Sitostanol formulation with emulsifier to reduce cholesterol absorption and method for preparing and use of same

Compositions useful to reduce cholesterol absorption. The compositions may be dosed in capsule or tablet form, or by adding either in liquid or dry powder form to foods and beverages. The compositions are an aqueous based homogeneous micellar mix which is dried to provide a mixture of finely-divided plant sterol, preferably sitostanol and lecithin. The mole ratio of the plant sterol, preferably sitostanol to lecithin, should be within the range of 1:0.1 to 1:10, preferably at least 1:2.

FIELD OF THE INVENTION 
This invention relates to a composition and method for reducing cholesterol 
absorption and serum cholesterol in humans. It represents an improvement 
over my parent application. 
BACKGROUND OF THE INVENTION 
Phytosterols are plant sterols structurally similar to cholesterol that 
have been known for many years to reduce cholesterol absorption and serum 
cholesterol levels while not being absorbed themselves. Lowering of 
circulating cholesterol and low density lipoprotein cholesterol is an 
important part of a strategy to prevent and treat cardiovascular disease 
and especially coronary heart disease. Cholesterol absorption is a 
critical component of whole body cholesterol metabolism. Cholesterol 
derived from the diet and also from endogenous biliary secretion enters 
the intestine, and approximately 50% of the mixed intestinal load is 
absorbed, Bosner, M. S., Ostlund, R. E., Jr., Osofisan, O., Grosklos, J., 
Fritschle, C., Lange, L. G. 1993. The failure to absorb cholesterol 
quantitatively is therefore a key mechanism for the elimination of 
cholesterol from the body. 
Drugs commonly used to treat high cholesterol levels have little or no 
effect on cholesterol absorption. For example, the potent new 
hydroxymethylglutaryl coenzyme A reductase inhibitors have a primary 
action to reduce cholesterol synthesis rather than increase cholesterol 
elimination. Bile acid sequestrants such as the ion-exchange resin 
cholestyramine act within the intestine but do not bind cholesterol and 
may actually increase cholesterol absorption when given chronically. 
McNamara, D. J., N. O. Davidson, P. Samuel, and E. H. Ahrens, Jr. 1980, 
Cholesterol absorption in man:effect of administration of clofibrate 
and/or cholestyramine. J. Lipid Res. 21:1058-1064. Although 
orally-administered neomycin reduces cholesterol absorption effectively, 
it is toxic and has the disadvantage of requiring chronic administration 
of a potent antibiotic, Samuel, P. 1979. Treatment of hypercholesterolemia 
with neomycin--A time for reappraisal. N. Engl. J. Med. 301:595-597. The 
drug Cytellin.RTM., an aqueous suspension of mixed phytosterols, was 
produced by Eli Lilly Co. for treatment of elevated cholesterol, but it 
has not been sold since 1985. As seen, it is apparent that new inhibitors 
of cholesterol absorption would complement currently-available treatment 
for high serum cholesterol. 
Since phytosterols are natural products which are non-toxic and inexpensive 
byproducts of food processing, they may be important in the treatment of 
individuals with mildly-increased serum cholesterol, or for the general 
population in food products or dietary supplements. The use of 
phytosterols could reduce the need for systemically-absorbed drugs. 
Despite their potential attractiveness, the usefulness of phytosterols has 
been limited by small and erratic effectiveness and a large dosage 
requirement. For example, doses of 5-18 g sitosterol/day reduced serum 
cholesterol by 16-20%. Farquhar, J. W. and M. Sokolow, 1958. A 
dose-response study showed that 3-9 g/day of powdered sitosterol was 
needed to decrease serum cholesterol levels by 12%. Lees, A. M., H. Y. I. 
Mok, R. S. Lees, M. A. McCluskey, and S. M. Grundy. 1977. Plant sterols as 
cholesterol-lowering agents:clinical trials in patients with 
hypercholesterolemia and studies of sterol balance, Atherosclerosis 
28:325-338. To reduce the amount needed, recent experiments have used 
sitostanol instead of sitosterol because it appears to be more potent than 
other phytosterols and is non-absorbable, Sugano, J., H. Morioka, and I. 
Ikeda. (1977) A comparison of hypocholesterolemic activity of 
.beta.-sitosterol and .beta.-sitostanol in rats. J. Nutr. 107:2011-2019. 
