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Table of Contents:

1. What are Phytosterols? A General Introduction
2. The Good and the Bad Roles of Cholesterol
3. The Effect of Dietary Phytosterols en Cholesterol
4. Animal Trials
5. Human Efficacy Studies
6. Different Forms of Phytosterols
7. Recommended Daily Intake
8. Technical Data on Cholestatin
9. References

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1. What are Phytosterols? A General Introduction

The term "phytosterols" was first used in 1897 for sterols of plant origin. Phytosterols are cholesterol analogs, which means they have similar functions but different origins. Cholesterol is found in animals and humans while phytosterols occur only in plants. Cholesterol is a very important molecule in animals and humans, serving as a vital constituent of cell membranes and a precursor to various biomolecules. Phytosterols serve similar functions in plants.

Phytosterols are found in vegetables and nuts as well as other foods and are therefore part of our modern day diet but not in the quantities necessary to exert health benefits. The current western diet is estimated to include 200-300 mg phytosterols per day, while vegetarian and Japanese diets include a larger quantity, 300 - 500 mg per day1. It is theorized that primitive man's diet included over 1000 mg per day. It is commonly accepted that approximately 1 g per day is required to have a serum cholesterol lowering effect3. Phytosterol supplementation is essentially readjusting our daily intake to approximate that which our ancestors may have experienced.

Phytosterols differ from cholesterol in small but significant ways. The structure of phytosterols includes some branches that are not present on the cholesterol molecule.

Due to these structural differences, phytosterols are not well absorbed in animals or humans. The vast majority of the phytosterols ingested remain in the gastrointestinal (GI) tract. In healthy humans, absorption is limited to approximately 5% of the total beta-sitosterol and approximately 15% of the campesterol ingested. With daily phytosterol intakes of 160 to 360 mg/day, only 0.3 to 1.7 mg/dL phytosterols are found in the blood. Plasma levels of phytosterols have been shown to as much as double with supplernentation3'.This is a small quantity compared with 240 mg/dL greater, cholesterol levels that will be present in hypercholesterolemia. Phytosterols are eliminated from the body more rapidly than cholesterol via the biliary route.

The majority of phytosterols available today are derived from vegetable oil processing. Crops primarily processed for vegetable oil like canola, cottonseed, corn and soybean, are major sources of phytosterols. An alternative commercial source is tall oil, a by-product of paper mills.

2. The Good and the Bad Roles

Cholesterol is essential for life in animals and humans. A correct ratio of cholesterol to phospholipids is crucial for cell membrane fluidity. Cholesterol is also important due to its role in the synthesis of hormones, bile acid, and vitamins. Various active biomolecules are made in the body using cholesterol as a raw material. Cortisol, testosterone, estradiol and progesterone are examples of steroids made from cholesterol.

Unfortunately, cholesterol can also work against us. Stroke, heart attack, hardening of the arteries and even a form of gall stones are attributed to an excess of cholesterol.

Cholesterol is implicated in the deposition of plaque in arteries. Nutritional deficiencies and sudden intense stress in which the brood vessels don't have time to dilate are examples of conditions that can result in small tears in the lumen of the artery. A possible mechanism for the progression of atherosclerosis is that the body's natural response to these small tears is to release collagen into the blood stream to repair the tear. White blood cells, with oxidized cholesterol attached are also incorporated into the repair, causing the build up of what is called soft plaque. The presence of soft plaque results in a characteristic "fatty streak" which runs along the interior of an artery. This fatty streak is composed of smooth muscle cells filled with cholesterol and macrophages, which are trying to remove the cholesterol.

The next stage of atherosclerosis, fibrous plaque, involves the addition of proteins and other substances to the fatty streak. As the deposition increases in size, it begins to project into the artery. When occlusive atherosclerotic plaque is present, patients can experience effort angina claudication in which they experience pain or exhaustion during what should be moderate exertion.

Atherosclerotic plaque can rupture and cause clotting which, depending on the location of the rupture, may cause myocardial infarction or heart attack, coronary death, stroke, unstable angina, or critical leg ischemia.

