Product, containing glucomannan, xanthan gum and alginate for treatment of metabolic disorders

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry and represents clinical nutrition for prevention, treatment or relief of one or several symptoms, associated with impairment of metabolism or its disorder, which contains composition of polysaccharide high-viscosity dietary fibre, including viscous fibre mixture or its complex, consisting of from 48% to 90% in wt % of glucomannan, from 5 to 20 % in wt % of xanthan gum and from 5% to 30% in wt % of alginate, as well as, at least, one macroelement, selected from the group, consisting of protein carbohydrate and fat, where clinical nutrition is composed in order to provide dose of composition of polysaccharide high-viscosity dietary fibre from 20 g/day to 35 g/day for time period, effective for prevention, treatment and relief of one or several symptoms, associated with impairment of metabolism or its disorder.

EFFECT: invention ensures extension of arsenal of means, preventing, relieving or treating one or several symptoms, associated with impairment of metabolism or metabolic disease.

14 cl, 6 ex, 20 tbl, 48 dwg

 

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the priority according to the application No. 61/312,630, filed March 10, 2010 and application No. 61/357,658, filed June 23, 2010, the contents of which are incorporated herein by reference in full.

Field of the INVENTION

The invention relates to compositions of dietary fiber, medical nutrition products, compositions containing dietary fibers and their use to delay the onset and/or reducing the severity of metabolic syndrome and type II diabetes.

PRIOR art

Obesity and metabolic syndrome, the conditions that can lead to the development of type II diabetes, are becoming more and more common. The increase in visceral adiposity, serum glucose and insulin levels, along with hypertension and dyslipidemia, are a group of clinical conditions, which together are called metabolic syndrome (E. J. Gallagheretal., Endocrinol. Metab. Clin. NorthAm. 37:559-79 (2008)). It was found that the state data are the result of increasing the resistance of cells to insulin, and in many cases these symptoms are a precursor of type II diabetes. Currently, there are disputes about the exact diagnostic indicators that identify the metabolic syndrome, and drug p�aparaty for its treatment have not been approved, although related to dyslipidemia and hypertension is assigned to a special treatment with medicines. Type II diabetes is usually treated using different drugs to regulate sugars in the blood, and in more severe cases, insulin injections. However, diet and weight loss play a major role in correcting many of the metabolic abnormalities associated with metabolic syndrome and type II diabetes (Yipetal., ObesityRes. 9:341S-347S (2001)). The study showed that people suffering from metabolic syndrome, are at 50% greater risk of severe coronary attack (D. E. Moller et al., Annu. Rev. Meet. 56:45-62 (2005)). In this regard, any weight loss, fasting insulin and glucose will bring significant health benefits for those people who suffered from this disease.

It is known that eating foods with high-glycemic index leads to overeating and obesity (Ludwig et al., Pediatrics 103(3):E (1999)). Therefore, it is preferable that any substance used in the management of diabetic or pre-diabetic conditions as well as weight loss, had a low glycemic index. Most preferably, if such substances will lower the glycemic index of foods.

Reduction of carbohydrate intake is also necessary for the successful management of d�abioticheskie States. Counselling relating to nutrition benefits, but diabetics have a greater sense of hunger, because they experience more frequent condition of hypoglycemia (Strachan et al., Physiol. Behav. 80(5): 675-82 (2004)). Moreover, therapy, lowering levels of blood glucose in diabetic patients, are often associated with undesirable side-effect of increased body mass (Schultesetal., J. Clin. Endochnol. Metabol. 88(3): 1133-41 (2003)). It was reported that diets with a high content of soluble fiber can reduce the risk of diabetes through increased insulin sensitivity (Ylonen et al., Diabetes Care 26: 1979-85 (2003)). This may be due to the possible role of dietary fiber in the regulation of blood sugar. It was also reported that high viscosity food causes more saturation compared to low viscosity food (Marciani et al., Am. J. Physiol. Gastrointest. Liver Physiol. 280:01227-33 (2001)).

Thus, there is a need in the compositions of dietary fiber, which helps in managing metabolic syndrome, including diabetic condition by lowering the levels of blood sugar and promote satiety. The present invention addresses this and other needs.

Summary of the INVENTION

In one aspect, the invention proposes a medical nutrition product designed to prevent, treat or reduce the intensity of one or n�many symptoms, associated with metabolic disease or disorder. A medical nutrition product, in accordance with this aspect of the invention comprises a composition of a polysaccharide dietary fiber, high viscosity, containing a mixture of fibers ("VFB") high viscosity or complex ("VFC"), comprising from about 48% to about 90% (in/in) glucomannan, from about 5% to about 20% (W/W) xanthan gum and from about 5% to about 30% (W/W) alginate, and at least one mineral selected from the group consisting of a protein, carbohydrate and fat.

In another aspect, the present invention proposes a method of cooking a food product health food, containing the step of adding an effective amount of the dietary fiber composition has a high viscosity, containing a mixture of fibers of high viscosity (VFB) or complex ("VFC"), comprising glucomannan, xanthan gum, and alginate in food therapeutic food. In some embodiments of the invention the food product is a medical food designed to prevent, treat or reduce the intensity of one or more symptoms associated with a metabolic disease or disorder. In some embodiments of the invention the composition is a dietary fiber that is added to a food product health food, contains from about 48% to about 90% (V/V) CH�of komandan, from about 5% to about 20% (W/W) xanthan gum and from about 5% to about 30% (W/W) alginate.

In another aspect, the present invention proposes a method of preventing, treating or reducing the intensity of one or more symptoms associated with a metabolic disease or disorder. Method, in accordance with this aspect of the invention, comprises administering to the person who needs it, from about 25 mg/kg/day to about 1000 mg/kg/day of the composition is a highly viscous polysaccharide dietary fiber, containing a viscous fiber blend (VFB) or complex (VFC) containing from about 48% to about 90% (in/in) glucomannan, from about 5% to about 20% (W/W) xanthan gum and from about 5% to about 30% (in/in) alginate, effective during the period of time effective to prevent, treat or reduce the intensity of one or more symptoms associated with a metabolic disease or disorder in a subject.

In another aspect, the present invention proposes a method of reducing the intensity of at least one symptom associated with the progression of insulin resistance in a mammal suffering from a disease or at risk of developing diabetes type II. Method in accordance with this aspect of the invention contains an introduction �lecapitaine, which need it, from about 25 mg/kg/day to about 1000 mg/kg/day of the composition of polysaccharide dietary fiber, high viscosity, containing a mixture of fibers (VFB) high viscosity or complex (VFC) containing from about 48% to about 90% (in/in) glucomannan, from about 5% to about 20% (W/W) xanthan gum and from about 5% to about 30% (W/W) alginate, during the period, to constitute at least two weeks.