In subjects with severe hypercholesterolemia sitostanol at 1.5 g/day 
reduced serum cholesterol by 15%, Heinemann, T., O. Leiss, and K. von 
Bergmann (1986) Effect of low-dose sitostanol on serum cholesterol in 
patients with hypercholesterolemia. Atherosclerosis 61:219-223. However, 
sitostanol at 3 g/day had no effect in subjects with moderate 
hypercholesterolemia. Denke, M. A. (1995), Lack of efficacy of low-dose 
sitostanol therapy as an adjunct to a cholesterol-lowering diet in men 
with moderate hypercholesterolemia. Am. J. Clin. Nutr. 61:392-396. 
Several investigators have proposed ways to increase the solubility or 
bioavailability of phytosterols in order to make them more useful. Based 
on studies in rats and the finding that phytosterol esters are much more 
soluble in oil than the free sterols, it has been proposed to use 
phytosterol esters in oil to lower cholesterol absorption, Mattson, F. H., 
R. A. Volpenhein, and B. A. Erickson (1977), Effect of plant sterol esters 
on the absorption of dietary cholesterol. J. Nutr. 107:1139-1146. U.S. 
Pat. No. 5,502,045 describes the use of sitostanol ester in oil for the 
treatment of hypercholesterolemia in humans. It was found that 2.8 g 
sitostanol/day given as sitostanol ester in margarine reduced LDL 
cholesterol by 16%. Miettinen, T. A., P. Puska, H. Gylling, H. Vanhanen, 
and E. Vartiainen (1995), Reduction of serum cholesterol with 
sitostanol-ester margarine in a mildly hypercholesterolemic population. N. 
EnglandJ. Med. 333:1308-1312. However, the use of sitostanol ester 
dissolved in dietary fat has the disadvantage of requiring the 
administration of 23-50 g/day of dietary fat and of being 21% less 
effective at reducing cholesterol absorption in humans compared to the 
unesterified sterol. Mattson, F. H., S. M. Grundy, and J. R. Crouse, 
(1982), Optimizing the effect of plant sterols on cholesterol absorption 
in man. Am. J. Clin. Nutr. 35:697-700. 
Additional workers have investigated ways to improve the usefulness of 
unesterified phytosterols. In International patent Publication WO 95/00158 
a complex of sitosterol and the unabsorbable dietary fiber pectin reduced 
serum cholesterol by 16.4% when given to hypercholesterolemic humans in a 
dose of 2.1 g/day. However, no measurements of an effect on cholesterol 
absorption were made, and the complex was only about 50% soluble even at 
strongly alkaline pH, suggesting that the bioavailability of the 
sitosterol component was limited. 
U.S. Pat. No. 5,244,887 describes the use of stanols including sitostanol 
in food additives to reduce cholesterol absorption. In U.S. Pat. No. 
5,244,887, for preparation of the additives, sitostanol is dissolved with 
an edible solubilizing agent such as triglyceride, an antioxidant such as 
tocopherol, and a dispersant such as lecithin, polysorbate 80, or sodium 
lauryl sulfate. However, no data were given to guide one in the selection 
of the most effective components and their amounts or specific methods of 
preparation. Effectiveness in reducing cholesterol absorption was also not 
determined. The preferred embodiment consisted of 25% by weight stanols in 
vegetable oil, but the solubility of sterols in oil is only 2%. 
U.S. Pat. No. 5,118,671 describes the production of sitosterol-lecithin 
complexes for pharmaceutical use but does not consider oral use for 
cholesterol lowering. 
Cholesterol is absorbed from an intestinal micellar phase containing bile 
salts and phospholipids which is in equilibrium with an oil phase inside 
the intestine. Delivery of phytosterol as a solid powder or aqueous 
suspension is not preferred because of the limited rate and extent of 
solubility in intestinal liquid phases. Esterification of the phytosterol 
with delivery through the oil phase of foods is an alternative route but 
has the disadvantage of use of edible oils as the carrier. 
Accordingly, it is an object of the present invention to provide a delivery 
system for plant sterols, particularly sitostanol, which avoids an oil 
phase and which provides bioavailable sitostanol at a level which reduces 
cholesterol absorption as much as 37%, while at the same time using an 
excellent taste emulsifier in as low amounts as possible. 