Cholesterol does not just float freely through our blood stream. It is transported by lipoproteins. There are different lipoproteins, classified by density and they have different functions (Figure 3).Very Low Density Lipoprotein (VLDC) is a precursor to Low Density Lipoprotein (LDL). LDL is responsible for transporting cholesterol to the various tissues within the body. High Density Lipoprotein (HDL) is involved in the reverse transport of cholesterol, that is, it carries excess cholesterol from tissues within the body, including veins and arteries, back to the liver where it can be disposed of.

The primary villain in atherosclerosis is LDL cholesterol because it is believed to be more likely deposited on the walls of arteries. LDL cholesterol is harmful after being attacked by free radicals, homocysteine or other unstable molecules. The quantity of oxidized cholesterol in plaque is an indication of the vulnerability of that plaque to rupture.

HDL cholesterol is considered "good" cholesterol. Low levels of HDL are directly related to increased incidence of heart disease. Reverse cholesterol transport describes the function of HDL cholesterol. This is the removal of cholesterol from tissues other than liver cells and the delivery of the cholesterol to the liver and other tissues that can metabolize the cholesterol7.

One predictor .of coronary heart disease (CHD) in women, according to one study", was the ratio of total cholesterol to HDL. If a woman's total cholesterol is about four times or so of her HDL level, her risk of heart disease skyrockets to up to five times that of her normal counterpart.

Guidelines for acceptable levels of these forms of cholesterol are presented in Table1

3. The Effect of Dietary Phytosterols On Cholesterol

As early as the 1930's it was shown that serum cholesterol levels could be effected by phytosterol supplementation work in the 1950's confirmed the ability and numerous efficacy studies since, in animals and humans, have proven that phytosterols are highly effective in lowering cholesterol levels.

Cholesterol in the GI tract can be of two sources; dietary which is in the foods we eat and endogenous which is manufactured by our bodies, Both are excreted into the intestines from the gall bladder with bile after synthesis or absorption, Without intervention, most of this cholesterol will be reabsorbed in the intestines. For cholesterol to be absorbed, it must be incorporated into micelles and transported to the inner lining of the GI tract.

Phytosterols are poorly absorbed so the bulk of ingested phytosterols remain in the GI tract. Phytosterols decrease the transport of cholesterol to absorption sites by excluding it from the transport mechanism. There are two proposed mechanisms by which phytosterols decrease serum cholesterol levels. Both mechanisms are based on the similar physical and chemical properties of phytosterols and cholesterol.

One mechanism is believed to work through precipitation or crystallization of cholesterol and phytosterols. As the concentration of cholesterol and/or phytosterols increases in the intestine, both become less well dissolved and begin to drop out of solution in crystalline form. Crystalline cholesterol cannot be absorbed. Since cholesterol is the only one absorbed to any great extent, its absorption is dramatically reduced.

In the second proposed mechanism the phytosterols are believed to compete with the cholesterol for transportation to the wall of the intestine, the brush border, where absorption occurs2. Because cholesterol is less soluble in water than oil, it has to be transported to the site of absorption in micelles. Micelles are formed from bile salts, phospholipids, tri, di- and mono- glycerides, fatty acids, free cholesterol and fat soluble micronutrients. Phytosterols are even less soluble in water than cholesterol so they have a stronger affinity for the micelles. This “competition" for the micelles prevents the cholesterol from reaching the absorption site.

4. Animal Efficacy Studies

More than 50 years of research has proven that phytosterols are highly effective in lowering cholesterols levels hi different animals.

A 2003 study by Wang et al. investigated the effects The phytosterol group was fed the control diet with of Cholestatin on the lipid levels in Syrian Golden hamsters. The hamsters were fed a control diet that contained 0.25% cholesterol and 5% total fat with a contained 0.25% cholesterol and 5% total fat with a polyunsaturated to saturated fatty acids ratio of 0.4. The phytosterol group was the control diet with addition of Cholestatin (Degussa BioActives, 1,000 mg/kg). Supplementation with Cholestatin prevented a rise in plasma cholesterol levels that would be expected with a cholesterol enriched diet (Figure 4).

A statistically significant difference was found between the plasma total and HDL cholesterol levels in hamsters supplemented with phytosterols compared to those that were not. It is important to note that in the hamster model for cholesterol studies, the hamsters carry over 75% of plasma cholesterol as HDL cholesterol. While in humans a decreased HDL is not desired and does not occur as a result of phytosterol supplementation, reduction in HDL cholesterol in hamsters is a desired response to phytosterol supplementation.