In another aspect, the present invention proposes a method of determining the component Sugars in the sample containing at least one polysaccharide. Methods according to this aspect of the invention contain: (a) hydrolysis of a sample containing at least one polysaccharide with acid in order to obtain a hydrolysate; (b) separation of hydrolysis products in the hydrolysate using a chromatographic method; (C) detection of hydrolysis products separated in stage (b); and (d) comparison of hydrolysis products detected at the stage (C) with one or more of the reference standards to determine the component Sugars in the sample.

Description of the DRAWINGS

The foregoing aspects and many of the inherent advantages of this invention will become more understandable, since they are better understood from the following detailed description in conjunction with the accompanying drawings�, in which:

FIGURE 1A graphically depicts the effect of diet containing VFC, cellulose, or inulin on body weight (g) over time during the eight-week study on diabetic Zucker rats as described in example 1;

FIGURE 1 graphically depicts the effect of diet containing VFC, cellulose, or inulin, the consumption of foodstuffs (g/day) over time during the 8-week study in diabetic Zucker rats as described in example 1;

FIGURE 2A graphically depicts the effect of diet containing VFC, cellulose, or inulin, the levels of fasting blood glucose (mg/DL) over time during the 8-week study in diabetic Zucker rats as described in example 1;

FIGURE 2B graphically depicts the effect of diet containing VFC, cellulose, or inulin on serum levels of fasting insulin (ng/ml) over time during the 8-week study in diabetic Zucker rats as described in example 1;

FIGURE 2C graphically depicts the effect of diet containing VFC, cellulose, or inulin, glucose levels in the blood are not fasting (mg/DL) over time during the 8-week study in diabetic Zucker rats as described in example 1;

FIGURE 2D graphic�Ki portrays the impact of diet, containing VFC, cellulose, or inulin, in terms of the homeostatic model assessment (HOMA), fasting (mg*IU/ml2) over time during the 8-week study in diabetic Zucker rats as described in example 1;

FIGURE 3A graphically depicts the performance of the generalized index of insulin sensitivity (CISI) regarding diabetic Zucker rats in the fasting state, fed, containing VFC, cellulose, or inulin, during the 8-week study, as described in example 1;

FIGURE 3B graphically depicts the performance of the generalized index of insulin sensitivity (CISI) regarding diabetic Zucker rats not fasting, fed, containing VFC, cellulose, or inulin, during the 8-week study, as described in example 1;

FIGURE 3C plots the indicators regarding NOMA diabetic Zucker rats not fasting, fed, containing VFC, cellulose, or inulin, during the 8-week study, as described in example 1;

FIGURE 4 graphically depicts serum levels of triglycerides measured in diabetic Zucker rats in the fasting state, fed, containing VFC, cellulose, or inulin, during the 8-week study, as described in example 1;

FIGURE 5A graphically depicts the effect of dietary p�Tania, containing VFC, cellulose, or inulin, diabetic Zucker rats after 8 weeks on the dilation of the renal tubule on the basis of histological figure of 0-5, where 5 is the most severe indicator as described in example 1;

FIGURE 5B graphically depicts the effect of diet containing VFC, cellulose, or inulin, diabetic Zucker rats after 8 weeks on the degeneration/regeneration of the renal tubule on the basis of histological figure of 0-5, where 5 is the most severe indicator as described in example 1;

FIGURE 5C graphically depicts the effect of diet containing VFC, cellulose, or inulin, diabetic Zucker rats after 8 weeks on mesangial expansion kidneys on the basis of histological figure of 0-5, where 5 is the most severe indicator as described in example 1;

FIGURE 6 graphically depicts the percentage of the area of immunoreactive insulin pancreatic islet present in diabetic Zucker rats treated with diet containing VFC, cellulose, or inulin, at the end of 8 weeks study that determined by staining using Anticriminal insulin antibody as described in example 1;

FIGURE 7A graphically depicts histological indicator regarding infiltration�in mononuclear inflammatory cells of the pancreatic islet present in diabetic Zucker rats treated with diet containing VFC, cellulose, or inulin, at the end of the 8-week study based on the histological figure of 0-5, where 5 is the most severe indicator as described in example 1;

FIGURE 7B graphically depicts histological indicator regarding cellular degeneration of pancreatic islet that is present in diabetic Zucker rats treated with diet containing VFC, cellulose, or inulin, at the end of the 8-week study based on the histological figure of 0-5, where 5 is the most severe indicator as described in example 1;

FIGURE 7C graphically depicts histological indicator concerning the amount of fibrosis pancreatic islet present in diabetic Zucker rats treated with diet containing VFC, cellulose, or inulin, at the end of the 8-week study based on the histological figure of 0-5, where 5 is the most severe indicator as described in example 1;

FIGURE 8A graphically depicts the effect of diet containing VFC, cellulose, or inulin, diabetic Zucker rats after 8 weeks on hepatic steatosis as measured by reduced staining with Sudan black in the histological basis until�Atelier, gap of 0-5, where 5 is the most severe indicator as described in example 1;

FIGURE 8B graphically depicts the effect of diet containing VFC, cellulose, or inulin, diabetic Zucker rats after 8 weeks on hepatic microvesicular the vacuolization on the basis of histological figure of 0-5, where 5 is the most severe indicator as described in example 1;

FIGURE 8C graphically depicts the effect of nutrition containing VFC, cellulose, or inulin, diabetic Zucker rats after 8 weeks on hepatic macrovesicular the vacuolization on the basis of histological figure of 0-5, where 5 is the most severe indicator as described in example 1;

FIGURE 9 graphically depicts the impact of VFC or pulp to increase body weight and serum triacylglycerols (TAG) in rats Sprag-doli treated with sucrose, during the 43-week study, as described in example 2;

FIGURE 10A graphically depicts the impact of VFC or control (skim milk powder) at the levels of PYY in plasma for all healthy adult study participants during a 3-week study period (V1=start of study day 0; V2=day 14; V3=21 days), as described in example 4;

FIGURE 10B graphically depicts the impact of VFC or control (dry about�sirenne milk) on the levels of PYY in plasma in healthy adult study participants with a BMI< 23 during the 3-week study period (V1=start of study day 0; V2=day 14; V3=21 days), as described in example 4;

