MXPA98003404A - The use of a daidzein material in the preparation of compositions to reduce the concentration of cholesterol ldl and to increase the concentration of cholesterol hdl in the blood to reduce the risk of aterosclerosis and vascu disease - Google Patents

Abstract

A method is provided for altering the concentration of cholesterol constituents in human blood: a daidzein material is administered to a human to increase the concentration of HDL cholesterol and to decrease the level of LDL cholesterol in the blood; can be administered as a pharmaceutical composition, or in a dietary supplement, including dietary supplements based on soy protein: the use of daidzein to increase the concentration of HDL cholesterol and to lower the concentration of LDL cholesterol in the blood reduces the risk of atherosclerosis and vascular disease, providing more HDL cholesterol beneficial to health and reducing the level of LDL cholesterol inducer of atherosclerosis

Description

THE USE OF A DAIDZEIN MATERIAL IN THE PREPARATION OF COMPOSITIONS TO REDUCE THE CONCENTRATION OF CHOLESTEROL LDL AND TO INCREASE THE CONCENTRATION OF CHOLESTEROL HDL IN THE BLOOD TO REDUCE THE RISK OF ATEROSCLEROSIS AND VASCULAR DISEASE BACKGROUND OF THE INVENTION The present invention relates to the discovery that daidzein and its metabolites, o-demethylangolensin and dihydrodaidzein, are useful for altering the concentration of cholesterol constituents in a human blood by increasing the concentration of high density lipoprotein cholesterol and decreasing the concentration of low density lipoprotein cholesterol. Changes in the concentration of high and low density lipoprotein cholesterol in the blood reduce the risk of atherosclerosis and vascular disease. Cardiovascular disease is one of the main causes of morbidity and mortality, particularly in the United States and Western European countries. Several causative factors are involved in the development of cardiovascular disease, including hereditary predisposition to disease, gender, lifestyle factors such as smoking and diet, age, hypertension and hyperlipidemia, including hypercholesterolemia. Several of these factors, particularly hyperlipidemia and hypercholesterolemia, contribute to the development of atherosclerosis, a leading cause of vascular and heart disease. The high concentration of cholesterol in the blood is one of the main risk factors for vascular disease and coronary heart disease in humans. The high concentrations of high density lipoprotein cholesterol (hereafter "coleeterol LDL") and total cholesterol are directly related to an increased risk of coronary heart disease. Cholesterol and Mortalitv: 30 Years of Follo-Up from the Framingham Study, Anderson, Castel1i, & Levy, JAMA Vol. 257, pp. 2176-80 (1987). Although high levels of total cholesterol and LDL cholesterol are risk factors for developing atherosclerosis and vascular diseases, a deficiency of high density lipoprotein cholesterol (hereafter "HDL cholesterol") has recently been recognized as a risk factor for developing these conditions. Several clinical tests support a protective role of HDL cholesterol against atherosclerosis. A study has shown that for every increase of lmg / dl in HDL cholesterol in the blood, the risk of coronary vascular disease decreases by 3% in women. High-density Lipoprotein Cholesterol and Cardiovascular Disease: Four Propective American Studies, Gordon, Probsfield, and Garrison et al., Circulation, Vol. 79, pp. 8-15 (1989). The ingestion of plant protein materials in place of animal protein in the diet is associated with a lower risk of coronary heart disease, which may reflect decreases in serum cholesterol levels. It is known that vegetable protein materials, particularly soy protein materials, reduce the levels of total cholesterol and LDL cholesterol in the blood of animals. A recent meta-analysis of the effects of soy protein assimilation on serum lipids in humans has shown that dietary soy protein is significantly related to the decrease in total cholesterol and LDL cholesterol concentrations in human serum without significantly affecting the concentrations of HDL cholesterol. Meta-Analysis of the Effects of Soy Protein Intake on Serum Lipids, Anderson, Johnstone, and Cook-Newell, N.Engl J. Med., Vol. 333, No. 5, pp. 276-82 (1995). Phytoestrogens in soy protein are recognized as a potentially significant factor in the hypercholesterolemic effects of soy protein. Phytoestrogens, such as those found in soybeans, are compounds that are structurally similar to estrogen and that are derived from plants. Estrogen itself has been determined as a significant cardioprotective factor. Postmenopausal women who take estrogen replacement therapy have been shown to have a reduced risk of coronary heart disease and myocardial infarction. A mechanism by which estrogen is believed to reduce the risk of coronary heart disease is to inhibit the development of atherosclerosis through the estrogenic reduction of the concentration of atherogenic compounds such as LDL cholesterol in the blood. Nevertheless, the administration of estrogen to estrogen-deficient women has been associated with an increased risk of developing breast and endometrial cancer, limiting the utility of estrogen as a vascular and cardioprotective agent. Phytoestrogens - particularly isoflavones derived from soy and garlic such as genistein, daidzein, glycitein, their glycosidic derivatives, biocanin A and forononetin - exhibit estrogenic properties in certain tissues of mammals and humans, and exhibit antiestrogenic properties in other tissues by competitively inhibiting the binding of estrogen at estrogen receptor sites. Unlike estrogen, these isoflavone estrogen phytoes are not associated with an increased risk of cancer, and can actually inhibit the development of breast and uterine cancers. Recent studies have determined that these isoflavones decrease the concentrations of total cholesterol and LDL cholesterol in the blood of animals, and therefore inhibit or delay the development of atherosclerosis. The effect of these isoflavones on blood cholesterol levels in humans has become less clear, as indicated by the meta-analysis; however, it is believed that isoflavones decrease the total cholesterol and LDL cholesterol concentrations in the blood.