Vaskonen et al., 2002, induced severe hypercholesterolemia in hypertensive rats which generally have low cholesterol by feeding a typical human diet; high fat, high cholesterol and high salt. The increase in total and LDL cholesterol could be attenuated by the addition of phytosterols and minerals, Animals supplemented with phytosterols and minerals showed an increase in total and PDL cholesterol but the increases were only 45% and 35% of that seen in animals not supplemented. Phytosterols and minerals also protected against vascular and renal damage compared to animals fed only the human type diet.

Moghadasian et al., 1999, concluded From their study with mice prone to atheriosclerotic lesions that supplementation of a cholesterol enriched diet with phytosterols lowers plasma cholesterol levels and slows progression of atheriosclerotic lesions. The phytosterols used in this study were derived from pine trees but chemically, there is no difference in the four primary phytosterols, regardless the plant from which they were extracted. While the control mice developed plasma cholesterol levels of 42 mmol/L, the phytosterol treated mice only developed plasma cholesterol levels of 26.6 mmol/L; 36.7% lower.

Male and female Golden Syrian hamsters (n=120) studied by Ntanios et al., were fed a diet with 0.25% cholesteroP. While the control group was only fed the base diet, four other groups were fed the base diet with 1) 0.5% tall oil phytosterols, 2) 1.0% tall oil phytosterols, 3) 0.5% soybean phytosterols or 4) 1.0% soybean phytosterols. The results of this study showed that both tall oil phytosterols and soybean phytosterols were able to reduce cholesterol levels when compared to the control group. At 0.5% soybean and tall oil phytosterols decreased cholesterol levels by -30% and -1 5% respectively when males and females were considered together. At 1.0%, soybean and tall oil phytosterols decreased cholesterol levels by -34% and -31 % respectively when males and females were considered together. It is interesting to note that at the lower concentration, soybean derived phytosterols were more effective in lowering total cholesterol (Figure 5).

Both sources of phytosterols contained essentially the same amount of beta-sitosterols. Tall oil phytosterols contained approximately 20% sitostanol while soybean phytosterols contained 4% more campesterol and 15% more dihydrobrassicasterol.

In a study by Laraki et al., 1998, adult Wistar rats (n=48) were assigned to one of four groups and fed either 1) basal diet plus 12 mg/d cholesterol, 2) basal diet plus 24 mg/d cholesterol, 3) basal diet, 24 mg/d cholesterol and 24 mg/d phytosterols or 4) basal diet, 24 mg/d cholesterol and 96 mg/d phytosterols. The animals were kept on their respective diets for three weeks after which Laraki et al., found that when compared to the low cholesterol group (l),the high cholesterol group (2) had 37% higher total cholesterol and 56% higher LDL cholesterol. Having been fed the same high cholesterol diet plus 24 mg/d phytosterols, group 3 had a total cholesterol level that was 13.6% lower than group 2. Group 4, having received 96 mg/d phytosterols had total and LDL cholesterol levels 22% and 22.2% lower than group 2 respectively. In this study phytosterols were shown to effectively reduce cholesterol levels even when consumed with a diet that contained cholesterol.

Three groups of Wistar rats (n=12) were fed a diet containing 1.0% cholesterol for 18 days3? In addition to the standard cholesterol containing diet, one group of the rats was supplemented with 5.0% phytosterols. In a fashion similar to previous studies, Hirai et al, 1984, found that the total cholesterol in the phytosterol supplemented group was greatly reduced (-68.4%) from the total cholesterol levels found in the non-supplemented animals. Reductions in LDL and VLDL were also associated with phytosterol supplementation.

Sugano et at., 1982, showed that dietary supplementation for 35 days with beta-sitosterol was shown to greatly reduce the amount of cholesterol in the livers of male Wistar rats fed a diet including 10% butter fat. Levels of 0.1%, 0.5% and 2.0% sitosterol resulted in decreases of 1 7.5%,23.3 % and 3 1.5 % respective1 y. In the same study it was shown that JAC ddy mice could have a 54% decrease in liver cholesterol when fed a similar diet with 0.5% sitosterol.

