generally 5-10.degree. C. above the melting point of the 
highest melting ingredients in the case of a solid or semi-solid oil, or 
where the oil phase is liquid, a suitable temperature as determined by 
routine experimentation). Water-soluble components are dissolved in the 
aqueous (water) phase and oil-soluble components are dissolved in the oil 
phase. To create an oil-in water emulsion, the oil phase is vigorously 
mixed into the aqueous phase to create a suitable dispersion and the 
product is allowed to cool at a controlled rate with stirring. A 
water-in-oil emulsion is formed in the opposite fashion i.e. the water 
phase is added to the oil phase. When hydrophillic colloids are a part of 
the system as emulsion stabilizers, a phase inversion technique may be 
employed whereby the colloid is mixed into the oil phase rather than the 
aqueous phase, prior to addition to the aqueous phase. In using the 
oil-based composition of the present invention, which is semi-solid, it is 
preferred to add the composition to the oil phase prior to heating. 
Microemulsions, characterized by a particle size at least an order of 
magnitude smaller (10-100 nm) than standard emulsions and defined as "a 
system of water, oil and amphiphile which is a single optically isotropic 
and thermodynamically stable liquid" (14), may also be formed comprising 
the composition of the present invention. In a preferred form, the 
microemulsion comprises a surfactant or surfactant mixture, a 
co-surfactant,(usually a short chain alcohol) the oil-based composition of 
the present invention, water and optionally other additives. 
This system has several advantages as a delivery system for the oil-based 
composition of the present invention. Firstly, microemulsions tend to be 
created spontaneously, that is, without the degree of vigorous mixing 
required to form standard emulsions. From a commercial perspective, this 
simplifies the manufacturing process. Secondly, microemulsions may be 
sterilized using microfiltration techniques without breaking the 
microstructure due to the small diameter of the microdroplets. Thirdly, 
microemulsions are highly thermodynamically stable. Fourthly, 
microemulsions possess high solubilizing power which is particularly 
important as they allow for an increased solubilization of the poorly 
hydrosoluble phytostanol esters. 
Surfactant or surfactant mixtures which are suitable for use in the 
formation of microemulsions can be anionic, cationic, amphoteric or 
non-ionic and possess HLB (hydrophile-lipophile balance) values within the 
range of 1-20, more preferably in the ranges 2-6 and 8-17. Especially 
preferred agents are non-ionic surfactants, selected from the group 
consisting of polyglycol ether derivatives of aliphatic or cycloaliphatic 
alcohols, saturated fatty acids and alkyphenols, water-soluble 
polyethyleneoxy adducts onto polypropylene glycol and alkyl polypropylene 
glycol, nonylphenol polyethoxyethanols, castor oil polyglycol ethers, 
polypropylene/polyethylene oxide adducts, 
tributylphenoxy-polyethoxethanol, polyethylene glycol, 
octylphenoxy-polyethoxyethanol, lanolin alcohols, polyoxyethylated (POE) 
alkyl phenols, POE fatty amides, POE fatty alcohol ethers, POE fatty 
amines, POE fatty esters, poloxamers (7-19), POE glycol monoethers 
(13-16), polysorbates and sorbitan esters. 
There are many methods known and used by those skilled in the art for 
making microemulsions. In a preferred method of forming microemulsions of 
the present invention, a surfactant, a co-surfactant and the oil-based 
composition (pre-dissolved in a suitable proportion of an appropriate oil) 
is mixed and then titrated with water until a system of desired 
transparency is obtained. 
In a further preferred embodiment, the formation of microemulsions may be 
achieved by mixing the oil-based compositions with hydrotropic agents and 
food-grade surfactants (refer to 15). 
Solutions and Dispersions 
The oil-based composition of the present invention may be dissolved or 
dispersed in a suitable oil vehicle and used in this form, for example, in 
general food usage, in basting meats and fish, and for incorporation into 
animal feeds. 