Another objective of the present invention is to provide a water soluble 
composition which provides the sitostanol, not dissolved in fat, but 
rather combined with a preferred emulsifier (Sodium Stearoyl 
2-lactylate)(SSL) in an aqueous vesicular complex which can enter directly 
into the intestinal micellar phase and is therefore highly bioavailable. 
Another objective of the present invention is to provide a composition of 
preferred enhanced solubility that contains a plant sterol, preferably 
sitostanol mixed with an emulsifier even better than phospholipids, namely 
SSL, which has water solubility in excess of 90%. 
Another objective of the present invention is to provide a method for 
reducing cholesterol absorption from food products containing cholesterol 
by mixing finely divided water soluble powder of an aqueous homogeneous 
micellar mix of sitostanol and SSL with a food product which is to be 
ingested. 
A yet further objective of the present invention is to provide a method of 
manufacturing a dry, finely divided water soluble powder which contains a 
plant sterol, preferably sitostanol, and lecithin, which is highly water 
soluble, so that when in contact with an aqueous system it will provide an 
aqueous vesicular complex which can enter directly into the intestinal 
micellar phase to inhibit cholesterol absorption. 
The method and manner of achieving each of the above objectives, as well as 
others, will become apparent from the detailed description of the 
invention which follows hereinafter. 
SUMMARY OF THE INVENTION 
A composition for inhibiting cholesterol absorption from the intestine is 
described. The composition comprises phytosterols, preferably sitostanol, 
dispersed in an aqueous base emulsifier, preferably SSL. The mole ratio of 
sterol to emulsifier should be 1:0.1 to 1:10, preferably 1:0.9 to 1:0.5. 
The phytosterol-emulsifier complex is prepared by high shear mixing, for 
example by vortexing, mixing, sonicating or passing through a small 
orifice of a phytosterol:emulsifier mixture in water. The dispersed 
material is then either used as is or dried, for example, by 
lyophilization or spray-drying. The complex can be used in liquid form 
prior to any drying, or it can be dried as indicated, and then on contact 
with liquid it again forms an aqueous vesicular complex which can enter 
directly into the intestinal micellar phase. No fat is used as a carrier, 
and surprisingly the system, even when dried, does not change its physical 
structure from the micelles that contain vesicles, the majority of which 
contain some plant sterol and some lecithin. 
DETAILED DESCRIPTION OF THE INVENTION 
As previously mentioned, the current invention differs from prior art uses 
of plant sterols and sitostanol in many significant ways. 
First, the dose needed to reduce cholesterol absorption is lower than 
previously reported, namely 25-300 mg of sitostanol. Second, the preferred 
formulation does not contain triglycerides or oils. The phytosterol is not 
dissolved in fat, but rather is combined with phospholipid to form an 
aqueous vesicular complex which can enter directly into the intestinal 
micelle phase. Third, the mix can be prepared in solid form by drying an 
aqueous sitostanol/emulsifier vesicular formulation with retention of 
solubility in artificial bile. Fourth, the mix is effective when consumed 
separately from cholesterol-containing foods. Fifth, the mix can be added 
to non-cholesterol-containing and fat-free foods and beverages. Sixth, the 
mix is prepared in a manner to prevent self-association of sitostanol as 
occurs when it is dried from organic solvents containing sitostanol and 
solubilizing agents. The mix herein referenced has the advantage of a high 
degree of bioavailability as assayed with artificial bile in vitro. This 
is significant and something that cannot be achieved with fat carrier 
systems. 
The composition is useful for reducing cholesterol absorption in humans at 
doses between 10 and 1000 mg, and a preferred dose is 100-300 mg. The dose 
is less than required by current protocols. The composition may be used in 
capsule or tablet form as a drug or dietary supplement. Alternatively, it 
may be used in foods as a food additive or substance generally recognized 
as safe for human consumption. 
In preparation of the composition useful for reducing cholesterol in highly 
bioavailable form, the first step is to provide an aqueous homogeneous 
micellar mix of the plant sterol with the preferred emulsifier of choice. 
The preferred method is to use sitostanol because only small amounts are 
absorbed in the small intestine, but on the other hand, this plant sterol 
shows high inhibition of cholesterol absorption. Similar compounds are 
also suitable, including sitosterol, campesterol, campestanol, 
stigmasterol. Moreover, lignans, such as sesamin, and saponins are also 
useful for this purpose, but sitostanol is preferred. 