In a study by Chandler et al., 1979, to examine the effect of beta-sitosterol, male white leghorn chicks were given either a base diet or base diet with T.O% cholesterol for 14 days with 1.0% beta-sitosterol". Results showed a significantly lowered liver and plasma cholesterol in the subjects supplemented with beta-sitosterol and cholesterol. The lowest levels were found in those subjects given both cholesterol and beta-sitosterol. This "synergistic" effect is due to the down regulatory effect that dietary cholesterol has on cholesterol synthesis.

A '1969 shady by Bartov et al., fed a base diet, a base diet plus cholesterol, or base diet plus cholesterol and phytosterols to chicks for a 14 day period-The results showed that phytosterol supplementation could prevent a rise in plasma (1 38 mg/dL v.s.413 mg/dL ) and liver cholesterol levels (4.4 mg/g v.s.32.7 mg/g) that would result from cholesterol supplementation. With 3% phytosterol supplementation, total cholesterol was 66% lower and liver cholesterol was 86% less than the cholesterol only group.

Best and Duncan, 1958, fed male Holtzman rats a base diet of mouse mash, cottonseed oil and cholesterol. Groups of rats on this diet were supplemented with free sitosterol and sitosterol esters. Both the free sitosterol and sitosterol esters decreased liver cholesterol levels. While the sitosterol esters did decrease liver cholesterol levels, the palmitate and proprionate esters were not as effective as the free sitosterol. The authors postulated that this was due to the rate of hydrolysis of the esters.

Peterson et al., 1954, performed a six week feeding study using 3 week old chicks to examine the effects of phytosterols on a diet containing cottonseed oil and free and esterified cholesterol. Cholic acid supplementation dramatically increased plasma cholesterol levels when administered in the absence of phytostero1s. Phytosterols were able to counter-act the effect of the cholic acid and when administered in the absence of cholic acid, phytosterols decreased plasma and liver cholesterol levels via interference with cholesterol absorption as shown by feeding radiolabeled cholesterol.

In a study utilizing male rabbits, Pollack, 1953 conducted a 14 day study of the effect of phytosterols on cholesterol absorption. Rabbits fed base diet plus cholesterol exhibited elevated blood cholesterol levels and 90% of these rabbits had evidence of atherosclerosis at the end of the study. Rabbits fed various levels of mixed phytosterols but no cholesterol, showed no change in blood cholesterol levels over controls however, this group also showed no evidence of atherosclerosis. Groups fed cholesterol and phytosterols (3,5,6,o r 7 g/d) showed no evidence of cholesterol induced atherosclerosis.

Peterson, et al, 1952, dosed groups of 44 chicks with cholesterol (Cho) or cholesterol and phytosterols (Cho&PS). Figure 6 shows that the cholesterol levels in chicks dosed with the combination had significantly lower cholesterol levels. Levels in the, Cho group were seven times higher than controls while the Cho & PS levels were only twice as high as the control group.

In addition to the decreased cholesterol absorption atherosclerotic lesions, while lowest in the control group were significantly lower in the Cho & PS group than in the group dosed with cholesterols alone (Figure 7).This is true for lesions formed in the thoracic And abdominal aortas. More recent research has reinforced this down regulation of atherosclerotic Lesions using mice. In mice treated with phytosterols , The aortic lesion area was less than half that found In the non phytosterol treated mice. Additionally, a substantial reduction in all the lesional components Was observed, indicating a slowing of atherosclerotic Progression in those animals treated with phytosterols.

This takes on additional significance in light of information that indicates that coronary atherosclerosis begins at a young age in humans and a number of Teenagers and young adults may have the disease but have no symptoms.

5. Human Efficacy Studies

Phytosterols have been investigated in pilot studies numerous gold standard (double-blind, placebo- controlled) human clinical trials. Phytosterols were administered in functional foods (e.g.spreads, chocolate) or as dietary supplements (capsule, chewable tablets).

Phytosterols were shown to reduce serum cholesterol in a 2002 study performed by Vanstone et.al. In randomized , cross over double blind study, fifteen subjects were fed 1.8g per day with a typical North African diet. Control subjects were fed the typical North African diet alone. With phytosterol supplementation, total plasma control levels decreased by 7.8% (P<O.OT) while LDL decreased by 1 1.3% {P<0.03).