Self-Emulsifying Systems 
The oil-based composition may be mixed with appropriate excipients, for 
example, surfactants, emulsion stabilizers (described above) and the like, 
heated (if necessary) and cooled to form a semi-solid product capable of 
forming a spontaneous emulsion on mixing with water. This semi-solid 
product may be used in numerous other forms such as filler material in 
two-piece hard or soft gelatin capsules, or may be adapted for use in 
other delivery systems. 
Solvent-Derived Composition 
As described above, the solvent-derived composition differs from the 
oil-based composition in that the esterification and hydrogenation steps 
occur in a solvent and not an oil. This solvent may be any suitable 
organic or non-organic solvent without limitation, including water. After 
hydrogenation of the esterified phytosterol and subsequent isolation and 
purification, this solvent-derived composition may be used effectively 
alone or in a physically modified form to lower serum cholesterol. In 
physically modifying the solvent-derived composition, the ultimate goal is 
the same as discussed above with respect to the oil-based composition, 
that is, the enhancement of solubility and dispersability, of the 
composition so as to ensure even distribution of the esterified 
phytostanols throughout the food, beverage, pharmaceutical or 
nutraceutical to which they are added. 
Generally, the preparation of the solvent-derived composition comprises 
selecting one or more phytosterols and esterifying the phytosterols in 
suitable solvent (aqueous organic or a combination of both) by either of 
two preferred methods. In a first method, the selected phytosterol(s) 
derived from, for example, vegetable oil, is added to an appropriate acid 
anhydride, such as acetic anhydride and then heated, cooled and stirred. 
In a second method, the phytosterol(s) is dissolved in an appropriate 
solvent such as acetic acid, acetic anhydride and the like. The esterified 
product is then hydrogenated by any one of the techniques known and 
applied in the art. After the hydrogenation step, it is preferred in this 
embodiment that isolation techniques be employed to obtain a solid powder 
through precipitation, filtration and drying or by other conventional 
work-up techniques. 
Thereafter, the solvent-derived composition, in powder form, may be 
incorporated directly into foods, beverages, pharmaceuticals, 
nutraceuticals and the like or alternatively, may be physically modified 
as described below to enhance the solubility and dispersability of the 
composition. It is to be understood that the techniques of solubilizing or 
dispersing the oil-based composition to form emulsions, solutions and 
dispersions and self-emulsifying systems may be adapted and applied to the 
solvent-derived composition. Likewise, the solubilizing techniques 
described below as being of preferred use with respect to the 
solvent-derived composition may equally be used with the oil-based 
composition of this invention. Additional techniques of enhancing the rate 
and degree of solubility of the solvent-derived composition include, 
without limitation: reducing particle size by mechanical grinding 
(milling, micronisation etc..), lyophilizing, spray drying, controlled 
precipitating, or a combination thereof; forming solid dispersions, 
suspensions, hydrated lipid systems, inclusion complexations with 
cyclodextrins, using hydrotopes and formulations with bile acids and their 
derivatives. 
Reducing Particle Size 
Many techniques of particle size reduction are suitable for use within the 
present invention including, inter alia, dry milling, micropulverization, 
fluid energy grinding, controlled precipitation, lyophilisation and 
spray-drying. Each of these techniques is well known in the art and will 
not be discussed in any detail other than to provide reference to 20 and 
21, the former showing preferred processes of spray-drying and the latter 
summarizing the other techniques listed above. 
It has been found that reducing the particle size to under 500 um and most 
preferably under 20 um allows suitable dispersability/solubility of the 
composition in the carriers and dosage forms described further below. 
Solid Dispersions 
An alternative means of increasing the solubility/dispersability of the 
solvent-derived composition involves the use of solid dispersion systems. 
These dispersions may include molecular solutions (eutectics), physical 
dispersions or a combination of both. 