The preferred phospholipid of my parent case was lecithin, and the most 
preferred phospholipid system useful to enhance the bioavailability was a 
mix of lecithin and lysolecithin. Where the mix was used, it was preferred 
that the mole ratio of lecithin to lysolecithin be at least 1:0.2, 
preferably 1:0.5. It has now been found that SSL provides even better 
emulsifier results. 
In this first step, the aqueous homogeneous mixture of the plant sterol and 
the emulsifier are homogeneously mixed to provide a micellar mix. The 
preferred mixing form is a high shear mixing. By way of example, 
vortexing, sonicating, passing through a small orifice such as a French 
press or other mixing means may be employed. The most preferred mixing is 
sonication. This disperses the material and enhances the formation of a 
micellar mix that contains vesicles, the majority of which contain some 
plant sterol and some emulsifier. 
Generally, with respect to sonication, any method that is commonly used for 
preparation of emulsions can be used to prepare homogeneous mixtures of 
the plant sterol and the emulsifier, either alone or in combination. For 
example, Waring blenders, or other high shear mixers can provide 
acceptable results. Microfluidizers can be used. In this latter procedure, 
the plant sterol and the emulsifier are forced through ceramic capillaries 
under high pressure. Where the preferred sonication technique is used, a 
time within the range of 1.5 minutes to about 4 minutes for sonication is 
sufficient. On small scale experiments, sonication is typically performed 
in about 1.5 minutes. 
The drying process is not critical, so long as it does not destroy the 
vesicular complex formed between the plant sterol and the emulsifier. 
Generally, nondrastic drying procedures are preferred such as vacuum 
drying, freeze drying or low-temperature ambient air drying. Where heat is 
employed, the temperature at atmospheric conditions should not exceed 
0.degree. C. 
As earlier explained, the dosage of the dry powder may be within the range 
of 10 to 1000 mg per day, and a preferred dose being 25 to 300 mg per day. 
The most preferred doses to achieve significant cholesterol absorption 
reduction levels are achieved at a dose range of from 100 mg to 300 mg one 
to four times daily.

The following examples are offered to further illustrate, but not limit the 
process of the present invention. 
In these first five examples sitostanol is used as an example of a 
phytosterol and lecithin of a phospholipid as per my parent application. 
In Example 6 SSL is used and the improvement can be seen. 
Phytosterols, as used here, mean sterols such as sitostanol, sitosterol, 
campesterol, stigmasterol, saponins, lignans, aromatic and isoprenoid 
natural products, and their derivatives and reduction products. 
Phospholipids, as used here, means glycerophospholipids and sphingolipids, 
as well as their derivatives, such as lysophospholipids. Sodium 
stearoyl-2-lactylate (SSL) means this compound or its chemical equivalent 
reaction products of an alkali or alkaline earth metals and fatty acids 
and lactic acid. 
EXAMPLE 1 
Sitostanol, tracer amount of [.sup.3 H]sitostanol, and other compounds that 
are found in the gut or that are commonly used as food additives were 
mixed together in chloroform solution at a fixed mole ratio. An aliquot, 
containing 1.2 .mu.Mol of sitostanol, was transferred to an evacuation 
tube and the solvent was removed under reduced pressure (&lt;50 mtorr). The 
experiment was initiated by adding 0.5 mL of artificial bile (8 mM sodium 
taurocholate containing 5 mM soy lecithin and 0.15 mM NaCl, pH 7.4) 
followed by rotation at 8 rev/min for 30 min at 37.degree. C. The tube was 
then centrifuged for 1 minute at 17,000.times.g to precipitate any solid 
material, the supernatant was removed and added to scintillation fluid for 
measurement of radioactivity, and the percent of radioactivity in the 
artificial bile supernatant was calculated. Table I below summarizes the 
solubility of sitostanol mixtures in the presence of artificial bile salt. 