Phytosterols were shown to reduce serum cholesterol in a 1999 study conducted by Jones et.al. Sixteen hypercholesterolemic male subjects were fed 1.7 g per day phytosterols in conjunction with a precisely controlled metabolic diet. Sixteen control subjects were fed the controlled diet alone. Significant cholesterol reductions were seen in both the control and phytosterol groups however the phytosterol group showed a significantly larger (P<0.001) decrease. The greater reduction in LDL due to phytosterols also caused an improved LDL/HDL ratio in those supplemented with phytosterols (Figure 8).

Pelletier, et.aI!4 dosed twelve patients with 740 mg phytosterols pea day for 28 days (Figure 9). A significant reduction was seen in total cholesterol (pc.001) and LDL cholesterol (p<0.001), A significant increase in HDL cholesterol (pe.001) was also observed.

In a 1993 study, Becker et al., dosed a group of children with familial hyper-cholesterolemia with a sterol pastil (a lozenge) providing 2 g sitosterol per day, LDL-cholesterol had been reduced by 19.5% at 3 months.

Lees, et al, 1977, studied the effect of soybean and tall oil derived phytosterols on cholesterol levels in humans. The results of the effort included the finding that the efficiency of plant sterols can vary greatly from patient to patient, Soybean sterols as a suspension or powder at 18 g per day decreased serum cholesterol levels by 12%.Tall oil sterol powder was slightly more effective than tall oil sterol suspension decreasing Total Cholesterol by 12% and 7% respectively.

A study by Kudchodkar et al, in 1976 showed that phytosterol administration decreased plasma cholesterol levels on average by 9%. Excretion of cholesterol increased 34% to 102% indicating that the absorption of cholesterol in the GI tract was reduced.

Earlier work on phytosterol efficacy that by Beveridge al., 1964, indicated that statically significant results could be achieved by phytosterol doses of 0.87 g/d. Ninety-two university students were treated for 7 days with a run-in diet that had 45% of the total calories derived from butter. Eighty-five students finished an 8 day treatment with various phytosterol levels added to the high fat diet. Total cholesterol reductions of 12.7%, 15.7%, 24.8% and 35.4% were observed at doses or 0.87, 1.3, 5.5 and 20.4 g per day respectively (Figure 10).

Feeding 12 to 18g of phytosterols per day in three equal doses prior to meals was found to result in an average plasma cholesterol decrease of 17% in a study of 15 men who had previously suffered an heart attack. This decreased cholesterol level was for the six months feeding phase of the study. The plasma cholesterol level increased promptly after cessation of phytosterol supplementation

Some very early work by Pollack in 1953 reported a significant reduction in plasma cholesterol levels at doses of 2.5, J.0 and 10.0 g phytosterols per day. The reduction was reported to be minimal when initial plasma cholesterol levels were below 200 mg/dL. This researcher also reported a rapid return to pre-supplementation plasma cholesterol levels after subjects went off of the supplement. In 1999 two researchers studied the efficacy of an alternative phytosterol dosage farm, chewable tablets (Cholestatin, Degussa BioActives, chewable tablets containing 400 mg of soybean derived phytosterols in a tropical flavored tablet).

Dr. Walsh tested 22 patients for one month, assigning each to either the placebo or active group. Patients were instructed to eat one tablet prior to each meal unless the meal was going to be rich in cholesterol in which case two tablets were taken. After one month, all participants were tested and taken off of the tablets for a two week wash-out period. Patients were then assigned to the group they were not in for the first month of the study 0.e. participants that had been taking placebos were now taking active and vice-versa). Results from this study indicated a 10% reduction in total cholesterol and a 14% reduction in LDL cholesterol (Figure 11).

Dr. Keenan, in a less rigorous study, completed a study with seven participants using Cholestatin Chewable tablets. All participants were assigned to the active group and were instructed to take one tablet prior to each meal for 30 days. This study resulted in a 5% decrease in total cholesterol. A result more in-line with other phytosterol research was the 10% decline in LDL cholesterol levels. Dr. Keenan did note a 6% decrease in the total Cholesterol to HDL ratio, showing that the ratio of “bad” to “good” cholesterol had been effected in a beneficial way.