For example, solid dispersions may typically be prepared by utilizing 
water-soluble polymers as carriers. Without limitation, these carriers may 
include, either alone or in combination: solid grade polyethylene glycols 
(PEG's), with or without the addition of liquid grade PEG's; 
polyvinylpyrrolidones or their co-polymers with vinyl acetate and 
cellulose ethers and esters. Other excipients, such as additional members 
of the glycol family e.g. propylene glycol, polyols, e.g. glycerol etc.. 
may also be included in the dispersions. 
Solid dispersions may be prepared by a number of ways which are familiar to 
those in the art. These include, without limitation, the following 
methods: 
(a) fusing the ingredients, followed by controlled cooling to allow 
solidification and subsequent mechanical grinding to produce a suitable 
powder. Alternatively, the molten (fused) dispersion may be sprayed into a 
stream of cooled air in a spray drier to form solid particles (prilling) 
or passed through an extruder and spheroniser to form solid masses of a 
controlled particle size. In a further alternative, the molten dispersion 
is filled directly into two-piece hard gelating capsules; 
(b) dissolving the ingredients in a suitable solvent system (organic, mixed 
organic, organic-aqueous) and then removing the solvents e.g. by 
evaporating at atmospheric pressure or in vacuo, spray drying, 
lyophilizing and the like; or, in a variation of the foregoing, and 
(c) dissolving the ingredients in a suitable solvent system, subsequently 
precipitating them from solution by the use of an immiscible solvent in 
which the ingredients have little or no solubility, filtration, removing 
the solvent, drying and optionally grinding to provide a suitable powder 
form. 
Other commercially available agents for enhancing solubility of the 
phytosterol composition through the formation of solid dispersions are 
considered to fall within the purview of this application. For example, 
the commercial excipient marketed under the trade-mark Gelucire.TM. by 
Gattefosse comprising saturated polyglycolised glycerides may readily be 
used herein. 
Suspensions 
Suspensions, which may be used to enhance the solubility and/or 
dispersability of the solvent-derived composition, comprise a solid, 
perhaps finely divided, internal phase dispersed in an oily or aqueous 
external phase (the vehicle). In addition, the solid internal phase may be 
added to an emulsion as described above during its formation to produce a 
delivery system having properties common to both suspensions and 
emulsions. 
Numerous excipients, which are commonly used in the art, may be suitable 
for producing a suspension within the scope of the present invention. 
Typically, a suspension comprises an oily or aqueous vehicle, the 
dispersed (suspended) internal phase, dispersing and/or wetting agents 
(surfactants), pH adjustment agents/buffers, chelating agents, 
antioxidants, agents to adjust ionic strength (osmotic agents) colours, 
flavours, substances to stabilize the suspension and increase viscosity 
(suspending agents ) and preservatives. 
Appropriate vehicles include, but are not limited to: water, oils, 
alcohols, polyols, other edible or food grade compounds in which the 
phytosterol composition is partially or not soluble and mixtures thereof. 
Appropriate dispersing agents include, but are not limited to: lecithin; 
phospholipids; nonionic surfactants such as polysorbate 65, octoxynol-9, 
nonoxynol-10, polysorbate 60, polysorbate 80, polysorbate 40, poloxamer 
235, polysorbate 20 and poloxamer 188; anionic surfactants such as sodium 
lauryl sulfate and docusate sodium; fatty acids, salts of fatty acids, 
other fatty acid esters, and mixtures thereof. 
Agents/buffers for pH adjustment include citric acid and its salts, 
tartaric acid and its salts, phosphoric acid and its salts, acetic acid 
and its salts, hydrochloric acid, sodium hydroxide and sodium bicarbonate. 
Suitable chelating agents include edetates (disodium, calcium disodium and 
the like), citric acid and tartaric acid. Suitable antioxidants include 
ascorbic acid and its salts, ascorbyl palmitate, tocopherols (especially 
alpha-tocopherol), butylated hydroxytoluene, butylated hydroxyanisole, 
sodium bisulfite and metabisulfite. Suitable osmotic agents include 
monovalent, divalent and trivalent electrolytes, monosaccharides and 
disaccharides. Suitable preservatives include parabens (Me, Et, Pr, Bu), 
sorbic acid, thimerosal, quaternary ammonium salts, benzyl alcohol, 
benzoic acid, chorhexidine gluconate and phenylethanol. Colours and 
flavours may be added as desired and may be selected from all nature, 
natural-identical and synthetic varieties. 