FIGURE 10C plots the impact of VFC or control (skim milk powder) at the levels of fasting ghrelin in healthy adult study participants during a 3-week period (V1=start of study day 0; V2=day 14; V3=21 days), as described in example 4;

FIGURE 11A graphically depicts the comparison of flow curve form of pellets VFB (referred to as a three-component mixture of 1 ("TM1") and processed (e.g., granulated) VFC (PGX®) at 0.5% (V/V) as described in example 6;

FIGURE 11 graphically depicts a comparison of flow curve form of pellets VFB (referred to as a three-component mixture of 1 ("TM1") and processed (e.g., granulated) VFC (PGX®) at 0.2% (V/V) as described in example 6;

FIGURE 11C plots the comparison of flow curve form of pellets VFB (referred to as a three-component mixture of 1 ("TM1") and processed (e.g., granulated) VFC (PGX®) at 0.1% (V/V) as described in example 6;

FIGURE 12A graphically depicts a comparison of the power-law K form of pellets VFB (TM1), processed (e.g., granulated) VFC (PGX®) and xanthan gum as described in example 6;

FIGURE 12B graphically depicts a comparison of the power-law η agranular�Anna VFB (TM1), processed (e.g., granulated) VFC (PGX®) and xanthan gum as described in example 6;

FIGURE 13A graphically depicts the flow curve of brandy glucomannan at 0.1%, 0.2% and 0.5% (V/V), as measured at 25°C as described in example 6;

FIGURE 13C plots the curve of the flow of xanthan gum at 0.1%, 0.2% and 0.5% (V/V), as measured at 25°C as described in example 6;

FIGURE 13C plots the curve of the flow of sodium alginate at 0.1%, 0.2% and 0.5% (V/V), as measured at 25°C as described in example 6;

FIGURE 14A plots the curve of the flow of unheated water solutions (0.5% concentration) of three-component mixtures containing brandy glucomannan, xanthan gum and sodium alginate containing brandy glucomannan (km) and xanthan gum (XG) at a constant ratio (KM:XG=4,12:1) and variable amounts of sodium alginate(0%, 2%, 5%, 8%, 11%, 13%, 17%, 21%, 24%, 27%, 30% and 33%), measured at 25°C as described in example 6;

FIGURE 14B graphically depicts the curve of the flow of aqueous solutions (0.5% concentration), heated for 1 hour ternary mixtures containing brandy glucomannan, xanthan gum and sodium alginate containing brandy glucomannan (km) and xanthan gum (XG) at a constant ratio (KM:XG=4,12:1) and variable amounts of sodium alginate (0%,2%, 5%, 8%, 11%, 13%, 17%, 21%, 24%, 27%, 30% and 33%), measured at 25°C as described in example 6;

FIGURE 14C graphically depicts the curve of the flow of aqueous solutions (0.5% concentration), heated for 4 hours three-component mixtures containing brandy glucomannan, xanthan gum and sodium alginate containing brandy glucomannan (km) and xanthan gum (XG) at a constant ratio (KM:XG=4,12:1) and variable amounts of sodium alginate(0%, 2%, 5%, 8%, 11%, 13%, 17%, 21%, 24%, 27%, 30% and 33%), measured at 25°C as described in example 6;

FIGURE 15A plots the dependence is proportional To the sodium alginate in the mixture regarding unheated or heated (one hour) 0.5% aqueous solutions of mixtures of brandy glucomannan, xanthan gum and sodium alginate in a constant ratio KM:XG (4,12:1) and variable amounts of alginate (from 0% to 33%), as described in example 6;

FIGURE 15B graphically depicts the dependence of n is proportional to the sodium alginate in the mixture on unheated and heated (one hour) 0.5% aqueous solution of mixtures of brandy glucomannan, xanthan gum and sodium alginate in a constant ratio KM:XG (4,12:1) and variable amounts of alginate (from 0% to 33%), as described in example 6;

FIGURE 16A plots the concentration distribution of apparent sedimentation g*(s) vs s regarding glucomannan when the concentration of n�the load of 2 mg/ml and I=0,0, when the speed of rotation of the rotor 45 OOO rpm, temperature = 20,0°C. the Ordinate is expressed in stripes in Svedberg (S) and the abscissa in units of Svedberg, as described in example 6;

FIGURE 16 graphically depicts the concentration distribution of apparent sedimentation g*(s) vs s regarding sodium alginate at a concentration of load of 2 mg/ml and I=0,0, if the speed of rotation of the rotor 45 OOO rpm, temperature = 20,0°C. the Ordinate is expressed in stripes in Svedberg (S) and the abscissa in units of Svedberg, as described in example 6;

FIGURE 16C graphically depicts the concentration distribution of apparent sedimentation g*(s) vs s regarding xantana with stress-concentration 2 mg/ml and I=0,0, if the speed of rotation of the rotor 45 OOO rpm, temperature = 20,0°C. the Ordinate is expressed in stripes in Svedberg (S) and the abscissa in units of Svedberg, as described in example 6;

FIGURE 17A plots of the concentration distribution of apparent sedimentation on rough/granular VFB (referred to as "TM1") with ionic forces of 0-0. 2 M, as described in example 6;

FIGURE 17 graphically depicts the concentration distribution of apparent sedimentation on rough/granular VFB (referred to as "TM1") with ionic forces of 0-0. 01 M, as described in example 6;

FIGURE 17C graphically depicts the concentration distribution to�Judaica sedimentation on processed/granulated VFC (PGX ®with ionic forces of 0-0. 01 M, as described in example 6;

FIGURE 17D graphically depicts the concentration distribution of apparent sedimentation on processed/granulated VFC (PGX®with ionic forces of 0-0. 2 M, as described in example 6;

FIGURE 18A graphically depicts the effect of ionic strength (expressed in units of molar concentration M) on the amount of material with a sedimentation coefficient of the > 3,5 S on rough/granular VFB (TM1) as described in example 6;

FIGURE 18B graphically depicts the effect of ionic strength (expressed in units of molar concentration M) on the amount of material with a sedimentation coefficient of the > 3,5 S regarding processed/granulated VFC (PGX®as described in example 6;

FIGURE 19A graphically depicts the distribution of the sedimentation coefficient on unheated mixtures containing a constant ratio of glucomannan:xantana (KM:XG=4,12:1) and varying concentrations of alginate (from 0% to 33%), as described in example 6; and

FIGURE 19B graphically depicts the distribution of the sedimentation coefficient on a heated (1 or 4 hours) mixtures containing a constant ratio of glucomannan: xantana (KM:XG=4,12:1) and varying concentrations of alginate (from 0% to 33%), as described in example 6.