Despite progress in the development of compounds and methods to lower total cholesterol and LDL cholesterol in the blood of humans, there remains a need to develop additional compounds that can safely provide these effects on blood cholesterol levels to reduce the risk of developing atherosclerosis and vascular disease. There is an additional need to develop compounds and methods to increase the levels of HDL cholesterol in the blood of a human to provide the cardioprotective effects of this cholesterol.

BRIEF DESCRIPTION OF THE INVENTION A method is provided for altering the concentration of cholesterol constituents in a human blood to reduce the risk of atherosclerosis and vascular disease. A material containing daidzein is administered to a human in an effective amount to increase the concentration of HDL cholesterol and to decrease the concentration of LDL cholesterol in the human blood. In one embodiment of the invention, daidzein is administered to a human in a dietary supplement of soy protein material. In another embodiment of the invention, daidzein is administered to a human in a pharmaceutical composition. In a further embodiment, the administration of the daidzein-containing material causes an increase in the concentration of o-demethylangolensin in the human blood. In another aspect, the invention is a method for altering the concentration of cholesterol constituents in a human blood to reduce the risk of atherosclerosis and vascular disease, wherein a daidzein material is administered to a human in an effective amount to increase the concentration of HDL cholesterol in the human blood.

DESCRIPTION OF THE PREFERRED MODALITIES The present invention is based on the discovery that the concentration of daidzein (formula I below) and the metabolites of daidzein, particularly o-demethylangolensin (formula 2), in the blood of a hand that consumes amounts of daidzein, are significantly correlated with an increase in the concentration of HDL cholesterol in the blood, as well as a decrease in the concentration of non-HDL cholesterol, and are more significantly correlated with these changes in the concentration of cholesterol in the blood than other isoflavones present in soy. The invention encompasses the therapeutic use of daidzein in humans to increase the concentration of HDL cholesterol and to decrease the concentration of LDL cholesterol in the blood, thus inhibiting the development of atherosclerosis and vascular disease.

Formula 1 Daidzein is a naturally occurring substance found mainly in plants such as legumes, garlic, and the root of the kudzu vine (pueraria root). Sources of common daidzein legumes include soybeans, chickpeas and various types of beans and peas. Garlic sources of daidzein include red garlic and garlic underground. Soy is a particularly preferred source of daidzein. Daidzein can be isolated from plant sources in which it occurs naturally or can be prepared synthetically. Daidzein can be isolated from red garlic as described by Wong (J. Sci. Food Agr., Vol. 13, p.304 (1962)) or can be isolated from the fungus Micromonospora halophytica provided by Ganguly and Sarre (Chem. & amp; amp; Ind. (London), pp. 201 (1970)), both references are incorporated herein. Daidzein can be prepared synthetically by the methods provided by Baker et al. (J.Che. Soc., P 274 (1933)), Wesley et al. (Ber. Vol. 66, P. 685 (1933)), Mahal et al. (J. Chem. Soc., P.1769 (1934)), Baker et al. (J. Chem. Soc, p.1852 (1953)), or Farkas (Ber.