Suitable solubilizing agents include all food grade oils such as plant 
oils, marine oils (such as fish oil) and vegetable oils, monoglycerides, 
diglycerides, triglycerides, tocopherols and the like and mixtures 
thereof. 
Hydrated Lipid Systems 
In a further embodiment of the present invention, the 
solubility/dispersability of the solvent-derived composition may be 
enhanced by the formation of phospholipid systems such as liposomes and 
other hydrated lipid phases, by physical inclusion. This inclusion refers 
to the entrapment of molecules without forming a covalent bond and is 
widely used to improve the solubility and subsequent dissolution of active 
ingredients. 
Hydrated lipid systems, including liposomes, can be prepared using a 
variety of lipid and lipid mixtures, including phospholipids such as 
phosphatidylcholine (lecithin), phosphodiglyceride and sphingolipids, 
glycolipids, cholesterol and the like. The lipids may preferably be used 
in combination with a charge bearing substances such as charge-bearing 
phospholipids, fatty acids, and potassium and sodium salts thereof in 
order to stabilize the resultant lipid systems. A typical process of 
forming liposomes is as follows: 
1) dispersion of lipid or lipids and the solvent-derived composition of the 
present invention in an organic solvent (such as chloroform, 
dichloromethane, ether, ethanol or other alcohol, or a combination 
thereof). A charged species may be added to reduce subsequent aggregation 
during liposome formation. Antioxidants (such as ascorbyl palmitate, 
alpha-tocopherol, butylated hydroxytoluene and butylated hydroxyanisole) 
may also be added to protect any unsaturated lipids, if present; 
2) filtration of the mixture to remove minor insoluble components; 
3) removal of solvents under conditions (pressure, temperature) to ensure 
no phase separation of the components occur; 
4) hydration of the "dry" lipid mixture by exposure to an aqueous medium 
containing dissolved solutes, including buffer salts, chelating agents, 
cryoprotectorants and the like; and 
5) reduction of liposome particle size and modification of the state of 
lamellarity by means of suitable techniques such as homogenization, 
extrusion etc.. 
Any procedure for generating and loading hydrated lipid with active 
ingredients, known to those skilled in the art, may be employed within the 
scope of this invention. For example, suitable processes for the 
preparation of liposomes are described in references 18 and 19, both of 
which are incorporated herein by reference. Variations on these processes 
are described in U.S. Pat. No. 5,096,629 which is also incorporated herein 
by reference. 
U.S. Pat. No. 4,508,703 (also incorporated herein by reference) describes a 
method of preparing liposomes by dissolving the amphiphillic lipidic 
constituent and the hydrophobic constituent to form a solution and 
thereafter atomizing the solution in a flow of gas to produce a pulverent 
mixture. 
Cyclodextrin Complexes 
Cyclodextrins are a class of cyclic oligosaccharide molecules comprising 
glucopyranose sub-units which may be used to form inclusion complexes with 
the solvent-derived composition. The molecular shape of cyclodextrin is a 
torus having a hydrophobic centre and relatively hydrophilic outer 
surface. In aqueous solutions, both the inner and outer surfaces attract 
water and the hydrogen bonds within the cavity of the torus attract, 
thereby distorting the cyclodextrin. This distorted configuration 
represents a high energy state which will readily accept a "guest" 
molecule such as the esterified and subsequently hydrogenated phytosterol 
of this invention via non-covalent bonding. Since the esterified and 
subsequently hydrogenated phytosterol formed within the process of this 
invention is quite hydrophobic and insoluble, it will readily form such a 
complex with cyclodextrin. The complex so formed often confers properties 
of improved solubility, dispersability, stability (chemical, physical and 
microbiological), bioavailability and decreased toxicity on the guest 
molecule (here, the composition of the present invention). 