DETAILED DESCRIPTION

In the present invention p�adlouni food additives, products medical nutrition and more effective ways to delay the occurrence of, slowing the progression and/or reduce the intensity of at least one symptom of a metabolic disease or disorder, such as metabolic syndrome, type I diabetes, type II diabetes, pancreatic disease and/or hyperlipidemia.

In this context, the term "metabolic syndrome" refers to one or more of the following symptoms: increased visceral adiposity, serum glucose and insulin levels, along with hypertension and dyslipidemia (E. J. Gallagher et al., Endocrinol. Metab. Clin. NorthAm. 37:559-79 (2008)). Metabolic syndrome is the name of a group of symptoms that occur together and are associated with an increased risk of developing coronary heart disease, stroke and type II diabetes. Symptoms of metabolic syndrome include excess weight around the waist (Central or abdominal obesity), high blood pressure, high triglycerides, insulin resistance, low cholesterol, high density lipoprotein (HDL) and tissue damage caused by high glucose. It is believed that insulin resistance is the main cause of metabolic syndrome.

In this context, the term "reducing the intensity of at least one of the symptoms of metabolic Zab�the indicated or disorder" includes symptomatic therapy for attenuating, removing or masking the symptoms of the disease or disorder and therapies to prevent, decrease, stop or reverse the progression of the severity of the condition or symptoms to be treated. In this regard, the term "treatment" includes medical therapeutic treatment of the established condition or symptoms and/or prophylactic administration of a medicinal product, if necessary.

In this context, the term "treating" also encompasses, depending on the condition of a subject who is in need, prevention of metabolic disease or disorder, or preventing one or more symptoms associated with the pathology of metabolic disease or disorder, including the emergence of a metabolic disease or disorder or any symptoms associated with them, as well as reducing the severity of metabolic disease or disorder, or the prevention of recurrence of one or more symptoms associated with a metabolic disease or disorder.

In this context, the term "medical nutrition product" refers to a product which is designed for human consumption or enteral administration under the supervision of a physician and which is intended for special dietary treatment of a disease or condition for which distinctive �iMovie needs based on recognized scientific principles, are established by medical evaluation.

In this context, the term "glucomannan" refers to water-soluble dietary fiber with residues of β-(1,4)-linked D-mannose and β-(1,4)-linked D-glucose in a ratio of about 3:1 and different α-linked terminal galactose groups. It is usually extracted from the root of the cognac plant Amorphophallus konjac, but it can also be extracted from other plant sources.

In this context, the term "xanthan gum" refers to the heteropolysaccharide containing glucose, mannose, potassium or sodium glucuronate, acetate and pyruvate.

In this context, the term "alginate" refers to a mixed polymer mannuronate acid and guluronic acid.

In this context, the term "mixture" fiber refers to a fiber blend.

In this context, the term "viscous fiber blend" ("VFB") refers to a mixture of glucomannan, xanthan gum, and alginate.

In this context, the term "viscous fiber complex" ("VFC") refers to an interactive matrix of the three components of glucomannan, xanthan gum, and alginate in which the components are processed in a manner (e.g., granulation), which allows their interaction with the aim of creating a new ingredient, not a mixture of three separate components by creating a second�link and tertiary interactions (connections and networks) between raw ingredients which prevent the individual components from the existence of properties that each of them would show in its pure state.

Products medical nutrition

In one aspect, the present invention proposed medical nutrition products, developed to prevent, treat or reduce the intensity of one or more symptoms associated with a metabolic disease or disorder, such as metabolic syndrome, type I diabetes or type II diabetes mellitus, exocrine pancreatic insufficiency, including patients suffering from chronic pancreatitis and/or hyperlipidemia. A medical nutrition product, in accordance with this aspect of the invention comprises a composition highly viscous polysaccharide dietary fiber, containing a viscous fiber blend (VFB) or complex (VFC) containing from about 48% to about 90% (in/in) glucomannan, from about 5% to about 20% (W/W) xanthan gum and from about 5% to about 30% (W/W) alginate, and at least one mineral selected from the group consisting of protein, carbohydrate and fat.

As described in the application for a US patent pending, No. 11/400,768, filed on 7 April 2006, and in the application for a US patent pending, No. 11/830,615, filed July 30, 2007, the contents of which are included in this document in the order the links above�, developed a composition highly viscous polysaccharide dietary fiber, containing a mixture of fibers (VFB) or complex (VFC), produced by combining from about 48% to about 90% (in/in) glucomannan, from about 5% to about 20% (W/W) xanthan gum and from about 5% to about 30% (W/W) alginate, sold under the name "PolyGlycopleX®or PGX®"that has a very high water-holding capacity and gel-forming property. The components of the polysaccharide components of this composition fibers complement each other and act synergistically with the aim of creating strong interactions that lead to the level of viscosity, which is three to five times higher than any other currently known polysaccharide. As described in examples 5 and 6 in this document, it was determined that after processing (e.g. pelleting) three components - glucomannan, xanthan gum and alginate - interact to create a new ingredient (complex ("VFC")) and not a mixture of 3 separate components through the creation of secondary and tertiary interactions (zones of connections and networks between raw ingredients, which prevent the individual components from the existence of properties that each of them would show in its pure state.

The song you�Okavango dietary fiber causes a significant increase in the viscosity of the contents in the gastrointestinal tract at a lower gravimetric quantity than the gravimetric quantity that would be necessary with other soluble fibers. This highly concentrated property allows this composition to the fibers to cause significant physiological effects at doses that are significantly lower than other soluble fibers, making it thus more convenient to include significant quantities of this material in food products.

In one embodiment of the invention, the polysaccharides used in the production of a mixture of viscous fiber (VFB), processed by granulation with the goal of producing interactive matrix of the three components (i.e., complex (VFC)). In this context, "granulation" refers to any process of increasing size, in which small particles are gathered together into larger, permanent clusters. Granulation may be accomplished through mixing equipment for mixing operations by compaction, extrusion or globulinovoy. The composition of dietary fiber can be granulated using different sizes of mesh. The term "cell sieve" refers to a particle size that is defined by its ability to pass through a screen having openings of a given size. The size of the mesh used in this paper are equivalent�alenty Tyler as outlined in table 21-12 Chemical Engineers Handbook (5th edition, publishing house Perry &Chilton). The more granulation (i.e., the smaller the size of the mesh cell) song/complex dietary fiber, the more time is required to obtain the desired viscosity. In some embodiments, the composition/complex granulated dietary fiber when using the combined mesh by separating granular material according to the size of the particles, and then re-uniting the separated granules with one particle size to obtain a predetermined viscosity profile. For example, the combined size of the mesh cell, comprising from 30 to 60, obtained by combining the granules constituting 30 mesh (about 600 microns), granules, constituting about 40 mesh (about 400 microns), and granules comprising about 60 mesh (250 micron).