TABLE 1 
______________________________________ 
Composition of Sterol Mixture Dried from Chloroform 
Soluble Sterol, % 
______________________________________ 
Sitostanol Alone 2.3 
Sitostanol + Tween-20 1:1 by weight 7.8 
Sitostanol + Taurocholate 1:1 by weight 57.7 
Sitostanol + Monoolein + Diolein 1:1:1 by weight 14.5 
Sitostanol + Lecithin 1:1 by weight 38.2 
Sitostanol + Lysolecithin 1:1 by weight 8.0 
Sitostanol + Lecithin + Lysolecithin 1:1:0.5 by weight 97.9 
______________________________________ 
As shown in line 1 of Table 1, sitostanol alone is poorly soluble in 
artificial bile salt (2.3%), and the addition of Tween-20, a polysorbate 
emulsifier used in foods, increases the solubility slightly to 7.8% (line 
2). Sitostanol solubility can be enhanced 25-fold, from 2.3% to 57.7%, if 
it is dried in the presence of an ionic detergent, such as the bile salt 
sodium taurocholate (line 3). Since bile salt is a component of the 
digestive process, other compounds that are found in the gastrointestinal 
system were also tested. Monoolein and diolein are the products of dietary 
fat digestion, but as shown in line 4, they only produced a modest 
enhancement of solubility, 2.3% to 14.5%. Bile contains lecithin, and this 
phospholipid increased sitostanol solubility from 2.3% to 38.2% (line 5). 
However, the reaction product of phospholipase A.sub.2 hydrolysis of 
lecithin, lysolecithin, produced a slight increase in sitostanol 
solubility, 2.3% to 8.0% (line 6). Surprisingly, when lecithin and 
lysolecithin were mixed together with sitostanol, the resulting solid 
mixture produced almost complete solubility of the sterol, 97.9% (line 7). 
Taken together, these data indicate that solid sitostanol does not readily 
dissolve in artificial bile, but that it can be made soluble to a varying 
degree by including other compounds in solid mixture. Moreover, a compound 
(lysolecithin) that by itself has little effect on sitostanol solubility 
can have a marked outcome when it is used in combination with other agents 
(lecithin). 
EXAMPLE 2 
Sitostanol, tracer amount of [.sup.3 H]sitostanol and lecithin, were mixed 
together in chloroform. Two aliquots containing 1.2 .mu.Mol of sitostanol 
were removed and the chloroform solvent was removed under vacuum as 
described in Example 1. One aliquot was used without further preparation 
and to the other 500 .mu.l water was added and the sample mixture was 
sonicated for 5 minutes on 40% power with a Fisher Sonic Dismembrator 
Model 300 equipped with a microtip. The sample was then frozen with dry 
ice acetone and lyophilized to remove water. It is essential to maintain 
the temperature of the sample below freezing in order to prevent 
precipitation of sitostanol from the mixture. The solubility of each of 
these samples in artificial bile was then determined as described in 
Example 1, and the results are shown in the Table below. 
TABLE 2 
______________________________________ 
Sample Drying Method Soluble Sterol, % 
______________________________________ 
Sitostanol dried from chloroform 
2.3 
Sitostanol/Lecithin (1:1 mole ratio) dried from 38.2 
chloroform 
Sitostanol/Lecithin (1:1 mole ratio) sonicated in water, 89.7 
lyophilized 
______________________________________ 
The data show the importance of lecithin in solubilizing sitostanol. 
However, the method of drying the sitostanol/lecithin mixture also affects 
the subsequent dissolution of the sterol. When the mixture is dried from 
chloroform, 38.2% of the sterol is solubilized by artificial bile. In 
contrast, when the mixture is sonicated and then lyophilized, 
solubilization increases to 89.7%. This shows that dispersing 
sitostanol/lecithin in aqueous medium followed by removal of water is a 
preferred method for preparing sitostanol/lecithin mixtures. 
EXAMPLE 3 
The effectiveness of variable amounts of lecithin to solubilize sitostanol 
was studied as in Example #1, except that after rotation at 37.degree. for 
30 min residual sedimenting sitostanol was re-extracted twice by vortexing 
with 0.5 ml additional artificial bile and recentrifuging. The following 
results were obtained: 
TABLE 3 
______________________________________ 
Sitostanol:Lecithin Mole Ratio 
Soluble Sterol, % 
______________________________________ 
1:1 53.1 
1:2 67.9 
1:10 67.6 
______________________________________ 
These data show that even with repeated extraction and addition of a 
tenfold excess of lecithin, a significant amount of sitostanol (32%) 
remained insoluble. When [.sup.3 H]phosphatidylcholine was added as a 
tracer instead of [.sup.3 H]sitostanol, the amount of lecithin solubilized 
was 93.3%. This indicates that lecithin was nearly quantitatively 
extracted from the dried sitostanol/lecithin complex, whereas a limiting 
amount of sitostanol remained. Thus, methods to solubilize sitostanol in 
artificial bile must take into consideration the existence of residual 
insoluble sitostanol. Drying sitostanol/lecithin mixtures from a more 
polar solvent such as ethanol or a less polar solvent such as hexane gave 
similar results. 