Recent examples of clinical trials with Phytosterol containing Functional Foods

A novel dosage form was used to study the effect of phytosterols on plasma lipids when delivered in dietary chocolate. Seventy subjects were dosed with either placebo or active for four weeks in conjunction with a low-fat, low-cholesterol diet. At the conclusion of the study, those who were taking the active exhibited decreased total (-6.4%) and LDL cholesterol (-10.3%) (Figure 12).

Hypercholesterolemic subjects (n='l55) were given a placebo control spread for a six week run in period after which the subjects were divided into three groups (Christiansen et al., 2001).The control group received the placebo spread, a second group received a spread with 1.5% phytosterols and a third group received a spread with 3.0% phytosterols. The result of this study was that the groups receiving either 1.5% or 3.0% phytosterols experienced a significant reduction in Total and LDL-cholesterol levels. Compared to control, both treatment groups showed similar reductions leading the researchers to conclude that 1.5g/d phytosterols would be sufficient to reach the maximum reduction (Figure 13).

Possible Additional Benefits to Phytosterol Supplementation

While the effects of phytosterols in lowering cholesterol levels are well studied, there are indications that phytosterols may have additional benefits in areas such as benign prostatic hyperplasia. In addition, there is evidence suggesting that beta-sitosterol can suppress in vivo carcinogenesis. Human prostate cancer cells were studied in vitro comparing the effects of cholesterol v.s. beta- sitosterol. It was found that the presence of beta-sitosterol decreased cell growth by 24% and increased the rate at which the cancerous cells died by four-fold.

There are reports of further beneficial effects of phytosterols, alone and in combination with other naturally occurring compounds. Phytosterols are discussed as having possible immunomodulatory and anti-inflammatory activities as well as being anti-ulcer and anti-diabetic agents. Additional research will be necessary to substantiate these potential benefits.

6. Different Forms of Phytosterols

There are four forms of phytosterols on the market, all being promoted for their ability to lower cholesterol 1evels: free phytosterols, free phytostanols, esterified phytosterols and esterified phytostanols. Phytostanols occur to a slight extent naturally, but phytostanol products available commercially are phytosterols that have been hydrogenated to eliminate any double bonds present in the molecule. Esters of phytosterols and phytostanols are compounds to which a component of fat, a fatty acid, has been attached to make what is called an ester.

The cholesterol lowering activity of phytosterol or phytostanol esters results from the presence of the phytostanol or phytosterol. Upon ingestion, the esters are hydrolysed, resulting in a free fatty acid and free phytosterol or phytostano119

The Executive Summary of the Third Report of the National Cholesterol Education Program (NCEP)'" published in May of 2001 recommends phytosterols or phytostanols at the same dose, 2 g per day.

An association in the United Kingdom, the Institute of Food Science &Technology published an information statement in June, 2000, stating their belief that phytosterols and phytostanols are both effective at reducing serum cholesterol. A dose of approximately 1 g per day, is suggested for both forms. The PDR for Nutritional Supplements (1st. ed.) has concluded that phytosterols and phytostanols are equally capable of re-educing cholesterol levels. More recent work has also shown free phytosterols and free phytostanols to lower LDL cholesterol in an equivalent fashion. Given that phytosterol and phytostanol esters are reduced to the free phytosterols and phytostanol after ingestion, it is not surprising that phytosterol esters and phytostanol esters have also been reported as equally efficacious.

Not unexpectedly, free phytosterols have been shown to be as effective as phytosterol esters and phytostanol esters9 These studies allow us to conclude that all four forms are equal when provided in an appropriate formulation.

In a recent review of the efficacy of the four forms of phytosterols, Dr. Peter Jones of McGill University, a well known researcher on the effects of phytosterols on serum cholesterol levels, stated "With the supplementation of 2 g per day of either sterols or stanols, esterified or free, one can expect an average reduction of 0.5 mmol/L or 10-15% in LDL reduction."