The proportions of glucomannan, xanthan gum, and alginate in the mixture/complex viscous dietary fiber (VFB/C), characteristic of the product health food, may be from about 48% to about 90% of glucomannan (for example, from about 60% to about 80% or from about 60% to about 90%, or from about 65% to about 75%, or from about 50% to about 80%, or from about 50% to about 70%, or about 70%), from about 5% approximately 20% of xanthan gum (e.g., from about 10% to about d� 20% or from about 11% to about 13%, or from about 13% to about 17%, or approximately 13%, or approximately 17%) and from about 5% to about 30% of alginate (e.g., from about 10% to about 20% or from about 13% to about 17%, or approximately 13%, or approximately 17%). In some embodiments of the invention the proportions of glucomannan, xanthan gum, and alginate in food compositions contained in the product of medical nutrition, account for approximately 70% of glucomannan, from about 13% to about 17% xantana and from about 13% to about 17% alginate.

In some embodiments of the invention, the medical nutrition products are developed with the aim of providing a total daily human consumption from 1.0 g to 100 g of a mixture of viscous fiber or complex (VFB/C), comprising from about 48% to about 90% (in/in) glucomannan, from about 5% to about 20% (W/W) xanthan gum and from about 5% to about 30% (W/V) alginate (VFB/C), for example, from about 5 g to about 50 g VFB/C per day, for example, from about 10 g to about 35 g VFB/C per day, from about 12 g to 35 g VFB/C per day or, for example, from about 15 g to 35 g VFB/C per day, for example, from about 20 g to 35 g VFB/C per day, for example, from about 12 g to about 25 g VFB/C per day, for example, from about 15 g to about 25 g VFB/C per day. In some embodiments of the invention, the medical nutrition products are designed to provide daily doses VFB/C the person drawing up�owns about 25 mg/kg/day to about 1000 mg/kg/day, for example, from about 50 mg/kg/day to about 600 mg/kg/day, e.g., from about 100 mg/kg/day to about 500 mg/kg/day, e.g., from about 200 mg/kg/day to about 400 mg/kg/day.

Products medical foods of the invention can optionally contain additional components, such as proteins or amino acids, carbohydrates, lipids, vitamins, minerals and cofactors, natural and artificial flavors, dyes or other coloring additives and conservatives. The term "vitamins" includes, among other things, thiamine, Riboflavin, nicotinic acid, Pantothenic acid, pyridoxine, Biotin, folic acid, vitamin B12, lipoic acid, ascorbic acid, vitamin a, vitamin D, vitamin E and vitamin K. the term "vitamins" are also included cofactors and coenzymes, for example, coenzymes, including tiaminpirofosfata (RTR), FMN (FMM), fad (FAD), nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), coenzyme A (COA), pyridoxal phosphate biocytin, tetrahydrofolic one acid, coenzyme, lipoyllysine, 11-CIS-retinal and 1,25-dihydroxycholecalciferol. The term "vitamins" also includes choline, carnitine, and alpha, beta and gamma carotenes. The term "mineral" refers to inorganic substances, metals and the like required for RA�ion power includes, among other things, calcium, iron, zinc, selenium, copper, iodine, magnesium, phosphorus, chromium, manganese, potassium and the like, and mixtures thereof. The mineral may be in the form of a salt, oxide or chelated salt.

In some embodiments of the invention, the products of medical foods of the invention additionally contain one or more lipids. In the context of in accordance with this embodiment of the invention, the lipid is defined as a substance, such as fat, oil or wax that dissolves in alcohol but not in water. In this context, the terms "fat" and "oil" are used interchangeably and contain fatty acids. In some embodiments of the invention, the lipid for use in the composition contains fat, selected from the group consisting of milk fat (e.g. milk fat, fat, oil), animal fat (e.g., lard) or vegetable fat (e.g., coconut oil, cocoa butter, palm oil or margarine).

In some embodiments of the invention, the lipid for use in medical nutrition products of the invention contains an edible oil or mixture of oils. Such oils include vegetable oils (such as canola oil, soybean oil, palm kernel oil, olive oil, safflower oil, sunflower oil, Flaxseed (linseed oil), corn oil, cottonseed oil, peanut oil, nut�OE oil, almond oil, grapeseed oil, evening primrose oil, coconut oil, borage oil, black currant oil); oil from sea foods (e.g., fish oils and fat from fish liver) or mixtures thereof.

In some embodiments of the invention, the lipid for use in medical nutrition products of the invention contains oils that contain medium chain triglycerides, such as coconut oil, palm kernel oil, or medium chain triglycerides in purified form.

In some embodiments of the invention in the products medical foods of the invention proposed is the only source of calories or nutrients to a patient. In some embodiments of the invention, the medical product supply in accordance with the invention is designed to provide the primary source of fiber in human nutrition. In some embodiments of the invention, the medical product supply in accordance with the invention is designed to provide a sole source of fiber in human nutrition and labeled and/or inputted by the doctor accordingly.

Products medical foods that are consumed as part of a complete, balanced diet, are usually developed with the aim to replace one or more meals during the day, thereby reducing the amount of fibers isopycnic products. Due to the fact that the products are medical foods are introduced under a doctor's supervision, it is unlikely that patients will consume more dietary supplements containing fiber.

Products medical foods of the present invention are intended for use by a separate population of patients who are on treatment and under a doctor's supervision. Products medical foods of the invention can be entered mammal, for example, a person suffering from disease or increased risk of development of metabolic condition, to prevent, treat or reduce the intensity of one or more symptoms associated with a metabolic disease or disorder, such as metabolic syndrome (also known as syndrome X syndrome and insulin resistance), diabetes type I, diabetes type II, obesity, non-alcoholic hepatic steatosis (fatty liver), pancreatic disease, and hyperlipidemia, as further described in this document.