Cyclodextrins are cyclic oligosaccharides composed of dextrose units joined 
through a 1-4 bond such as alpha, beta and gamma cyclodextrin, 
carboxymethyl-beta-cyclodextrin, carboxymethyl-ethyl-beta-cyclodextrin, 
diethyl-beta-cyclodextrin, dimethyl-beta-cyclodextrin, 
methyl-beta-cyclodextrin, random methyl-beta-cyclodextrin, 
glucosyl-beta-cyclodextrin, maltosyl-beta-cyclodextrin, 
hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin and 
sulfobutylether-beta-cyclodextrin. In other words, the external hydroxyl 
substituents of the cyclodextrin molecule may be modified to form 
derivatives having improved solubility in aqueous media and to have other 
desired advantages such as decreased toxicity etc. Other types of chemical 
modification known to those skilled in the art are also within the purview 
of the present invention. 
There are a number of ways to produce a cyclodextrin complex, however, 
three basic ways are described herein. It may be necessary to dissolve the 
cyclodextrin and the molecules of the present composition in an aqueous or 
mixed aqueous-organic solution, followed possibly by heating; or by 
kneading, slurring or mixing the cyclodextrin and guest molecule in a 
suitable device with the addition of an appropriate quantity of aqueous, 
organic or mixed aqueous-organic liquid, optionally with heating; or by 
physically admixing the cyclodextrin and guest molecule using a suitable 
mixing device. Isolation of the inclusion complex so formed may be 
achieved by co-precipitation, filtration and drying; 
extrusion/spheronisation and drying; subdivision of tie moist mass and 
drying; spray drying; lyophilization or by other suitable techniques 
depending on the process used to form the cyclodextrin complex. A further 
optional step of mechanically grinding the isolated solid complex may be 
employed. 
These cyclodextrin/phytosterol composition complexes enhance the solubility 
and dissolution rate and increase the stability of the composition formed 
within the scope of the present invention. For a review of cyclodextrin 
complexation, please refer to 22. 
Complexation with Bile Salts 
Bile acids, their salts and conjugated derivatives, suitably formulated, 
may be used to solubilize both the oil-based and solvent-derived 
compositions of the present invention, thereby improving the solubility 
and dispersion characteristics of these compositions. Examples of suitable 
bile acids include: cholic acid, chenodeoxycholic acid, deoxycholic acid, 
dehydrocholic acid, and lithocholic acid. Examples of suitable bile salts 
include: sodium cholate, sodium deoxycholate and their other salt forms. 
Examples of suitable conjugated bile acids include: glycochenodeoxycholic 
acid, glycholic acid, taurochenodeoxycholic acid, taurocholic acid, 
taurodeoxycholic acid and their salts. 
A suitable system for solubilizing both the oil-based or solvent-derived 
compositions of the present invention consists of the composition plus one 
or more bile acids, salts or conjugated bile acids. Further materials may 
be added to produce formulations having additional solubilization 
capacity. These materials include, but are not limited to: phospholipids, 
glycolipids and monoglycerides. These ingredients may be formulated either 
in the solid phase or by the use of suitable solvents or carrier vehicles, 
with appropriate isolation and, optionally, particle size reduction using 
techniques described hereinabove. 
Since bile acids and their derivatives have an unpleasant taste and may be 
irritating to the mucous membranes of the stomach and upper regions of the 
gastro-intestinal tract, a suitable enteric coating may be applied to the 
solid formulation particulates, using techniques known to those skilled in 
the art. Typical enteric coatings include, inter alia: cellulose acetate 
phthalate, cellulose acetate trimellitiate, hydroxyproplmethylcellulose 
phthalate, hydroxyproplmethylcellulose acetate succinate, poly 
(vinylaceate phthalate), acrylate polymers and their derivatives (e.g. 
appropriate members of the Eudragit series), ethylcellulose or 
combinations thereof. Additional excipients may be added to the coating 
formulation to modify membrane functionality or to aid in the coating 
process (e.g. surfactants, plasticisers, channeling agents, permeability 
modifiers and the like). Coating formulation vehicles may comprise aqueous 
or organic systems, or mixtures of both. 