Vol. 90, p. 2940 (1957)), references that are incorporated herein. Daidzein is commercially available and can be purchased, for example, from Indofine Chemical Company, Inc., P.O. Box 473, Somerville, New Jersey, 08876. In a preferred embodiment, daidzein is isolated from a soy material. Soy materials from which daidzein can be isolated include: soybeans, dehulled soybeans, soybean meal, soybean meal, soybeans, soy flakes (whole and defatted), soy molasses, soy protein molasses, soy, soy serum, soy whey protein and soy protein isolate. In one embodiment, the isoflavones that include daidzein are extracted from soybeans, dehulled soybeans, soybean meal, soybean powder, soybeans, soy flakes, soy protein concentrate, soy whey protein or soy protein isolate. , preferably soybean meal, soybean powder, soybeans or soy flakes, with an organic extractor of low molecular weight, preferably an alcohol, ethyl acetate, acetone, or ether and most preferably aqueous ethyl alcohol or methyl alcohol. Most preferably, the extraction material has a pH of about the isoelectric point of the soy protein (pH of about 4 to 5) to minimize the amount of soy protein extracted by the extraction material. The extraction material containing the isoflavones is separated from the insoluble soy materials to form an extract rich in isoflavone, and the daidzein separated from the other isoflavones and impurities in the extract by contacting the extract with a material that absorbs the isoflavones in the extract, and eluting the isoflavones adsorbed outside the absorbent material with a solvent that causes the isoflavones to be differentially eluted from the adsorbed material. In a preferred embodiment, daidzein is separated from the other isoflavones and impurities in the extract by means of a conventional reversed phase separation of high performance liquid chromatography ("HPLC"). After extracting the isoflavones from the soy material and separating the extract from the insoluble soy materials, the extract is filtered to remove insoluble materials that could clog a column of HPLC. A column of CLAR is prepared by packing a conventional HPLC column commercially available with an adsorbent material in the form of particles that will bind the daidzein releasably, and that can bind releasably the other isoflavones and impurities in the extract in a specific manner of compound. The adsorbent material can be any reverse phase HPLC packing material, however, a preferred packing material can be chosen by the criteria of charge capacity, separation effectiveness and cost. Said packaging material which is preferred are 16 μm lOOfi Kro asyl C18 globules available from Eka Nobel, Nobel Industries, Sweden. The filtered extract is passed through the packed HPLC column until all the agglutination sites in the column are completely saturated with isoflavones, which is detected by the appearance of isoflavones in the effluent that comes from the column. The HPLC column can then be eluted with a solvent to carry out the separation. In a preferred embodiment, the eluent is a polar solvent such as ethanol, methanol, ethyl acetate or acetonitrile, and is preferably an aqueous alcohol having an alcohol content of between about 30% and about 90%, preferably about 50% and most preferably the alcohol is ethanol. The daidzein material, other isoflavone materials and impurities are collected separately from the column effluent. The daidzein fraction of the effluent can be identified from the other fractions according to conventional techniques of analytical chemistry and HPLC. Of the aglucone isoflavone materials, the effluent fraction containing daidzein elutes from the first column, followed by a glycitein fraction and followed by the more polar genistein. The daidzein fraction of the eluent can be collected from the column, and the volatile content of the solvent (eg, alcohol) can be removed by evaporation. The daidzein material can be recovered directly if all the solvent is removed by evaporation, or it can be recovered by cooling the remaining solvent (eg, water) and centrifuging or filtering the remaining solvent. In a particularly preferred embodiment, the isoflavone glucoside of daidzein -daidzin-, and the isoflavone conjugates of daidzein -6'-0-malonyl daidzin ("6'-0-Mal daidzin") and 6'-0- acetyl daidzin ("6'-0-Ac daidzin") - are converted to daidzein before the separation of daidzein from the other isoflavones to increase the amount of daidzein material recovered. The conversion of the isoflavone and daidzein glucoside conjugates to daidzein can be carried out on the soybean substrate from which the daidzein will be extracted before extraction, or it can be carried out on the isoflavone-rich extract after the separation of the extract from the insoluble soy materials. Preferably, the isoflavone conjugates of daidzein are converted to the isoflavone glucoside daidzin by forming an aqueous alkaline solution of the soy substrate from which the isoflavone will be extracted and having a pH of from about 8 to about 13, preferably a pH of 9 to 11, and treating the aqueous alkaline solution at a temperature of about 25 ° C to about 75 ° C for a period of time sufficient to carry out the conversion of at least a majority of the isoflavone conjugates of daidzein in daidzin, preferably about 30 minutes to about 5 hours. Most preferably, the conversion of the isoflavone conjugates from daidzein to daidzin is carried out at a pH of about 11 at a temperature of about 35 ° C over a period of about 45 minutes. Substantially all of the isoflavone glucoside daidzin can be converted to daidzein, preferably after converting the isoflavone conjugates of daidzein to daidzin. Daidzin is converted to daidzein by contacting daidzin as an enzyme capable of cutting a 1,4-β-glucoside bond - preferably an enzyme & amp;-glycosidase commercially available, an enzyme a or & galactosidase, a pectinase enzyme, a lactase enzyme or a gluco-a -lase enzyme- at a pH at which the enzyme is active, typically from about 3 to 9, and at a temperature of about 25 ° C to 75 ° C, most preferably from about 45 ° C to about 65 ° C for a period of time sufficient to carry out the conversion, typically from about 1 hour to about 24 hours, most preferably from about 1 hour to about 3 hours. After the conversion of the isoflavone conjugates of daidzein and daidzin to daidzein, daidzein can be extracted from the soybean substrate and separated from the extract as described above. The conversion of the isoflavone conjugates of daidzein and daidzin to daidzein significantly increases the amount of daidzein recoverable by the separation process, since a significant amount of the isoflavone conjugates of daidzein and daidzin are present in the soy materials. Daidzein can be administered to a human in a pharmaceutical formulation to lower the concentration of LDL cholesterol and increase the concentration of HDL cholesterol in the blood to reduce the risk of atherosclerosis and vascular disease. Pharmaceutical formulations incorporating daidzein obtained by any of the above methods or purchased from a commercial strong can be prepared by methods known in the art. For example, daidzein can be formulated into tablets, capsules, powders, suspensions, solutions for parenteral administration including intravenous, intramuscular and subcutaneous administration, and in solutions for application on patches for transdermal application with common and conventional vehicles, binders, diluents and excipients. .