In some embodiments of the invention, the product of medical foods of the invention is administered to a subject in need of this at least once a day. In some embodiments of the invention, the product of medical foods of the invention is injected twice daily, preferably once in the morning and once in the afternoon/evening. Normal treatment regarding �of roductos therapeutic feeding will continue for at least two weeks to eight weeks or longer. Depending on factors such as the medical condition to be treated and the patient's response, the treatment can be extended as long as the patient will not diminish the intensity of at least one symptom of the disease or disorder. The product of medical foods of the present invention will usually be consumed two servings per day as a meal replacement or snack between meals. In some embodiments of the invention, the product of medical foods of the invention is administered to the subject as the only source of food three to four times per day, as part of the controlled from a medical point of view diet very low in calories. Typical utilization of nutrition with very low calories content is used in the treatment of obesity to achieve rapid weight loss and reduction of cardiometabolic risk factors.

Methods for preparing therapeutic food

In another aspect, the present invention proposes a method for preparing a medical nutrition product, containing the step of adding an effective amount of a dietary fiber composition containing viscous fiber mixture (VFB) or complex (VFC), comprising glucomannan, xanthan gum and alginate, a product of medical foods. In some embodiments of the invention, the method of prigotovlenie� product therapeutic feeding includes the step of adding an effective amount of the composition of dietary fiber, containing complex fiber (VFC), developed from a viscous mixture of fibers (VFB), comprising glucomannan, xanthan gum and alginate, a product of medical foods.

In some embodiments of the invention the product is a medical food designed to prevent, treat or reduce the intensity of one or more symptoms associated with a metabolic disease or disorder. In some embodiments of the invention the composition is a dietary fiber that is added to the product of medical nutrition, contains a mixture of fibers (VFB) or complex fiber (VFC), developed from a mixture of fibres (for example, granulated VFB) containing from about 48% to about 90% (in/in) glucomannan (for example, from about 60% to about 80% or from about 60% to about 90%, or from about 65% to about 75%, or from about 50% to about 80%, or from about 50% to about 70%, or about 70%), from about 5% to about 20% (W/W) xanthan gum (e.g., from about 10% to about 20% or from about 11% to about 13%, or from about 13% to about 17%, or approximately 13%, or approximately 17%) and from about 5% to about 30% (W/V) alginate (for example, from about 10% to about 20% or from about 13% approximately 17%, or approximately 13%, or approximately 17%). In some embodiments of the invention the proportions of glucomannan, xanthan gum, and alginate in the mixture of fibers or in the comp�EXE fiber, created from a mixture of fiber, dietary fiber composition, which is added to the product of medical nutrition, contains about 70% of glucomannan, from about 13% to about 17% xantana and from about 13% to about 17% alginate.

In some embodiments of the invention, the amount of dietary fiber composition containing viscous fiber mixture (VFB) or complex (VFC), added to the medical nutrition product designed for the treatment or prevention of metabolic diseases or disorders, from approximately 5% to approximately 20% of the total weight of the product of medical nutrition. In some embodiments of the invention, the amount of dietary fiber composition or complex (VFB/C) is added to the product of medical nutrition, contains from about 1 g to 100 g per day, e.g. from about 5 g to 50 g per day, from about 10 g to 35 g per day, e.g. from about 12 g to 35 g per day, e.g. from about 15 g to 35 g per day, for example, from about 20 g to 35 g per day, for example, from about 12 g to about 25 g per day, e.g. from about 15 g to about 25 g per day, based on the consumption of two servings per day. Products medical foods of the invention are customarily used at least once a day, preferably two or three times a day. A medical nutrition product, in accordance with the present invention is intended for oral�about the introduction.

The composition of the dietary fiber containing the fiber mixture or complex, can be combined with any type of medical nutrition product, including solid, liquid or semi-solid foods medical foods. Typical solid foods medical foods include, inter alia, cereals (e.g., rice, cereal (hot or cold)), granola, oatmeal, baked goods (e.g. bread, cookies, muffins, pies, and other), pasta (including noodles made from rice or other cereals), meat (e.g., poultry, beef, lamb, pork, seafood) and dairy products (e.g. milk, yoghurt, cheese, ice cream and butter). Typical liquid or semi-liquid products medical foods include, inter alia, drinks, meal replacements, fruit juices, soups (including dry soup mix), dietary supplements and smoothies.

The composition of the dietary fiber containing the fiber mixture or complex, you can add in the product of medical foods before consumption, using any suitable method. For example, the dietary fiber composition can be baked in the medical nutrition product, can be mixed with the product of medical foods or sprinkle on medicinal product supply.

Products medical foods of the invention are packaged in standard�nye dose markings clearly indicating that the product is intended for use in the management for special metabolic disease or disorder under medical supervision.

Ways to prevent, treat or reduce the intensity of one or more symptoms associated with a metabolic disease or disorder

In another aspect, the present invention proposes a method of preventing, treating or reducing the intensity of one or more symptoms associated with a metabolic disease or disorder, such as metabolic syndrome, type I diabetes, type II diabetes, obesity, non-alcoholic hepatic steatosis (fatty liver), pancreatic disease, and hyperlipidemia. Method in accordance with this aspect of the invention provides an introduction to someone else in need of this, an effective dose of a high-viscosity composition of polysaccharide dietary fiber, viscous mixture containing fiber (VFB) or complex (VFC) containing from about 48% to about 90% (in/in) glucomannan (for example, from about 60% to about 80% or from about 60% to about 90%, or from about 65% to about 75%, or from about 50% to about 80%, or from about 50% to about 70%, or about 70%), from about 5% to about 20% (W/W) xanthan gum (e.g., from about 10% to about 20% or from about 11% �rimero to 13%, or from about 13% to about 17%, or approximately 13%, or approximately 17%) and from about 5% to about 30% (W/V) alginate (for example, from about 10% to about 20% or from about 13% to about 17%, or approximately 13%, or approximately 17%). In some embodiments of the invention the proportions of glucomannan, xanthan gum, and alginate in the mixture of fiber or complex, constitute about 70% of glucomannan, from about 13% to about 17% xantana and from about 13% to about 17% alginate.

In some embodiments of the invention, the method includes the introduction of the dietary fiber composition containing viscous fiber mixture (VFB) or complex (VFC, for example, granulated VFB) containing from about 48% to about 90% (in/in) glucomannan, from about 5% to about 20% (W/W) xanthan gum and from about 5% to about 30% (W/W) alginate, someone who needs it, at a dose, of from 1.0 g to 100 g VFB/C per day, for example, from about 5 g to about 50 g VFB/C per day, for example, from about 10 g to about 35 g VFB/C per day, from about 12 g to 35 g VFB/C per day or, for example, from about 15 g to 35 g VFB/C per day, for example, from about 20 g to 35 g VFB/C per day, for example, from about 12 g to about 25 g VFB/C per day, for example, from about 15 g to about 25 g VFB/C per day.