Hydrotopic Complexation 
Compounds which are capable of opening up the water structure associated 
with hydrophobic (lipophilic) and other molecules are referred to as 
hydrotopes. These compounds may be used to enhance the aqueous solubility 
of poorly water-soluble substances such as phytosterols, phytostanols and 
their esters. Examples of hydrotopes include, inter alia, sodium benzoate, 
sodium hydroxybenzoates, sodium salicylate, nicotinamide, sodium 
nicotinate, sodium gentisate, gentisic acid ethanolamide, sodium toluates, 
sodium aminobenzoates, sodium anthranilate, sodium butylmonoglycolsulfate, 
resorcinol and the like. 
Complex formation, which is non-covalent in nature, may be achieved by 
mixing appropriate ratios of the solvent-derived composition and the 
hydrotope or mixtures thereof in a suitable liquid vehicle, which may be 
aqueous, organic or a combination of both. Additional excipients such as 
surfactants, polyol, disaccharides etc.. may be added to facilitate 
complexation or to aid in dispersability. The resultant complex is 
isolated as a dry powder by any process known in the art (co-precipitation 
and drying, evaporation of the liquid vehicle, spray drying, 
lyophilization etc..). Particle size may be reduced by any standard 
technique such as those described previously herein, if desired. The 
resultant hydrotope complex may be used without further modification or 
may be compounded into a variety of other formulations or vehicles as 
required. 
Methods of Use 
The composition of the present invention, either in oil-based or 
solvent-derived form, and whether treated to enhance 
solubility/dispersability or not may be used as an effective agent to 
lower serum cholesterol in animals, particularly humans. It is to be 
understood, however, that this composition is equally suited for 
administration to other animals, for example, in the form of veterinary 
medicines and animal foods. 
1) Pharmaceutical Dosage Forms 
It is contemplated within the scope of the present invention that the 
composition of the present invention may be incorporated into various 
conventional pharmaceutical preparations and dosage forms such as tablets 
(plain and coated) for use orally, bucally or lingually, capsules (hard 
and soft, gelatin, with or without additional coatings) powders, granules 
(including effervescent granules), pellets, microparticulates, solutions 
(such as micellar, syrups, elixirs and drops), lozenges, pastilles, 
ampuls, emulsions, microemulsions, ointments, creams, suppositories, gels, 
and transdermal patches, modified release dosage forms together with 
customary excipients and/or diluents and stabilizers. 
The composition of tie present invention, adapted into the appropriate 
dosage form as described above may be administered to animals, including 
humans, orally, by injection (intra-venously, subcutaneously, 
intra-peritoneally, intra-dermally or intramuscularly), topically or in 
other ways. Although the precise mechanism of action is unclear, the 
composition of the present invention, administered intra-venously, lowers 
serum cholesterol. It is believed that the phytosterol composition may 
have, in addition to the role as an inhibitor of cholesterol absorption in 
the intestine, a systemic effect on cholesterol homeostasis through bile 
acid synthesis, enterocycte and biliary cholesterol excretion, bile acid 
excretion and changes in enzyme kinetics and cholesterol transport between 
various compartments within the body (PCT/CA97/00474 which was published 
on Jan. 15, 1998). See also paper to Peter Jones (under publication). 