EXAMPLE 4 
The effect of sonicated sitostanol/lecithin vesicles on human cholesterol 
absorption was compared to that of solid sitostanol dosed in the presence 
of sonicated lecithin. Sitostanol was dehydrated by twice dissolving in 
chloroform and evaporating, and was then ground to a powder in a mortar 
and pestle. To prepare the sitostanol/lecithin vesicles in a 1:3 mole 
ratio, 2.00 gm of sterol was added to 11.3 gm of purified soy lecithin in 
a 150 mL glass beaker. Chloroform was added with stirring to solubilize 
both components, and the solvent was then removed by incubating in a sand 
bath at 65.degree. C. Soy lecithin (11.3 g) without sitostanol was 
prepared in the same manner. When all the solvent was removed, the beakers 
were placed in a lyophilization jar, and the residual chloroform was 
removed under vacuum for at least 24 hr. The solid in each beaker was then 
broken up with a spatula, 120 mL of deionized water was added, and the 
suspension was stirred vigorously for one hour. Vesicles were prepared by 
sonicating the contents of each beaker with a Branson Sonifier (setting 7) 
equipped with a small tip. During sonication, the beaker was immersed in a 
room temperature water bath. Vesicles containing lecithin alone were 
formed in 15-30 min, but those containing both sterol and lecithin 
required 30-45 min. The samples were then centrifuged at 10,000.times.g 
for 10 min and passed through a 5 g filter. The mean diameter of the 
vesicles determined on a Zetasizer that had been calibrated with a 250 nm 
standard was 204.7 nm for lecithin vesicles and 247.2 nm for the 
sitostanol/lecithin vesicles. The concentration of sitostanol was measured 
enzymatically. After preparation and characterization, the vesicles were 
stored overnight in a refrigerator at 4.degree. C. The next day samples 
were diluted to 60 ml with water and 500 mg lemon flavored Crystal Light 
(Kraft Foods, Inc. ) was added. Three U.S.P. stomach capsules were filled 
with a total of 1 g sitostanol powder or 1 g glucose placebo for each 
subject. 
Six normal subjects underwent three cholesterol absorption tests in random 
order separated by 2 weeks. For each test a National Cholesterol Education 
Program Step 1 diet was consumed for 8 days beginning on day 1 of the 
study. On day 4, a standardized test breakfast was consumed consisting of 
240 mL orange juice, 240 mL whole milk, 21 gm corn flakes and a 60 gm 
bagel saturated with 40 mg [26,26,26,27,27,27-.sup.2 H.sub.6 ] cholesterol 
tracer dissolved in 2.5 mL corn oil. Each subject also consumed a drink 
containing either sitostanol/lecithin vesicles or lecithin vesicles and 
three capsules containing either sitostanol powder or glucose placebo. The 
concentration of deuterated cholesterol tracer in plasma cholesterol on 
days 7 and 8 was measured by negative ion methane chemical ionization gas 
chromatography/mass spectrometry. Reduction in cholesterol absorption was 
determined by dividing the mean deuterated cholesterol concentration on 
days 7 and 8 by that observed during the test that contained only lecithin 
vesicles and glucose capsules and expressing it as a percent. The 
following results were obtained: 
TABLE 4 
______________________________________ 
Treatment Given Reduction in Cholesterol Absorption 
______________________________________ 
1000 mg sitostanol powder 
11.3 .+-. 7.4% (p = 0.2) 
700 mg sitostanol/lecithin vesicles 36.7 .+-. 4.2% (p = 0.003) 
______________________________________ 
These results show that, compared to placebo, 1000 mg sitostanol powder did 
not reduce cholesterol absorption significantly. This is consistent with 
previous reports showing that only multi-gram sitostanol doses reduce 
cholesterol absorption. However, 700 mg sitostanol/lecithin vesicles 
reduced cholesterol absorption by 37%, showing that properly formulated 
sitostanol is active and bioavailable. 