In some embodiments of the invention, the method includes introducing a mixture of dietary fibers (VFB) or complex (VFC) MLE�opiausiu, for example someone who needs it, at a dose of approximately 25 mg/kg/day to about 1000 mg/kg/day, e.g. from about 50 mg/kg/day to about 600 mg/kg/day, e.g., from about 100 mg/kg/day to about 500 mg/kg/day, e.g., from about 200 mg/kg/day to about 400 mg/kg/day for a time period effective to prevent, treatment or reduce the intensity of one or more symptoms associated with a metabolic disease or disorder in a subject.

In some embodiments of the invention, the dietary fiber composition containing a mixture of fibers (VFB) or complex (VFC), is administered to the subject in the form of a medical nutrition product, as described in this document. In some embodiments of the invention, the dietary fiber composition is administered to a subject in need of this at least once a day. In some embodiments of the invention, the dietary fiber composition of the invention is injected twice daily, preferably once in the morning and once in the afternoon/evening. The usual mode of treatment in accordance with this aspect of the invention will continue for at least two weeks to 16 weeks or longer. Depending on factors such as the medical condition to be treated, and the reaction of the patient, the treatment can be extended until, until the patient is reduced INTA�intensity of at least one symptom of a metabolic disease or disorder.

In one embodiment, the implementation of the present invention a method of reducing the intensity of at least one symptom associated with the progression of insulin resistance in humans suffering from a disease or at increased risk of developing type II diabetes. Method in accordance with this aspect of the invention provides an introduction to someone else in need of this, about 25 mg/kg/day to about 1000 mg/kg/day (e.g., from 100 mg/kg/day to 500 mg/kg/day or 350 mg/kg/day to about 450 mg/kg/day) high viscosity of the composition of polysaccharide dietary fiber containing the fiber mixture or complex (VFB/C), comprising from about 48% to about 90% (in/in) glucomannan, from about 5% to about 20% (W/W) xanthan gum and from about 5% to about 30% (W/W) alginate, during a period of time effective to reduce at least one symptom in the progression of insulin resistance, for example, reduced levels of glucose in the blood. In some embodiments of the invention, the method includes the introduction of the dietary fiber composition within a time period of at least two weeks to 16 weeks or longer.

According to the American Association for the study of heart disease and the National Institute of diseases of the heart, lung and blood diagnosed.� metabolic syndrome in the presence of the subject has three or more of the following signs: blood pressure equal to or higher than 130/85 mm Hg; sugar (glucose) in blood equal to or higher than 100 mg/DL; large waist circumference (men: 40 inches or more; women: 35 inches or more); low cholesterol high density lipoprotein (HDL) (men: less than 40 mg/DL; women: below 50 mg/DL); or triglycerides equal to or higher than 150 mg/DL. Therefore, in some embodiments of the invention a method of reducing the intensity of at least one symptom associated with the progression of insulin resistance in humans suffering from a disease or at increased risk of developing diabetes type II, provides an introduction to the subject an effective amount of VFB/C for a time period effective to (1) reducing sugar (glucose) in the blood of the subject to a level below 100 mg/DL; (2) reduction in waist circumference below 40 inches for men, or 35 inches for women; and/or (3) reduce the level of triglycerides to a level equal to or less than 150 mg/DL.

As described in examples 1-4, the effectiveness of VFC (for example, granulated VFB) is demonstrated to reduce the intensity of the development and progression of early stages of metabolic syndrome in mammals, including the ability to slow the progression of organ damage induced by glucose, reduce the accumulation of lipids in the liver, to preserve pancreatic beta-cells and povyshatelnoj to insulin compared with the control group.

Methods of analysis of a sample containing at least one polysaccharide.

In another aspect, the present invention proposes a method of determining the component Sugars in the sample containing at least one polysaccharide, such as composition of dietary fiber containing the fiber mixture or complex. Methods according to this aspect of the invention contain: (a) hydrolysis of a sample containing at least one polysaccharide with acid for the purpose of obtaining hydrolysate; (b) separation of hydrolysis products in the hydrolysate using a chromatographic method; (C) detection of hydrolysis products separated in stage (b); and (d) comparison of hydrolysis products detected at the stage (C) with one or more of the reference standards to determine the component Sugars in the sample.

In some embodiments of the invention, the sample contains at least one dietary fiber. In some embodiments of the invention, the sample contains sodium alginate. In some embodiments of the invention, the sample contains a mixture of fiber or complex containing alginate, glucomannan and xanthan gum.

Hydrolysis

In accordance with the methods of this aspect of the invention, the sample containing at least one polysaccharide is hydrolysed with acid for the purpose of obtaining the hydrolysis�TA. In some embodiments of the invention, the acid used for gidrolizirovanny sample represents trifluoroacetic acid (TFA).

In some embodiments of the invention, the sample contains alginate, a mixed polymer mannuronate acid and guluronic acid. In such embodiments of the invention stage of hydrolysis of the sample containing alginate, is carried out under conditions suitable for releasing and maintaining L-guluronic acid and D-mannuronate acid. For example, in one embodiment, the primary hydrolysis of alginic acid can be done using 95% sulfuric acid at a temperature of 3°C for 14 hours or 80% sulfuric acid at room temperature for 14 hours, as described by Fischer and Dahlem. In accordance with such embodiments to mixing in alginic acid mineral acid is cooled to a temperature of from -10°C to -5°C. the Viscous mass is thoroughly mixed to avoid formation of lumps. The mixture is then diluted with the help of crushed ice and water until the sulfuric acid solution will be approximately 0.5 N. the Solution is then heated for six hours in a boiling water bath, then neutralized with calcium carbonate. After filtration and washing of the precipitate calcium sulphate, water for washing the bright yellow filtrate is concentrated, then PR�goes through a cation exchange column and concentrated under reduced pressure to a liquid syrup. After further slow the concentration in dessicator and innoculate D-mannopyranoside when the melting point of 191°C, a certain amount of lactone crystallizes from the syrup, but only if the hydrolysed alginic acid contains more D-mannuronate acid than L-guluronic acid. After removal of the crystalline D-mannuronate remaining D-mannuronate and L-guluronate acid are separated by means of chromatographic methods, as described in this document.