2) Foods/Beverages/Nutraceuticals 
In another form of the present invention, the composition of the present 
invention may be incorporated into foods, beverages and nutraceuticals, 
including, without limitation, the following: 
1) Dairy Products--such as cheeses, butter, milk and other dairy beverages, 
spreads and dairy mixes, ice cream and yoghurt; 
2) Fat-Based Products--such as margarines, spreads, mayonnaise, 
shortenings, cooking and frying oils and dressings; 
3) Cereal-Based Products--comprising grains (for example, bread and pastas) 
whether these goods are cooked, baked or otherwise processed; 
4) Confectionaries--such as chocolate, candies, chewing gum, desserts, 
non-dairy toppings (for example Cool Whip.TM.), sorbets, icings and other 
fillings; 
5) Beverages--whether alcoholic or non-alcoholic and including colas and 
other soft drinks, juices, dietary supplement and meal replacement drinks 
such as those sold under the trade-marks Boost.TM. and Ensure.TM.; and 
6) Miscellaneous Products--including eggs, processed foods such as soups, 
pre-prepared pasta sauces, pre-formed meals and the like. 
Either the oil-based or the solvent-derived composition of the present 
invention may be incorporated directly and without further modification 
into the food, nutraceutical or beverage by techniques such as mixing, 
infusion, injection, blending, immersion, spraying and kneading. 
Alternatively, the composition may be applied directly onto a food or into 
a beverage by the consumer prior to ingestion. These are simple and 
economical modes of delivery. 
If it is desired to enhance the solubility or dispersability of the 
composition, whether oil-based or solvent-derived, prior to incorporation 
into the food, beverage or nutraceutical, this may be achieved by any of 
the techniques described herein, without limitation. 
Without limiting the generality of the foregoing, it is to be understood 
that depending on the vehicle of delivery, one of the oil-based 
composition or the solvent-derived composition may be more suitable and 
efficient for each particular food, pharmaceutical, beverage and 
nutraceutical use. For example, the solvent-derived composition presents 
as a solid or semi-solid material which is conveniently suited for use in 
the pharmaceutical dosage forms described above. Conversely, the liquid 
oil-based system is conveniently suited for many of the food, beverage and 
nutraceutical uses described above. 
For example, in the formation of emulsions and microemulsions which may 
readily be incorporated into margarines, butter, spreads, mayonnaise, 
dressings, yoghurt and the like, the oil-based composition may be more 
appropriate. Patents covering the preparation of margarines and yellow 
spreads include: U.S. Pat. Nos.: 5,118,522; 5,536,523; 5,409,727; 
5,346,716; 5,472,728; and 5,532,020, all of which are incorporated herein 
by reference. 
Conversely, lower fat content may be achieved in many foods by the 
incorporation of the solvent-derived composition. This may be particularly 
important for foods that already have a high fat content.

EXAMPLES 
Example 1 
Esterification of Phytosterols 
A phytosterol mixture (0.60 grams) derived form a vegetable oil was placed 
in a 25 ml one neck round bottom flask, equipped with a magnetic stirring 
bar, reflux condenser and a heating bath. Acetic anhydride (10 ml) was 
added and the reaction mixture was refluxed for 3.5 hours. The mixture was 
cooled down to room temperature and was stirred at this temperature for 24 
hours. The resulting white cake-like material was filtered out, washed 
with ethyl acetate (2 ml) and dried under vacuo for 24 hours to yield 0.66 
g crude mixture. 
Example 2 
Hydrogenation of Esterified Phytosterols 
An esterified vegetable phytosterol crude mixture (0.66 g) was placed in a 
100 ml one neck dry round bottom flask with a magnetic stirring bar. Ethyl 
acetate (30 ml), acetic acid (6.0 ml) and Adam's catalyst (PtO2) were then 
added. The air atmosphere was replaced with hydrogen atmosphere and the 
reaction mixture was stirred under hydrogen (atmospheric pressure) and 
room temperature for 6 hours. The reaction mixture was then filtered to 
remove the catalyst (quantitatively) and washed with ethyl acetate (2 ml). 
The resulting solution was evaporated under reduced pressure and dried 
under vacuo for 24 hours to yield 0.51 g of hydrogenated mixture (77% 
crude yield). 
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