7. Suggested Daily Intake

The average daily western diet is estimated to include 200 to 300 mg of phytosterols. This is not believed to be a high enough dose to elicit positive health effects. Recommended daily intake of free sterols range from 1 g per day to 2 g per day and higher. Efficacy has been shown at the lower doses and a daily dose of between one and two g is reasonable.

Phytosterols are available in many forms. Functional Foods containing phytosterols are available (spreads, salad dressings, chocolate) as well as dietary supplements such as tablets and soft gelatin capsules. As functional foods become more available, phytosterols may become incorporated into many of our favorite foods. Work has been done in the R&D Laboratory of Degussa BioActives adding phytosterols to hamburgers", pizzas and some snack foods without adversely affecting the taste or texture of the foods. Degussa BioActives continuously works on future functional food development efforts using free phytosterols.

8. Technical Data On Cholestatin

Cholestatin is a mixture of phytosterols. Being derived from a natural source, the exact ratio of phytosterols present is subject to variation within the specifications set for the product. The minimum percentages of the various phytosterols present in Cholestatin, and the minimum total percent of free phytosterols are shown in Table 4.

As shown in Table5, the mixture, Cholestatin, has a melting point that is the same as that reported for pure beta-sitosterol. The Cholestatin melting point was measured in our lab and is expected to exhibit some variation from lot to lot as its composition is subject to natural variation.


Phytosterols, like cholesterol, are a hard waxy substance. Properly stored Cholestatin will maintain its potency for many years. We have tested four-year-old material and have seen no significant decrease in phytosterol content.

The charts below show composite data for the stability of a Cholestatin product when placed in environmental chambers for stability testing in accordance with U.S. Pharmacopeia guidelines.

Long term (LT) stability testing is run at 25 c and 40% relative humidity. Under these conditions, Cholestatin was shown to maintain its potency for the duration of the test (48 months).

Short term (ST) stability tests are run at a higher temperature (40 "C) and higher relative humidity (60%) to pose a more severe challenge to the product being tested. The same phytosterol tablet as above, also maintained phytosterol potency during the 36 month duration in the short term stability test.

As stated previously, phytosterols are structurally related to cholesterol and when they degrade, products similar to the products of cholesterol degradation are produced. Primarily, oxides are formed. Phytosterol oxides have been identified in foods of plant origin". Conditions that cause cholesterol in food to oxidize; high temperature, exposure to light, ionizing radiation, processing and storage; can also be expected to facilitate oxidation of phytosterols. Given the low degree of intestinal absorption of phytosterols, phytosterol oxides are not expected to be taken up into the bloodstream to an appreciable extent. Phytosterol supplement tablets studied in Finland contained, at most, 0.1 5 mg of phytosterol oxides per six tablet dose. This is considered to be very low, since for example, the typical American meal can contain 11.5 mg of cholesterol oxides53. Studies to investigate the compounds resulting from the oxidation of phytosterols have identified the 7 and 7 hydroxy derivatives of sitosterol, stigmasterol and campsterol as well as 7-keto-sitosterol. A study using potato chips has shown that if phytosterols in food are subjected to 40°C for extended periods; referred to as abusive conditions, 5,6P-Epoxyp- Sitosterol can be formed.. Analysis of the potato chips did not show oxidation of phytosterols until after 95 days at 40°C. Over 80 oxidation products of cholesterol have been identified. It is accepted that there are more phytosterol oxides possible than have been described here but the vast majority of phytosterol oxides are expected to be present in minute quantities.


In vegetable oil (triglycerides), Cholestatin is soluble at about 1 %,whereas the solubility in mixtures of free fatty acids increases to around 5%.To provide pro ducts with high phytosterol content a matrix can be created that will hold the phytosterols in suspension. Phytosterols are almost insoluble in water. 9.

9. References

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14. • Peltier X, Belbraouet 5, Mirabel F, Mordret F, Perrin 11, Debry G. A Diet moderately enriched in phytosterols lowers plasma cholesterol concentrations in normocholesterofemic humans. Ann Nutr. Metab., 39: 291-295.1995.

15. • Moghadasian MH, Mc Manus BM, Pritchard PH, Frohlich 1J.Tall oil derived phytosterols reduce atherosclerosis in ApoE-deficient mice. Arterioscler Thromb Biol, 3711): 1 19-126.1997.

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