In another embodiment, the implementation stage of hydrolysis of the sample containing alginate, contains using trifluoroacetic acid (TFA). TFA has an advantage over mineral acids, being quite volatile, allowing its removal by lyophilization of the hydrolysate. For example, hydrolysis in 2M TFA at 100°C under nitrogen during a period of time lag of about eight hours to about 18 hours, was a suitable alternative hydrolysis in 1M H2SO4under the same conditions. (Hough et al.). It should be noted that hydrolysis time gap of 6-8 hours is usually sufficient for the destruction of polysaccharides composed of neutral Sugars, but the presence of precipitation uronic acid in significant proportions creates an additional difficulty, which�weapon in what connection glucosideuronic acid, in General, are much more resistant to acid hydrolysis than other glycosidic bonds. For polysaccharides, such as capsular polysaccharides of bacteria containing uronic acid in the range of from about 16% to 30% mol, hydrolysis in 2M TFA at 100°C under nitrogen for 18 hours was satisfactory in some cases (Hough et al.). However, in the presence of sugar residues are particularly susceptible to destruction by acid (e.g., D-ribose, D-xylose or L-rhamnose), the hydrolysis time is preferably limited to up to eight hours with a subsequent adjustment of the analytical results regarding the remaining sugar associated with uronic acid, the proportion allopurinol acid in the hydrolysate is determined by gel permeation chromatography on a tightly cross-linked gel.

In some embodiments of the invention the hydrolysis stage of the sample containing alginate, is carried out by incubating the sample with TFA during the time period of approximately 48 to 72 hours at a temperature ranging from about 95°to about 110°C. As described in example 6, the present inventors have determined that the hydrolysis using TFA for 72 hours was effective for hydrolytic release of Sugars from the sample containing alginate, n�the sample, containing VFB/C.

Chromatographic separation of the hydrolysate

In accordance with the methods of this aspect of the invention, the hydrolysis products in the hydrolysate is then separated using a chromatographic method, such as thin layer chromatography, gas chromatography (GLC) or liquid chromatography (LC), including the use of materials reversed phase C18. In some embodiments of the invention, the chromatographic method is able to separate the neutral sugar uronic acids, for example, using the Dionex chromatography.

The hydrolysis products separated by the chromatographic method, are detected and compared with one or more of the reference standards to determine the component Sugars in the sample. Typical detectors are suitable for detection of Sugars, include pulsed amperometric detector Dionex detector or evaporative light scattering (NT), or mass spectrometer connected to a high performance liquid chromatography (HPLC). Reference standards, such as samples with known components, can be used as control samples in parallel with the test specimen. Alternatively, the reference standard may be the known characteristics of one or more specific component Sugars (eg�emer, retention time/height/relative area) in the reference sample, analyzed by specific chromatographic method as described in example 5.

In some embodiments a method of the invention in accordance with this aspect of the invention contains gidrolizirovanny sample containing at least one polysaccharide, such as alginate, with the help of TFA; the separation of hydrolysis products in the hydrolysate using a chromatographic method such as HPLC system with C18 column; detection of hydrolysis products and a detector, such as NT or the mass spectrometer; and comparing the detected products with one or more of the reference standards to determine the component Sugars in the sample.

The following examples are only discussed below depict the best technical implementation of the invention, they should not be construed as limiting the invention.

EXAMPLE 1

This example describes the effects of dietary fiber composition containing a granulated mixture of viscous fiber (also called viscous fiber complex (VFC), sold under the name of PolyGlycopleX (PGX®)) on insulin resistance, body weight, viability of pancreatic β-cells and lipid profile in diabetic rats Zucker.

Rationale: the Male diabetic rats Zucker� (ZDF) (ZDF/Crl-Polymorphism fa/fa) was chosen as the animal model for use in this study, since this animal model is considered to be a superior model of hypertrophic obesity associated with type II diabetes and/or reduced insulin sensitivity at an early age (P. Daubioul et al., J. Nutr. 132:967-973 (2002); J. M. Lenhard et al., Biochem. & Biophys. Res. Comm. 324:92-97 (2004); J. N. Wilson, Atherosclerosis 4:147-153 (1984)). ZDF are mutants who have no brain latinova receptors. Leptin is a protein secreted by adipose tissue that signals appetite suppression. Consequently, these mutant rats lacking reverse alarm loss of appetite or induction of satiety. Rats ZDF consuming foods with very high speeds and very quickly become fat. This model, therefore, simulates people who are fat from overeating. Once ZDF rats become fat, they quickly become insensitive to insulin, just like people (also called metabolic syndrome). Rats ZDF are also gipolipidemicheskie, this rat model has demonstrated itself as a good model for the metabolic syndrome in humans. Over time diabetes in the ZDF model progresses like the progression in people becoming flourishing with loss of the population pancreat�ical β-cells (cells that secreting insulin). Proteins become glycosylated because of an excess of glucose, leading to problems of functioning of organs from ZDF and human rights, especially of the kidneys. High levels of glucose causes glycation of proteins, leading to diabetic nephropathy and vascular damage. In this study, were used ZDF at an early age (five weeks old) without a high fat diet to determine the likelihood of delay occurrence and/or reduce the severity of diabetes when administered granules of viscous fiber complex (VFC).

Standard marker of glucose degree of damage to proteins is glycosylated hemoglobin (HbA1 C), which increased in ZDF, which currently is one of the most important markers for the approval of drugs for humans. Measurement of albumin in urine is a standard marker of diabetic kidney damage. According to the management of the Department on control over products and medicines (FDA) for the treatment of diabetes are needed glycemic control and reduction of tissue damage caused by high glucose.

Ways Food for rats with a high content of fibers

Pellets of viscous fiber complex (VFC) (cognac/xanthan gum/alginate (70:13:17) (that is, the fiber mixture was processed by granulation to create complex sold�th called PGX ®) were included in the main food rats (D11725: Research Diets, new Brunswick, new Jersey). Alternative nutrition, used in this study included other types of fibers, as shown in TABLE 1 below. All the meals were designed to be as isoenergetically, providing different energy content of each source fiber (diet containing VFC and inulin, provided at 3.98 kcal/g and cellulose provided to 3.90 kcal/g).

Cellulose was chosen as the primary reference fiber which is insoluble and cannot be digested and considered an inert reference compound (J. W. Anderson et al, J. Nutr. 124: 78-83 (1994). Inulin is a fructose polymer of plant origin, which is water soluble and nevereverever and which has shown efficacy in some studies regarding the reduction of lipids and glycemic control in some studies; but the results are volatile (see R. