The method of selection of the composition of phytosterols, composition, therapeutic product

 

(57) Abstract:

Describes how the selection of the composition of phytosterols, including-sitosterol, campesterol and stigmasterol of soap in the form of a slurry obtained by processing wood chips, which includes the first stage mixing soap in the form of a pulp with a mixture solvent containing a ketone selected from the group having the General structure RCOR1where R and R1are alkyl groups, aliphatic hydrocarbon selected from C5-C10hydrocarbons and water, and does not contain alcohol, at a temperature of usually 25 to 150C, with the formation of a creamy precipitate, and the second stage cleaning creamy precipitate to obtain the composition of phytosterols. The technical result is to increase the degree of purification of phytosterols and the effectiveness of the composition in the treatment of dyslipidemia and atherosclerosis, including diseases of the coronary vessels of the heart. 5 C. and 8 C.p. f-crystals, 23 ill., 14 table.

The present invention relates to the production and selection of the composition of sterols from soap in the form of a slurry, to compositions per se and to the use of these compositions and their derivatives as agents for the prevention and treatment of primary and secondary dyslipidemia.

PRECEDE swesty as atherosclerosis. Atherosclerosis is the result of a number of hereditary (genetic) factors and environmental factors. The manifestation of these factors in our civilization, among which nutrition is the most important, leads to the development of atherosclerosis. The growth of atherosclerotic plaques, filled with cholesterol, limits ultimately the blood flow to the heart muscle or, alternatively, to the brain or legs depending on the position of plaques in the arterial line.

One of the major risk factors of atherosclerosis, which can be modified is the level of cholesterol in the blood. A number of prominent studies have shown that the level of cholesterol in the blood is clearly an important evidence of predisposition to risk a heart attack, and heart attacks. The relationship between the concentration of cholesterol in the blood and the risk of data breaches is constant (varying around the levels of cholesterol) in degree (the higher the level, the greater the likelihood of disease) in the absence of an apparent threshold (even by lowering the so-called low levels can further reduce the risk of disease). For example, in people older than 40 years, the level of blood cholesterol, the cholesterol below 5.0 mmol/L. The relationship is shown very clearly when the levels are more than 5.2 mmol/L. for Example, mortality among men with cholesterol levels of 8.0 mmol/l, was almost six times higher than among men with levels of 4.0 mmol/l, recent observations are consistent with earlier studies,

Other large clinical studies have clearly shown that by reducing high levels of cholesterol can reduce the risk of fatal and non-fatal heart attacks, heart, angina pectoris, changes in the electrocardiogram and coronary artery bypass operations. The most famous and the first such study was a clinical study of lipids in which research on primary coronary prevention showed that for every 1% reduction in the overall level of cholesterol in the blood, reducing the risk of coronary artery disease was 2%.

For any successful long-term preventive treatment of hypercholesterolemia should start with a relatively early age and continue no matter what. Despite the fact that a diet with low fat content is the cornerstone of such long-term treatment, up to 60% patientname character of a food with a high fat content. For many people, a bad cholesterol profile is complicated by the prevalence of additional risk factors for cardiovascular diseases such as high blood pressure, diabetes, obesity and Smoking.

In the last 10 - 15 years dietary modification for treatment of atherosclerosis and other cardiovascular diseases has greatly improved. In particular, researchers have found that plant sterols (phytosterols) are effective to reduce the level of cholesterol in plasma: Lees et al., Atherosclerosis, 28 (1977) 325-338; Kudehodkar et al., Atherosclerosis, 28 (1976) 239; Day Artery, 18(3):125-132 (1991).

Phytosterols are compounds such as styrene, synthesized in plants that have no food value for humans. In plants they are necessary for the functioning of cells by a mechanism similar to the mechanism of action of cholesterol in humans. The average power in the West contains 360 mg of phytosterols per day. Recently, plant sterols was drawn significant attention due to their possible anti-cancer properties and their ability to reduce cholesterol levels when making food for many mammals, including humans.

Chemical structure phytosterols are similar to cholesterol. Main panel), sitostanol, desmosterol, kaliastera, preferential, clionasterol and brassicasterol. Chemical structure of beta-sitosterol, campesterol and stigmasterol the following:

beta-sitosterol

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campesterol

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stigmasterol

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The mechanism of lowering blood cholesterol in animals with phytosterols is unknown, but, apparently, it involves the inhibition of cholesterol absorption from the proximal jejunum, through competition with cholesterol for specific binding sites. These studies also suggest that some phytosterols are not absorbed in the proximal jejunum (sitostanol), and if there is absorption (beta-sitosterol), then in very limited quantities.