EXAMPLE 5 
To demonstrate that sitostanol/lecithin reduces cholesterol absorption in a 
pharmacological dose-response fashion, it was given in reduced amount to 5 
of the 6 subjects of Example 4 during four additional cholesterol 
absorption tests. A dose of 300 mg sitostanol in sitostanol/lecithin 
vesicles was compared to lecithin placebo, and a dose of 150 mg sitostanol 
in sitostanol/lecithin vesicles was compared to another lecithin placebo. 
No capsules of solid sitostanol or placebo were given. The following 
results were obtained: 
TABLE 5 
______________________________________ 
Reduction in Cholesterol 
Treatment Given Absorption 
______________________________________ 
300 mg Sitostanol/Lecithin Vesicles 
34.4 .+-. 5.8% (p = 0.01) 
95 mg sitostanol/Lecithin Vesicles 5.6 .+-. 7.2% (not significant) 
______________________________________ 
Cholesterol absorption was reduced nearly as much by the 300 mg dose as the 
700 mg dose, indicating that this dose is saturating. This is consistent 
with previous work showing that phytosterols do not completely block 
cholesterol absorption. There is no significant effect on cholesterol 
absorption at a dose of 95 mg. 
EXAMPLE 6 
The Use of SSL (Sodium Stearoyl Lactylate) 
Since the parent patent application was filed in May, 1998, I have studied 
several emulsifiers used commonly in the food industry. The most useful 
for solubilizing sitostanol is SSL. SSL is potentially important because 
it is used in many foods including baked goods and has very acceptable 
taste and texture qualities when used with each food product. 
In the following experiments collectively referred to as Example 6, 
sitostanol containing trace amount of .sup.3 H-sitostanol was dried from 
chloroform with a solubilizing agent, sonicated in water at a 
concentration of 2.4 mM sitostanol, frozen and lyophilized. Solubility in 
artificial bile was determined by adding 8 mM sodium taurocholate 5 mM PC 
0.15 M NaCl 15 mM sodium phosphate pH 7.4 (artificial bile), rotating for 
30 min. at 37.degree. and centrifuging at 17,000.times.g for 1 min. The 
pellet was washed once and the combined sups and pellet were counted 
separately. The results of triplicate experiments are shown below. 
TABLE 6 
______________________________________ 
Mole 
Condition Ratio Percent Soluble 
______________________________________ 
1. Sitostanol + SSL 
1:0.90 95.0 .+-. 0.1 
2. Sitostanol + SSL 1:0.68 94.5 .+-. 0.1 
3. Sitostanol + SSL 1:0.45 92.8 .+-. 0.6 
4. Stiostanol + lecithin 1:1 89.1 .+-. 1.2 
5. Sitostanol + Precept 8160 1:1.2 95.7 .+-. 0.08 
Central Soya PC/LPC.sup.a 
6. Ethoxylated MG.sup.b 1:1 by wt 82.5 .+-. 4.0 
7. Sitostanol + Tween .RTM. 20.sup.c 1:1 by wt 51.3 .+-. 2.8 
8. Sitostanol + GMS.sup.d 1:1 Reaggregated after sonication 
9. Sitostanol + MO.sup.e 1:1 46.4 .+-. 1.2 
______________________________________ 
.sup.a PC/LPC = lecithin/lysolecithin 
.sup.b MG = monoglyceride 
.sup.c Tween .RTM. is registered trademark for polyoxyethylenesorbitan 
monolaurate 
.sup.d GMS = glyceryl monostearate 
.sup.e MO = 1monoolein 
In Table 6, trials 4-5 represent preferred compositions of my parent 
application. It can be seen that smaller amounts of SSL can be used to 
achieve comparable solubilities with an emulsifier of preferred taste. 
Thus, these experiments show that SSL is as effective as lecithin and 
lysolecithin in solubilizing sitostanol. Monoglycerides, ethoxylated 
monoglyceride, and polysorbate 20 are less effective. 
It can be seen from the above examples that the composition prepared in 
accordance with the process of this invention is bioavailable in vitro in 
bile, will significantly reduce cholesterol absorption, and that in 
general all of the objectives of the invention are achieved. 
It should be understood that certain modifications should be and will be 
apparent to those of ordinary skill in the art, and that such 
modifications to the precise procedures in compositions set forth herein 
are intended to come within the spirit and scope of the invention either 
literally or by doctrine of equivalents. In this light, the following 
claims are asserted.