Pharmaceutically acceptable inert carriers useful for forming pharmaceutical formulations according to the present invention include starch, mannitol, calcium sulfate, dicalcium phosphate, magnesium stearate, silicic derivatives and / or sugars such as sucrose, lactose and glucose. Binders include carboxymethylcellulose and other cellulose derivatives, gelatin, synthetic and natural gums including alginates such as sodium alginate, polyethylene glycol, waxes and the like. Diluents useful in the invention include a suitable oil, saline, sugar solutions such as aqueous dextrose or aqueous glucose, and glycols such as polyethylene or polypropylene glycol. Other excipients include lubricants such as sodium oleate, sodium acetate, sodium stearate, sodium chloride, sodium benzoate, talc and magnesium stearate, and the like; disintegrating agents including agar, calcium carbonate, sodium bicarbonate, starch, xanthan gum and the like; and adsorbent vehicles such as bentonite and kaolin. Dyeing and flavoring agents can also be added to the pharmaceutical formulations. Daidzein can also be administered to a human in a dietary supplement to lower the concentration of LDL cholesterol and increase the concentration of HDL cholesterol in the blood to reduce the risk of atherosclerosis and vascular disease. Dietary supplements that incorporate daidzein can be prepared by adding daidzein to a food in the process of preparing the food, regardless of the source of daidzein. Foods to which daidzein can be added include almost all foods. For example, daidzein can be added to foods that include, but are not limited to, meats such as ground meats, emulsified meats, marinated meats and meats injected with daidzein; beverages such as nutritional drinks, sports drinks, protein-fortified drinks, juices, milk, milk substitutes and beverages for weight loss; cheeses such as hard and soft cheeses, cream cheese and cottage cheese; frozen desserts such as ice cream, frozen milk, frozen low-fat desserts and non-dairy frozen desserts; yoghurts; soups; puddings; pastry products; Dressings for salads and dips and covers such as mayonnaise and potato dips. Daidzein is added to the food in a quantity selected to deliver a desired dose of daidzein to the food consumer. Daidzein can also be administered in a soy protein material rich in daidzein incorporated into a dietary supplement formulation to lower the concentration of LDL cholesterol and increase the concentration of HDL cholesterol in the blood to reduce the risk of atherosclerosis and vascular disease. One method for forming the daidzein-rich soy protein material from a commercially available defatted soy flake material is to extract the soy flake material with an aqueous alkaline solution, typically a calcium hydroxide solution or sodium hydroxide having a pH of from about 8 to about 10, preferably from about 9 to about 10, and separating the extraction material from the insoluble soy materials to form an aqueous extract containing daidzein, protein and other alkaline soluble components and Aqueous of the soy flake material. The extract is then treated with an acid, preferably a mineral acid, to lower the pH of the extract to about the isoelectric point of the protein, preferably to a pH of about 4 to about 5 and most preferably to a pH of about 4.4 to about 4.6, thus precipitating a protein rennet that captures a significant amount of daidzein from the extract. Preferably, the isoflavone conjugates of daidzein and daidzin are converted to daidzein as described above to increase the amount of daidzein captured in the precipitated protein rennet. The protein rennet is then separated from the extract, preferably by centrifugation, and dried to form the protein isolate. Preferably, unlike conventional processes for producing a protein isolate, the rennet is not washed with water or is washed with a minimum amount of water to minimize the loss of daidzein from the protein isolate.

Other methods for forming a soy protein material rich in daidzein include converting daidzin and daidzein isoflavone conjugates to daidzein as described above in a soy protein concentrate or in a soy whey protein material. The particular dose of daidzein that will be administered should be effective to reduce the concentration of LDL cholesterol and to increase the concentration of HDL cholesterol in the blood, and will depend on the route of administration and other risk factors to develop ateróeseleroeis or vascular disease, such as genetic predisposition to said disease and concentrations of cholesterol and lipids in plasma. The generally acceptable and effective daily doses may be from about 10 to about 1000 mg / day, typically from about 30 to about 500 mg / day and most preferably from about 50 to about 300 mg / day. Daidzein should be administered in an amount effective to increase the concentration of o-demethylangolensin in the blood of a human to whom daidzein is administered. Preferably, daidzein is also administered in an amount effective to increase the concentration of dihydrodaidzein in the blood of a human to whom daidzein is administered. O-demethylangolensin and dihydrodaidzein are both metabolites produced by the human body in the catabo of daidzein. See A Urinary Profile St? Dy of Dietary Phytoestrogens. The Identification and Mode of Metabo of New Isoflavanoids, Joannou et., J. Steroid Biochem. Molec. Biol .. Vol. 54, No. 3/4, p. 167-84 (1995), incorporated herein by reference. In the present invention it has been found that after administration of daidzein, the concentrations of both o-demethylangolensin and dihydrodaidzein in the blood, particularly o-demethylangolensin, are significantly correlated with an increase in HDL cholesterol levels and with a decrease in LDL cholesterol levels in the blood. The following non-limiting formulations illustrate pharmaceutical and dietetic formulations that include daidzein, and which may be used in accordance with the methods of the present invention.

FORMULATIONS The following formulations 1-4 illustrate pharmaceutical formulations including daidzein. In the formulations, "active ingredient" means daidzein.

Formulation 1 Gelatin Capsules Hard gelatin capsules are prepared using the following ingredients: active ingredient 10-1000 mg / capsule; starch, NF 0-600 mg / capsule; flowable starch powder 0-600 mg / capsule; Silicone fluid 350 centistoques 0-20 mg / capsule. The ingredients are mixed, passed through a sieve and filled into capsules.

Formulation 2 Tablets Tablets are prepared using the following ingredients: active ingredient 10-1000 mg / tablet; microcrystalline cellulose 20-300 mg / tablet; starch 0-50 mg / tablet; magnesium stearate or stearic acid 0-15 mg / tablet; fumed silicon dioxide 0-400 mg / tablet; colloidal silicon dioxide 0-1 mg / tablet and lactose 0-100 mg / tablet. The ingredients are mixed and compressed to form tablets.

Formulation 3 Suspensions Suspensions are prepared using the following ingredients: active ingredient 10-1000 mg / 5ml; Sodium carboxymethyl cellulose 50-700 mg / 5 ml; sodium benzoate 0-10 mg / 5 ml; 5 ml purified water and flavoring and coloring agents as needed.

Formulation 4: Parenteral solutions A parenteral composition is prepared by stirring 1.5% by weight of active ingredient in 10% by volume of propylene glycol and water. The solution is made isotonic with sodium chloride and is sterilized. The following formulations 5-8 illustrate dietary supplements that can be formed using an isolated soy protein rich in daidzein. The isolated soy protein rich in daidzein in the following examples typically contains between about 1 to about 3 milligrams of daidzein per gram of soy protein.

Formulation 5 Ready-to-drink beverage A ready-to-drink beverage is formed from the following ingredients: Ingredient Percentage of composition, by weight Water 80-85 Isolated soy protein 10-15 Rich in daidzein Sucrose 5-8 Cocoa 0.1-1 Vitamins / Minerals 0.1-1 Flavor 0.1-1 Cellulose Gel 0.1-0.5 The ready-to-drink beverage can be served in 8-ounce servings containing approximately 20 grams of isolated soy protein including approximately 20 to approximately 60 milligrams of daidzein.

Formulation 6 Powdered drink A powder drink is formed from the following components: Ingredient Percentage of composition, by weight Isolated soy protein 85-90 daidzein rich Sucrose 8-15 Maltodextrin 1-5 Vitamins / minerals 0.5-2 Aspartame 0-0.5 Flavoring 0-0.5 Thirty grams of the powder drink formulation can be added to water to form a portion which contains about 20 grams of isolated soy protein which includes about 20 to about 60 milligrams of daidzein.

Formulation 7 Food bar A food bar is formed from the following components: Ingredients Percentage of composition, by weight Isolated soy protein 20-30 rich in daidzein Corn syrup 35-45 Rice syrup solids 7-14 Glycerin 1- 5 Cocoa 2-7 Coating of compound 15-25 The food bar can be served in 70 gram portions containing approximately 15 grams of soy protein having approximately 15 to about 45 milligrams of daidzein.

Formulation 8 Soy Yogurt A soy yogurt is formed from the following ingredients: Ingredients Percentage of composition, by weight Water 65-75 Soy protein isolated 5-15 rich in daidzein Sucrose 3-8 Corn starch 1-5 Dextrin 0.3-1 Cellulose gel 1-3 Cultivation (yogurt) 0.01-0.1 Fruit 10-20 Vitamins / minerals 0.05-0.3 Soy yogurt can be served in a 170 gram serving containing approximately 8 grams of soy protein that has about 8 to about 24 milligrams of daidzein.

The following non-limiting test example illustrates the methods of the present invention.

EXAMPLE 1 A study of the effect of the isoflavones genistein, daidzein and glycitein on the concentrations of HDL cholesterol, non-HDL cholesterol and total cholesterol in the blood of post-enopausal women during a period of 6 months is carried out. The concentrations of isoflavones and their metabolites are statistically correlated with the resulting cholesterol levels to determine if there is a significant relationship between them. Sixty-one postmenopausal hypercholesterolemic women who had completed menopause for at least one year since the last menstrual period and who had a total plasma cholesterol of between 6.2 and 7.8 mmol / l are selected for inclusion in the statistical study. Two weeks before the start of the study, each subject completes a two-day dietary food record and is interviewed by a registered dietitian to calculate the daily energy expenditure of each individual for a low-fat, low-cholesterol diet from step 1 of the National Education Program about Cholesterol. Each subject is given a booklet published by the American Heart Association that contains a long list of foods, along with a calculated "fat prescription" that meets the criteria of the basic diet. All subjects follow the basic diet for a period of at least 14 days. After this, baseline blood samples are taken on two separate days, and the subjects are randomly assigned to one of the three dietary treatment groups. All three groups continue on the basic diet and incorporate 40 grammes of a test protein into the diet. The test protein ee selects either isolated soy protein containing moderate levels of isoflavones (SuproR 675 from Protein Technologies International, St. Louis, Missouri, which contains 1.39 mg of ieoflavone / g protein, wherein the isoflavones are genistein, daidzein, glycitein and their respective glycosides and conjugates of malonyl and acetyl) designated here as the "ISP" group; Isolated soy protein containing higher levels of isoflavones (2.25 mg isoflavone / g protein, wherein the isoflavones are genistein, daidzein, glycitein and sue glucosides and respective malonyl and acetyl conjugates) designated here as the "ISP +" group; or casein / dehydrated milk without fat (0 mg of total isoflavones / g of protein, New Zealand Milk Products, Wellington, New Zealand) designated here as the "casein" group. The ISP and ISP + proteins are reinforced with calcium to provide 800 to 900 mg of calcium per day in the form of calcium phosphate, an amount consistent with the amount of calcium provided through the daily components for the casein group. The isoflavone content of the ISP, ISP-t and casein proteins is shown in Table 1 below.

TABLE 1 Isoflavone Isoflavone of ISP ieoflavones of ieoflavones (mg / lOOg product) ISP + (mg / lOOg of casein) product) (mg / lOOg of product) Daidzin 16.8 40.1 0 6'-0-Mal 31.5 54.4 0 daidzin o'-O-Ac 0 5, .2 0 daidzin Daidzein 4.3 3.6 0 Daidzein 30.5 58.5 0 total (units of aglycine) rng / g of 0.44 0.82 0 Genistin protein 35.3 66.4 0 6'-0-Mal 58.9 80.4 0 genistin 6'-0-Ac 0 0 0 genistein Genistein 6.2 4.5 0 Genistein 59 87.9 0 total (aglycine units) TABLE 1 (cont.) Leoflavone Isoflavone of ISP ieoflavone of isoflavones (mg / lOOg product) ISP + (mg / lOOg of casein) product) (mg / lOOg of product) mg / g of 0.84 1.23 0 protein Glicitin 3 5.7 0 6'-0-Mal 5.5 8.2 0 glycitein Glicitein 3 7.2 0 Glicitein 7.8 0 total (aglycine units) mg / g 0.11 0.21 0 protein A combination of products are consumed by the subjects to provide the 40 g daily of test protein. A variety of recipes for confectionery products incorporating casein isolates and soy protein are developed, and the products are produced and administered to the subjects. Ready-mix beverages and soups containing isolated soy protein and casein are also used. Throughout the study, all subjects are instructed by a registered dietitian about the maintenance of body weight, physical activity, dietary requirements and consumption of acceptable foods and beverages. Every 4 weeks, 3 random days are chosen to include 2 days of the week and 1 weekend day in which subjects are asked to complete registration of dietary intake. The daily nutrient reagents are analyzed using a nutritional analysis software program (Nutritionist IV, version 3.0, N-Squared Computing, Salem Park, OR). The body weight is measured weekly by weighing a balance equipped with a beam balance that is calibrated weekly. The calculation of the daily physical activity is done using activity reports that subject them to comply with their 3-day feed intake record. In two separate days, at the end of the two-week adaptation period (baseline) and every 6 weeks for the duration of the 24-week study, after a 12-hour fast, blood samples are taken from the subject in tubes. that contain heparin, EDT and that do not contain anticoagulant. Plasma and serum are separated by centrifugation at 1190 x g for 15 minutes at 4 ° C. The levels of HDL, HDL2, HDL3 (together with the "HDL cholesterol"), total plasma cholesterol ("TC"), non-HDL cholesterol (LDL cholesterol + very low density lipoprotein cholesterol ("VLDL cholesterol"), are measured. the majority of the cholesterol that is not HDL being LDL cholesterol) and apolipoprotein Al and ba from the samples collected as follows. The HDL and HDL3 lipoprotein fractions are immediately separated by Mn2 + heparin precipitation and the plasma serum samples are stored in separate aliquots for subsequent analysis. The levels of plasma cholesterol ("TC"), HDL, HDL3 and total apolipopro ein A and B are quantified by automated techniques (Boheringer Mannheim Hitachi 704 Auto Analyzer, Boehringer Mannheim, Indianapolis, IN, Sigma Diagnoetics, St. Louis, MO; Raichem, San Diego, CA). The accuracy of the plasma measurements is verified by the quality control program, either from the CDC or the International Federation of Cereal Chemists (IFCC) of known concentrations (Northwest Lipid Reeearch Laboratories, Seattle, WA). The non-HDL coleeterol is calculated by re-assaying HDL of TC. The values of HDL2 are derived from the difference between the HDL3 and HDL3 fraction - The effects of the dietary supplement on total coleeterol, HDL coleeterol, and non-HDL cholesterol, as well as dietary intake, body mass index and physical activity are evaluated using Multiple linear regression analyzes. The effects of the treatment are indicated using two coded variables, one comparing the ISP + diet with the casein diet, and the second comparing the ISP diet with the casein diet. In each analysis, the baseline value of the variable is included in the model as a covariate. The treatment of the interaction effects of covariates has been proved by the method outlined by Weigel and Narvaez (Controlled Clinical Triáis, Vol. 12, pp. 378-94 (1991)). If there are no significant interaction effects, the interaction terms are retrieved from the model. The calculations of the regression model of normality and homogeneity of variance of residuals are evaluated by inspecting the graphs of residuals against the predicted values. The detection of the temporary onset of effects is carried out sequentially proving the presence of significant treatment effects at 18, 12 and 6 weeks, proceeding to the most initial time in sequence only when significant effects have been identified in each subsequent period. In addition, differences between groups in nutrient intake, physical activity, and body mass index (ht / wt2) at each time point are compared using one-way variance analysis. The changes of the baseline within each group are evaluated using paired T tests. In addition, the variance analysis is carried out on all baseline measurements and on measurable subject characteristics to determine homogeneity between groups. All the procedures have been conducted by Statietical Analyeye Syetem (SAS Inetitute Inc., Cary, N.C.). An alpha level of 0.05 is used in all tests and statistics. The one-way variance analysis indicates that there are no significant differences in dietary intake for any of the selected nutrients except for the protein. The protein consumption is significantly increased to 24 weeks compared to the baseline in all three groups (ISP, ISP + and casein). There are no significant changes in the body mass index or physical activity among the three groups. The measures evaluated for the effect of the ISP diet, the ISP + diet and the casein diet on total cholesterol, non-HDL cholesterol, HDL cholesterol, HDL2 cholesterol and HDL3 cholesterol are set forth in Table 2 below, along with the value of a normal deviation of the results statistically analyzed. An average difference ("AMD") between the values for each diet and a probability value ("p-value") are included.TABLE 2 Cholesterol ISP ISP + casein AMD total p-value (mmo 1 / L) week 0 6.57 ± .85 6.47 ± .88 6.26 ± .67 week 6 6.23 ± .91 6.08 ± .80 6.03 ± .70 not meaning - week 12 6.34 ± .98 6.13 ± .75 6.10 ± .65 not meaning - week 18 6.1011.11 6.13 ± .85 5.90 ± .62 does not mean - week 24 6.18 ± .91 6.13 ± .91 6.08 ± .72 not meaning - TABLE 2 (continued) Cholesterol not ISP ISP + Casein AMD value p HDL (mmol / L) week 0 5.22 ± .91 5.09 ± 1.03 4.86 ± .78 week 6 4.91 ± .96 4.78 ± .98 4.78 ± .78 not significant. -week 12 4.97 ± .96 4.78 ± .96 4.81 ± .67 not significant -week 18 4.73 ± 1.14 4.73 ± .98 4.58 ± .72 does not mean -Week 24 4.76 ± .93 4.71 ± 1.09 4.76 ± .83 -0.28 0.03 Cholesterol ISP ISP + caffeine AMD value p HDL (mmol / L) week 0 1.34 ± .27 1.38 ± .32 1.38 ± .31 week 6 1.32 ± .28 1.31 ± .32 1.26 ± .23 0.08 0.01 week 12 1.37 ± .27 1.32 ± .33 1.29 ± .26 0.11 0.02 week 18 1.38 ± 1.31 1.40 ± .32 1.32 ± .24 0.09 0.02 week 24 1.42 ± .31 1.42 + .31 1.32 ± .30 0.12 0.01 TABLE 2 (continued) Cholesterol ISP ISP + Casein AMD value p HDL2 (mmol / L) week 0 .30+ .17 .36 ± .23 .36 ± .18 week 6, 29 ± .19 .34 ± .21 .31 ± .15 not significant. - week 12 .32 ± .21 .35 ± .25 .28 ± .18 not significant - week 18 .29 ± .19 .34 ± .25 .29 ± .14 not eignif. - week 24 .29 ± .18 .34 ± .20 .29 + .17 not eignif. - Cholesterol ISP ISP + Casein AMD value p HDL3 (mmol / L) week 0 1.04 ± .24 1.02 ± .19 1.02 ± .19 week 6 1.03 ± .18 .97 ± .18 .96 ± .13 not significant. - week 12 1.04 ± .19 .99 ± .16 l.Ol ± .15 does not mean - week 18 Í.IO ± .21 1.06 ± .18 1.03 ± .15 0.05 0.05 week 24 1.13 ± .24 1.09 ± .21 1.03 ± .20 0.08 0.03 The results of the study indicate that the protein diet groups containing isoflavone have significantly increased concentrations of HDL coleeterol and have concentrations of non-HDL cholesterol significantly decreased relative to the control diet containing casein protein that is non-isoflavone. There is an increase of 5.2% in the concentration of HDL cholesterol in the patients who have an ISP diet and an increase of 3.6% with the ISP + diet. The increase in the concentration of HDL coleeterol during the period of treatment in eujetoe that contains the isoflavone diets is significantly statistically increased over that of those subjects who consume the casein control diet that does not contain isoflavones (p <0.05) . The concentration of non-HDL cholesterol during the treatment period in subjects consuming isoflavone diete statistically decreases significantly in relation to those who consume the casein control diet (p <0.05). To further evaluate the results of the study, the concentration of each isoflavone compound and sue metabolite in the blood of the patient was compared with the increase in HDL cholesterol and the decrease in non-HDL cholesterol (LDL) to determine if there is a correlation between Specific isoflavone and its metabolites and changes in blood cholesterol concentrations. The metabolites of daidzein include o-demethylangolensin ("o-DMA"), dihydrodaidzein ("DHD") and equol. The linear correlation analysis of isoflavone values in plasma against plasma lipoprotein values ee carried out in the samples taken from the eujetoe. Spearman's classification-order correlation analysis is carried out to eliminate the need to assume normality and to reduce the effect of data points. A correlation is generated between the change in values from 0 to 6 meeee and ee mueetra in table 3 below. The eetaditically significant referendae are marked in the table as follows: * indicates that p < 0.10; ** indicates that p < 0.05; *** indicates that p < 0.01.

TABLE 3 Cholesterol Ecuol Daidzein o- Genis- Isoflavone DMA DHD Total Thein Total -0.02 -0.02 -0.07 -0.17 0.00 -0.01 ("TC") No HDL -0.05 -0.13 -0.25 ** -0.29 *** -0.11 -0.13 HDL 0.20 0.22 * 0.30 ** 0.23 * 0.26 ** 0.27 ** HDL2 0.04 0.13 0.17 0.12 0.14 0.13 HDL3 0.19 0.22 * 0.30 ** 0.22 * 0.27 ** 0.29 ** TC / HDL -0.11 -0.24 ** -0.37 *** -0.33 *** -0.24 * -0.27 ** HDL / NHDL 0.13 0.26 ** 0.41 *** 0.36 *** 0.26 ** 0.31 ** As shown in Table 3, daidzein and its metabolites, as a group, are strongly related both to the increase in HDL cholesterol and to the decrease in non-HDL cholesterol including LDL cholesterol in the blood of subjects after completing the study . o-DMA, the compound to which daidzein is ultimately metabolized is particularly strongly associated with changing the levels of cholesterol in the blood of subjects. The above example shows the usefulness of daidzein to alter the concentration of coleeterol constituents in the blood of a human, and particularly in the blood of a postmenopausal woman by increasing the concentration of HDL coleeterol and decreasing the concentration of LDL cholesterol in the blood . It should be understood that the foregoing are merely preferred embodiments of the invention and that various ca bioe and alterations can be made without departing from the spirit and broader aspects of the limb as set forth in the appended claims, which should be interpreted in accordance with the principles of patent law, including the Doctrine of Equivalents.

Claims (36)

NOVELTY OF THE INVENTION CLAIMS

1. - The use of a daidzein material in the preparation of compositions to alter the concentration of lipoprotein cholesterol in the blood of a human by lowering the concentration of low density lipoprotein cholesterol and increasing the concentration of high density lipoprotein coleeterol in the blood of said human, to reduce the risk of atherosclerosis and vascular disease.

2. The use according to claim 1, further characterized in that said human is a postmenopausal woman.

3. The use according to claim 2, further characterized in that said woman is hypercholesterolemic.

4. The use according to claim 2, further characterized in that said woman is atherosclerotic.

5. The use according to claim 1, further characterized in that said human is hypercholesterolemic.

6. The use according to claim 1, further characterized in that said human is atherosclerotic.

7. The use according to claim 1, further characterized in that the administration of said composition of daidzein material causes an increase in the concentration of o-deemethylangolene in said blood of said human.

8. The use according to claim 1, further characterized in that the administration of said composition of daidzein material causes an increase in the concentration of dihydrodaidzein in said blood of said human.

9. The use according to claim 1, further characterized in that said composition of daidzein material obtained provides between about 10 mg to about 1000 mg per day, said daidzein material to said human.

10. The use according to claim 9, further characterized in that said composition of daidzein material obtained provides between about 30 mg to about 500 mg per day, of said daidzein material to said human.

11. The use according to claim 9, further characterized in that said composition of daidzein material obtained provides between about 50 mg to about 300 mg per day, of said daidzein material to said human.

12. The use according to claim 1, characterized in that said composition of daidzein material is administered to said human in a dietary supplement of soy protein material.

13. The use according to claim 12, further characterized in that said soy protein material is a material enriched with daidzein.

14. The use according to claim 12, further characterized in that said composition of daidzein material is administered to a human in a drink containing said eoya protein material.

15. The process according to claim 12, further characterized in that said composition of daidzein material is administered to a human in a food bar containing said soy protein material.

16. The use according to claim 12, further characterized in that said composition of daidzein material is administered to a human in a yogurt containing said soy protein material.

17. The use according to claim 1, further characterized in that said composition of daidzein material is administered to a human as a pharmaceutical composition.

18. The use according to claim 17, further characterized in that said pharmaceutical composition is a pill or a capsule.

19. The use of a daidzein material in the preparation of compositions to alter the concentration of a coleeterol lipoprotein in the blood of a human being by increasing the concentration of high density lipoprotein cholesterol in the blood of said human, in order to reduce the risk of atherosclerosis and vascular disease.

20. The use according to claim 19, further characterized because said human is a postmenopausal woman.

21. The ueo according to claim 20, further characterized in that said woman is hypercholesterolemic.

22. The use according to claim 20, further characterized in that said woman is atherosclerotic.

23. The use according to claim 19, further characterized in that said human is hypercholesterolemic.

24. The ueo according to claim 19, characterized in that said human is atherosclerotic.

25. The use according to claim 19, further characterized in that the administration of said daidzein composition causes an increase in the concentration of o-demethylangolensin in said blood of said human.

26. The use according to claim 19, further characterized in that the administration of said daidzein composition causes an increase in the concentration of dihydrodaidzein in said blood of said human.

27. The use according to claim 19, further characterized in that said composition of daidzein material obtained provides between about 10 mg to about 1000 mg per day, of said daidzein material to said human.

28. The use according to claim 27, further characterized in that said composition of daidzein material obtained provides between about 30 mg to about 500 mg per day, of said daidzein material to said human.

29. The use according to claim 27, further characterized in that said composition of daidzein material obtained provides between about 50 mg to about 300 mg per day, of said daidzein material to said human.

30. The use according to claim 19, further characterized in that said composition of daidzein material is administered to said human in a dietary supplement of soy protein material.

31. The use according to claim 30, further characterized in that said soy protein material is a material enriched with daidzein.

32. The use according to claim 30, further characterized in that said composition of daidzein material is administered to a human in a beverage containing said soy protein material.

33. The use according to claim 30, further characterized in that said composition of daidzein material is administered to a human in a food bar containing said soy protein material.

34. The use according to claim 30, further characterized in that said composition of daidzein material is administered to a human in a yogurt containing said soy protein material. 35.- The use according to claim 19, characterized. in addition because said composition of daidzein material is administered to a human as a pharmaceutical composition. 36. The use according to claim 35, further characterized in that said pharmaceutical composition is a pill or a capsule.