Based on the results of these studies have extensively studied the use of phytosterols as a dietary Supplement to reduce cholesterol absorption. Lees et al., supra; Pollak, Pharmac.Ther., 31 (1985) 177-208; Raicht et al., Biochimica et Biophysica Acta, 388 (1975) 374-384.

Lee et al., supra, compared the effects of sitosterol from two sources, sterols, soy and pine oil for cholesterol in the plasma. It was found that preparations of plant Sterol effective in the treatment of patients with Gianicolo described the action of beta-sitosterol on the balance of Sterol and enzymes, limiting the rate of Sterol metabolism.

In General, it is accepted that phytosterols are a unique combination of safety during long-term therapy, the effectiveness and diversity in the treatment of the person. The following intention in respect of phytosterols is their isolation and purification from plant sources and identification of additional sources that are cost-effective, acceptable to develop on a large scale and which exhibit hypocholesterolemic effect.

Usually, phytosterols extracted from sources such as oil, corn oil, wheat germ, resin soybeans and corn oil resin. Similarly, as the source of phytosterols use the resin of pine oil, which is obtained in the process of production of paper from wood, particularly pine wood. In General, in this method, wood chips is maintained with caustic soda to obtain a slurry or "soap". The soap is then distilled to remove volatile materials, receiving resin in the form of residue. It is from this resin researchers have identified phytosterols.

These traditional sources of phytosterols has several significant disadvantages. Resin somoene materials and phytosterols. Although resin is inexpensive, because it is a waste of various manufacturing processes, selection of sterols with high molecular weight with good yields and high purity required for pharmaceutical use is very difficult.

U.S. patent N 3 840 570 owned by Jullian, describes a method of obtaining sterols from the resin of pine oil by extraction with a mixture of water-alcohol-hydrocarbon, followed by saponification and subsequent cleaning. Original material in this way is the resin of pine oil, which is extracted phytosterols and various impurities. It is recognized that any method of purification resin pine oil is especially difficult to separate from sterols admixture of alcohols and acids with a long hydrocarbon chain (which themselves are alcohols with high molecular weight).

Other methods for removal of sterols from the resin of pine oil are described in U.S. patent N 2835682 belonging Steiner and Fritz; U.S. patent N 2715638 belonging Albrecht and Herrlinger; U.S. patent N 2573891 owned by Christenson. It is important to note that in each of these known methods of cleaning the source material was resin pine oil, for which allocation problems discussed above.

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THE INVENTION

The present invention describes a method of cleaning and obtaining a composition of phytosterols from soap in the form of a slurry, which includes the extraction of the soap in the form of a creamy pulp sludge and purification of this residue with obtaining unique composition of phytosterols. More specifically, creamy precipitate is extracted from the soap in the form of a slurry with the use of the process of solvent extraction. Then, purification creamy sludge by crystallization get the composition of the present invention.

The present invention also includes a unique composition that is effective for prevention or treatment of dyslipidemia and which include beta-sitosterol, campesterol and stigmasterol. The composition of phytosterols, described in the present invention differ significantly from that found in plants, food or oils. In particular, the presence of stigmastanol, apparently, increases efficiency. These compositions can optionally contain such compounds, which may be the phytosterols or not. In particular, such compounds can include triterpenes, alcohols with a long hydrocarbon chain and the other soluble in alcohol organic suedelike or treatment of primary or secondary dyslipidemia and atherosclerosis, including diseases of the coronary heart vessels, peripheral vascular disease and heart attacks in humans and animals.

The unique composition of the present invention exhibit excellent results in the lowering of total (TC) and cholesterol in the blood and cholesterol in the blood, containing lipoprotein low-density (LDL). In addition, it has been unexpectedly found that compositions of the present invention maintain or increase the level of cholesterol in blood plasma, containing lipoprotein high-density (HDL) in various animal species. This feature of the present invention is particularly important, demonstrating the fact, shown in the study that, regardless of the levels of TC at lower level of HDL in plasma the risk of atherosclerosis increases. Phytosterols isolated from the resin of pine oil, soybeans and other sources, according to the present invention exhibit this unique effect of HDL.

Although it is known that some types of phytosterols from a resin cast of the soap obtained in the processing of wood chips, however, the composition of phytosterols never received from components of soap in the form of a slurry, which was obtained by the method of processing RevEmu, an unexpected effect of the compositions of the present invention is due, at least partially, the use of soap in the form of pulp as a starting material and due to the unique cleaning method.

BRIEF DESCRIPTION OF DRAWINGS

The following drawings illustrate various aspects of the invention, where:

Fig. 1 is an image of the spectrum of gas chromatography for one of the compositions (hereinafter Forbes-2) included in the scope of the present invention;

Fig. 2 is an image of the spectrum of Fig. 1 with retention time from 35 to 45 minutes;

Fig. 3 represents an image of the spectrum of Fig. 1 with retention time of 22 to 27 minutes;

Fig. 4 is a digital description of the range gas chromatography Fig. 1;

Fig. 5 represents an image of the spectrum of gas chromatography for the other compositions (hereinafter Forbes-3) included in the scope of the present invention;

Fig. 6 represents an image of the spectrum of Fig. 5 retention time from 32 to 48 minutes;

Fig. 7 is a numeric description of the range gas chromatography Fig. 5;

Fig. 8 is a bar chart illustrating the effect Forbes-1 and Forbes-2 at the concentration of TC in rats;

Fig. 9 is stadshuset column chart illustrating the action Forbes-1 and Forbes-2 at the concentration of HDL cholesterol in rats;

Fig. 11 is a bar chart illustrating the effect Forbes-3 TC in serum of hamsters;

Fig. 12 is a bar chart illustrating the effect Forbes-3 LDL cholesterol in the serum of hamsters;

Fig. 13 is a bar chart illustrating the effect Forbes-3 HDL cholesterol in the serum of hamsters;

Fig. 14 is a bar chart illustrating the effect of treatment with different diets on cholesterol levels in the plasma of males and females hamster;

Fig. 15 is a bar chart illustrating the effect of treatment with different diets on cholesterol levels in plasma in male hamster;

Fig. 16 is a bar chart illustrating the effect of treatment with different diets on cholesterol levels in the plasma of females hamster;

Fig. 17 is a bar chart illustrating the effect of treatment with different diets on the level of triglycerides in the plasma of males and females hamster;

Fig. 18 is a bar chart illustrating the effect of treatment with different diets on the ratio of HDL/APO B in male and he is and the level of total cholesterol in hamster on a 45 day study,

Fig. 20 is a bar chart illustrating the effect of treatment with diet on the correlation of cholesterol and sitostanol;

Fig. 21 is a bar chart illustrating the effect of treatment with diet on HDL in hamster on a 45 day study;

Fig. 22 is a bar chart illustrating the effect of treatment with diet on the ratio of no-apoa/EPOA the hamster on the 45th day of the study;

Fig. 23 is a bar chart illustrating the effect of treatment with diet on non-apoa/sterols have a hamster on a 45 day study.

PREFERRED EMBODIMENTS OF THE INVENTION

The method of the present invention includes the following stages:

(A) obtaining or preparing the source material, soap in the form of a slurry obtained plant material;

(B) extraction of the soap creamy precipitate when using a suitable solvent;

(C) purification of creamy precipitate to obtain the composition of phytosterols.

There are numerous possible soap in the form of pulp, made from plant material. In General, in the known method (method "Kraft") wood chips are treated with caustic is, the key, but not limited to, wood, fir, cedar, pine, spruce, oak, Hemlock and poplar. Most preferably, the chip supply from any Pacific Northwest American or European forests, in which there are various kinds of wood.

At the stage of extraction of the soap mixed with a ketone and water solution. For the extraction of sterols used a hydrocarbon solvent. This stage can be carried out when temperatures are usually from about 25oC to about 150oC, most preferably from about 50oC to about 100oC. Most preferably, this stage extraction continues over 15-24 hours. It is important to note that the use of alcohol is not required in the extraction step. The extraction process of the present invention carried out using a mixture of ketone-water-hydrocarbon solvent.

Ketone selected from the group having the General structure RCOR1where R and R1are alkyl groups. Preferably the alkyl groups are C1-C6groups. Most preferably, the ketone is 2-propanone (acetone). The hydrocarbon may be selected from the group containing all C5-C10the hydrocarbons. Most predpochte creamy residue or the residue from which emit using the cleaning composition of phytosterols. This cleaning stage can be carried out by crystallization, chromatographic separation, or in any other suitable way. Most preferably creamy precipitate is dissolved in alcohol, slowly cooled, then filtered and washed with cold alcohol. The precipitate is dried, the resulting product is a composition of phytosterols.

In a preferred method, at the stage of purification used the alcohol is selected from the group having the General structure: R-CHOHR, R - CH2OH and RCOH, where R is a C1-C4alkyl group. Most preferably the alcohol is methanol. Stage of cooling can be carried out at temperatures from 10oC and 0oC, most preferably from 3 to 4oC within 24 hours.

The composition of phytosterols obtained by the method of the present invention may be incorporated directly into food additives, drugs, vitamins and medicines for the current and prophylactic treatment of atherosclerosis and its consequences, heart attacks, heart attacks and peripheral vascular disease. In addition, one of the embodiments of the present invention includes described the example, these compositions can be enabled or alternate appointed together with agents that lower lipid levels, to reduce the necessary dosage and, therefore, toxicity data connections.

The composition of phytosterols present invention show a marked ability to modification of lipoproteins, even in lower concentrations of phytosterols than known compositions. Most unexpectedly, however, it was discovered these compositions to increase plasma levels of high density lipoprotein (HDL), an effect not previously associated with any other compositions of phytosterols derived from pine oil. It is assumed that the reason for this unique action, you may be using soap in the form of pulp as a starting material, or the condition of the presence of stigmastanol as an element of composition.

In a preferred embodiment, compositions of the present invention contain the following ratio of phytosterols: beta-siderastrea (1) : campesterol (0,2-0,4) : and stigmasterol (0.2 to 0.5). Most preferably, campesterol and stigmasterol together comprise at least 50% of the total number of beta-sitosterol. In most pravlenie with phytosterols, derived from soybeans (see table 1a).

The composition and purity of the other two extracts, included in the scope of the present invention, is presented in table 1b.

In each composition described herein may contain additional compounds that are phytosterols or not. For example, it was found that campestanol, other phytosterols, may be present in relatively small quantities. In addition, there may be fatty alcohols with straight-chain, such as behenyl (C22) and lignoceric (C24). To determine the nature of the data related compounds was conducted gas-liquid chromatographic analysis of each of the most preferred compositions of the present invention. Conditions for gas chromatography for phytosterols were as follows: initial temperature 80oC that was maintained for 1 minute; linear rise up to 120oC with a speed of 20oC / minute, which was maintained for 7 minutes; linear decrease to 24oC with a speed of 20oC / minute, which was maintained for 15 minutes; linear rise up to 269oC with a speed of 20oC / minute, which was maintained for 25 minutes. At the end of each stage, the rate is was 300oC and the detector temperature was 320oC. the flow Rate in the column was 1 ml / min and the flow rate in the valve opening was 4 ml per minute. The flow rate of the purge valve was 4.5 ml / min. Gaseous media consisted of helium. The results of the analysis of gas-liquid chromatography for the two most preferred compositions according to the present is shown in Fig. 1-7.

For the composition Forbes-2 known Sterol is evident in the area 35-45 minutes, Fig.1 and 2. The presence of beta-sitosterol is evident in the peak of 87, campesterol is evident in the peak 81 and stigmastanol in the peak 84. Peaks 65, 66 and 77 of Fig.2 refer to related compounds, which may be hypocholesterolemic action. It is also possible that these related compounds may have a synergistic effect on the action of known phytosterols in the composition. Similarly to Fig. 5 and 6, campesterol, stigmasterol and beta-sitosterol presents peaks 6, 7 and 8 respectively.

Another preferred composition, which is included in the scope of the present invention, includes the following components:

Campesterol 14,1%

Campestanol 3,5%

B-sitosterol 62.8% of the

stigmastanol 16.9% of

Preparing a mixture of 3 l of acetone and 1.5 l of water, to which was added the soap. The mixture was continuously extracted with 4.5 l of hexane at 50oC within 24 hours when using the evaporator volume of 18 l Then the product obtained by extraction, dried over sodium sulfate and left to evaporate. Received 460 g of residue or creamy precipitate.

Creamy precipitate was heated and stirred using magnetic stirrer and slowly added 460 ml of methanol. The mixture was boiled under reflux with stirring for 15 minutes and slowly cooled within 3-5 hours. The mixture was cooled at 3-4oC overnight and then filtered and washed twice with 150 ml of cold methanol. Finally, the mixture was stirred under vacuum for 2 days, getting 100 grams of a mixture with a purity of 82% (i.e., 82 g of phytosterols).

EXAMPLE 2. To estimate the effects of the composition of phytosterols in rats.

Ninety male Wistar rats (80-100 g) were divided into 3 experimental groups: use composition Forbes-1, use composition Forbes-2 and use the composition is derived from soy beans. Thirty rats in each group was further divided into 5 depending the e 10 days basic polyoxidonium feed (table 1), supplemented with different amounts of cholesterol and phytosterols (table 2). In each of the 5 diet group 2 rats were injected composition Forbes-1, 2 rats were injected composition Forbes-2 and 2 rats were administered phytosterols derived from soybean (Sigma).

At the end of the feeding period, rats were injected intraperitoneally deuterium oxide (0.4 ml) and was deprived of food and water for at least 2 hours. Then rats were anestesiologi the halothane gas. Blood samples were collected from the heart. Samples of liver, small intestine and muscle were rapidly removed, weighed, placed in liquid nitrogen and kept at -80oC prior to the determination of cholesterol synthesis. Total cholesterol and LDL cholesterol and HDL were determined using standard kits (Biopacific Diagnistic Inc).

The compositions of phytosterols on total cholesterol, LDL and HDL cholesterol is shown in Fig. 8, 9 and 10, respectively. It is evident from Fig. 9 shows, the apparent efficiency Forbes-1 and Forbes-2 reduction of LDL cholesterol, and Fig. 10 shows an increase in HDL cholesterol, in particular, under the action of Forbes-1. In Fig. 8 adding cholesterol (diet group 2) to the main power (group 1) leads to an increase in the concentration of circulating cholesterol. Progressive addition of increasing amounts of phytosterols (groups 3-5)s soybeans, that determined by regression analysis. Fig.9 shows that phytosterols Forbes-1 and Forbes-2 have the best efficiency for the reduction of LDL cholesterol than soybean phytosterols. Fig. 10 shows the improved ability of the preferred compositions of the present invention in increasing HDL, in particular Forbes-1, compared with soybean phytosterols.

EXAMPLE 3. To estimate the effects of the compositions of phytosterols on hamsters.

The present study was conducted to examine the functioning of the food compositions of the phytosterols present invention to caused by cholesterol contained in food, the increase in the concentration of serum cholesterol in hamsters.

In General, 40 male rats (80-100 g), placed individually in cages stainless steel, fed forage for rodents and acclimatized for 3 days in the atmosphere of the room (20-22oC, light period 17.00 - 05.00). Then the hamsters were divided into five groups of 8 animals each and fed for 34 days main polyoxidonium food (table 3), supplemented with varying amounts of cholesterol, and one of the compositions of the phytosterols present invention (Forbes-3) (table 4).

At the end of the feeding period the animals were undesirable the halothane gas. Blood samples were collected from the heart. Images of other tissues, including the liver, intestine and muscle were separated, weighed, placed in liquid nitrogen and kept at -80oC prior to the determination of cholesterol synthesis. Total cholesterol and LDL cholesterol and HDL were determined using standard kits. The results were statistically evaluated by ONEWAY analysis, the method of the mean deviation (SYSTAT).

Hamsters fed a food with high cholesterol had significantly higher total cholesterol and LDL cholesterol in serum than those who received usual food with cholesterol (0,025%). Adding phytosterols in the amount of 0.5% and 1% significantly reduced this increase is caused by high consumption of cholesterol (Fig. 11 and 12). The concentration of LDL cholesterol in group 5 was lower compared with its levels in hamsters that were given feed with normal cholesterol (Fig. 12). Moreover, the observed negative regression of the ratio of total cholesterol and LDL cholesterol levels of phytosterols (Fig. 13). Adding phytosterols calls for a small increase in HDL, but does not cause any significant difference (Fig. 13).

EXAMPLE 4. To estimate the effects of the composition of phytosterols on XOM is purified feed, containing 30% fat (polyunsaturated/saturated fat = 0,3) within 90 days. Feed 1 did not contain cholesterol. Feed 2-6 consisted of 0.25% (by weight) of dietary cholesterol. Feed 3 and 4 contained 0.5 and 1% phytosterols Forbes (more than 90% purity), respectively. Feed received from the source of ingredients every week. The levels of fat, phytosterols and cholesterol were determined using gas-liquid chromatography. All animals had free access to feed and water throughout the experimental period. Animals weighed weekly. Daily and average weekly food intake was also determined by weighing cups with food before and after each 24 hour period of feeding. After 90 days of feeding, animals were slaughtered when using halothane gas and took blood samples for analysis of lipoprotein profile. Determined the levels of total cholesterol, cholesterol in particles containing UPS, and HDL cholesterol, and triglycerides. Immediately before the killing was determined by the rate of synthesis and absorption of cholesterol, using the method of elimination of 14C-cholesterol from the intestines and incorporation of deuterium in cholesterol tissue, respectively. Also investigated the incorporation of phytosterols into the tissue of the intestine and other tissues. Ohio, Carcinogenicity and function of enzymes.

The results:

The results are presented in Fig. 14-18. Group data in Fig. often indicated by the number that corresponds described in the scheme of the experiment. Letters above the columns in the graphical diagram indicate significant differences between groups. Where the above letters, columns with the same letters are not significantly different, whereas columns with different letters are different at the statistical level of p<0,05.

Data on cholesterol levels according to gender is shown in Fig. 15 and 16. For samco the lapsed total cholesterol. Adding phytosterols Forbes in the amount of 0.5% leads to a tendency to lower cholesterol, but adding phytosterols Forbes in the amount of 1% reveals a statistically significant decrease of cholesterol to approximately the same level as in the control group, without adding cholesterol. When food was added phytosterols soybeans in the amount of 0.5 and 1% significant reduction of circulating levels of total cholesterol in males was not. For HDL observed different effects Forbes compared with soybeans, if feeding with Forbes does not change the level of HDL in the group treated only cholesterol (group 2), the feeding of soybeans leads to a significant reduction of HDL levels in both of the investigated levels (groups 5 and 6). Did not observe significant differences in particle UPS that contain cholesterol, but had a tendency to decrease when feeding cholesterol Forbes in the amount of 1% compared with other groups.

Data for females is shown in Fig.16. For total cholesterol and HDL cholesterol had a significant impact only the addition of cholesterol in food. Adding any type of source of phytosterols in a concentration of 1% leads to a significant and similar reduction to the/P> The levels of circulating triglycerides in hamsters, which consumed studied nutrition for 90 days, is shown in Fig. 17. There is an increase in the level of circulating triglycerides in female animals receiving the main power, supplemented with cholesterol and Forbes in the amount of 0.5%, compared to animals receiving only the main feed, but other differences between the two sexes within groups was not detected. Males have no power or influence trends to him on the concentration of triglycerides was not found.

In General, the order of results on the study of food on the males shown in table 5.

You can clearly see the advantage of the compositions Forbes (the present invention). In addition, a similar pattern was found for the ratio of HDL:UPS in males (Fig. 18):

Forbes 0,5% - 2

Forbes 0,1% - 1

Soy 0,5% - 4

Soy 1,0% - 3

It was also found that the ratio of HDL:LDL for songs Forbes was more almost twice than for only one B-sitosterol.

EXAMPLE 5. The effect of the composition of phytosterols on rabbits.

In this study, two rabbits were studied for more than 43 days on one of the compositions of the present invention (food from Forbes 1%) on progesterine both rabbits A and B within two weeks of the introduction of the composition of phytosterols (Forbes 1%). This action continues even after the termination of the introduction of the Forbes 1%. The effect of the composition of the present invention to reduce total cholesterol is delayed after the initial phase of introduction.

EXAMPLE 6. The effect of the composition of phytosterols in APO-E deficient mice.

Animals: Nineteen 5-week-old male APO-E deficient mice were obtained from Jackson Laboratory, USA. Animals are randomly divided into two groups of 9 animals in the control group and 10 animals in the experimental group with Forbes. After 5 days of adaptation period in mice the blood was collected from the tail vein into the capillary tube, and separated plasma by centrifugation of the blood. Determined the plasma lipids of mice.

Food: Food for mice with low-fat, low-cholesterol was obtained from Jamieson''s Pet Food Distributors Ltd., Vancouver, B. C. Phytosterols derived from pine oil, was extracted from the soap, pine oil, using the method of the present invention. Using gas-liquid chromatography evaluated the purity and composition of each individual phytosterols in the mixture of the final product. The final product was up to 95% purity and contained 69% of sitosterol, 15% of campesterol and 16% of stigmasterol. Food for mice rivali. Part of this is supplemented cholesterol food again was granulated, dried and used for feeding the control group of mice, and another part was added 2% (by weight) of phytosterols extracted from pine oil, re-granulated, dried and used for feeding the experimental group of mice.

Biomedical research: the Level of plasma cholesterol and triglyceride was determined using an enzymatic kit (Boehringer Mannheim), and HDL cholesterol previously published method using polyethylene glycol 6000.

Body weight and food consumption. The body weight of mice and food consumption were measured weekly (see tables 7 and 8).

Other detection: a new feed any side effects not found. All animals from both groups looked normal, have a normal behavior, including normal in relation to food. One mouse from the control group was found dead 11.07.95. As the body weight has not been saved, an autopsy was not performed. In another mouse from a group Forbes was discovered dehydration with weight loss. The animal was killed and the cause of his disease was detected anomaly bite (overgrowth of the teeth).

s average deviation.

The results:

The results obtained to date are summarized in tables 9, 10 and 11.

These results show that the group receiving the composition Forbes, shows a significant (33%) reduction in total cholesterol, a slight increase in triglycerides and a significant increase in HDL cholesterol (>100%).

EXAMPLE 7. The effect of the composition of phytosterols on hamsters - 45-day study.

Fifty GS hamsters kept in two weeks for the care of animals before feeding polyoxidonium forage within 45 days. They were divided into five groups feeding with 0.25% cholesterol and mixtures with one of the four plant phytosterols from soy beans, pine oil, pure sitostanol and artificial mixtures representing phytosterols pine oil. The control group received only 0.25% cholesterol. Food consumption was recorded during the study period on every third day. Body weight was measured every week and during the collection of tissue samples. Three days before killing hamsters GS was slightly anestesiologi diethyl ether and through the jugular vein was injected with 0.4 ml of Intralipid containing 0.18 mg13C-cholesterol. Immediately after inyesterday of 0.44 mg13C-cholesterol. Then GS hamsters were kept in their wire cages within 72 hours, providing food and water on demand.

On the day of killing each hamster GS in b/W was introduced 1 ml of deuterated water and left for one hour before the killing. Animals were anestesiologi diethyl ether, and collected blood samples by cardiopathy. Collected liver, gall bladder, small intestine, large intestine and heart was frozen with liquid nitrogen and kept frozen at -80oC. Their carcasses and faeces were stored at -20oC for further analysis of lipids and sterols.

Measurement of food intake, body weight and liver.

Eating hamsters were measured every three days. Statistical analysis showed no significant differences among the five groups in food consumption. Average daily food intake into five groups ranged from 8,84 to 9,34 g / day, p=0,4. The animals had a significant increase in body weight, about 25 to 40 g during the study period, the value of p<0,05 (paired t-test). The final measurement of body weight was in the range of from 112.5 to 154,3, No statistical significance was not noted among the various treated groups, p value=0,43.

Ve the liver compared with the control and other groups, treated with 0.25% cholesterol, and various phytosterols, respectively. In addition, the group treated soybeans, showed a significant difference similar to the difference of the group who received only sitostanol, in statistical data analysis using Newman-Keuls test. In General, all groups treated with plant sterols, had lower liver weight compared with the control group treated with a single cholesterol, the value of p=0.01. The GS hamsters treated with sterols oils of pine and sitostanol, had an average liver weight by 15% and 20% less than the control group, respectively. Natural oils of pine and artificially prepared pine oil had similar values in weight of the liver, suggesting that the compound is not found in soy beans, vegetable Sterol - sitostanol plays an important role in reducing the Sterol content in the liver.

Analysis of lipids:

I. Total cholesterol:

Total cholesterol was determined using a standard set of enzymatic reagents for the auto-analyzer VP. Blood samples were investigated twice in the final statistical analysis used the average of the two. For analyses of various lipids used methods Newman-Keuls and Bonferroni by the control. The average value for the control group was 226,9 mg/DL, and for one of the groups taking sitostanol was 151,2 mg/DL, a value of p=0.007. Phytosterols of pine oil and a mixture of synthetic oils of pine show a similar reduction of cholesterol in the plasma (17,5%), 118,4 mg/DL and 186,1 mg/DL, respectively. However, if we exclude from the analysis the value of one of the samples, out of scale, phytosterols oil pine (Forbes) showed a significant decrease in the values of total cholesterol (175,2 mg/DL), the value of p<0.02 in group pine oil, in comparison with the control (only 0.25% cholesterol) group. The correlation between the presence of sitostanol and low plasma level was significant, the value of p<0,0001, and r=0.46.

II. The HDL cholesterol:

The share of apoE lipoproteins present in the HDL cholesterol, with no significant change in the values of the five different groups, the value of p=0,18. There was a significant reduction of 15% of the mean values of HDL in the group treated with sitostanol. However, this element does not influence in a significant reduction in total cholesterol, which was 34%. Attitude not apoE lipoproteins to apoa the lipoproteins (HDL) were not significantly changed between groups. Confusing really is giving insignificant result in the value of the ratio is not apoa/EPOA (HDL). In General, the different types of phytosterols insignificant change the level of HDL cholesterol in the plasma of hamsters GS.

III. LDL and/or not Apoa sterols.

Sitostanol is effective to mitigate apoa sterols in the plasma. Been shown to decrease by 55% not apoE lipoproteins, the value of p= 0.02. Like their effect on total cholesterol oils of pine and synthetic blend oils of pine reduced the level of not Apoa sterols by 21%, respectively, which again testifies to the existing strict correlation between the contents of sitostanol in phytosterols and their useful effect in reducing levels of cholesterol in the plasma of hamsters GS. However, this decrease was not statistically significant because of the variability of the values of triglycerides.

IV. Triglyceride:

When using the ANOVA method for determining the amount of triglycerides they were not suitable for normal test. Hamsters GS scored in the absence of starvation (an important condition for future analysis of cholesterol synthesis, kinetics and absorption). Because of this situation, different values were excluded from the scale. According to ANOVA on Ranks levels of triglycerides did not show any statistical differences among groups. Plant sterols did not affect the level of altoadige of the invention, is the most preferred profile by increasing HDL cholesterol and lower total cholesterol. This effect on HDL is not observed in the artificial composition of oil of pine. Similarly, the total effect of sitostanol adversely, due to a significant increase in HDL (see table 12).

Although it is not clear, it appears that in respect of plant sterols relatively hydrophobic Sterol largely inhibit the absorption of cholesterol, whereas the relatively hydrophobic sterols have a greater impact on the level of HDL. The composition of phytosterols present invention are unique because they have both of these effects.

1. The method of selection of the composition of phytosterols, including-sitosterol, campesterol and stigmasterol of soap in the form of a slurry obtained by processing wood chips, which includes the first stage mixing soap in the form of a pulp with a mixture solvent containing a ketone selected from the group having the General structure RCOR1where R and R1are alkyl groups, aliphatic hydrocarbon selected from C5- C10hydrocarbons and water, and does not contain alcohol, at a temperature of usually 25 - 150oC,tion of phytosterols.

2. The method according to p. 1, characterized in that creamy residue purified by crystallization from ethanol to obtain the composition of phytosterols.

3. The method according to p. 1, characterized in that the soap in the form of pulp produced from wood chips, including chips of cedar, spruce, pine, fir, oak, Hemlock and poplar.

4. Composition, lowering cholesterol, containing not more than 70% by weight-sitosterol, at least 10% by weight of campesterol and optionally containing stigmastanol.

5. Composition, lowering the cholesterol containing campesterol and stigmasterol in the amount of at least 50% of the number-sitosterol.

6. Composition, lowering cholesterol, in which the ratio-sitosterol, campesterol and stigmasterol is 1.0 : 0.2 to 0.4 : 0.2 to 0.5, respectively.

7. The composition according to p. 6, characterized in that the ratio-sitosterol, campesterol and stigmasterol is 1.0 : 0,354 : 0,414 respectively.

8. The composition according to p. 6, characterized in that the ratio-sitosterol, campesterol and stigmasterol is 1.0 : 0,330 : 0,203 respectively.

9. The composition according to p. 6, containing the following ratio of phytosterols: 1,0-sitosterol to 0,268 of campesterol to 0,299 stigmastadiene concentration of cholesterol in serum.

12. The product is therapeutically effective for the prevention or treatment of primary and secondary dyslipidemia and atherosclerosis, including composition under item 4 and a pharmaceutically effective carrier.

13. The composition according to p. 10, which additionally contains triterpenes, alcohols with long chain organic compounds, soluble in alcohols.

 

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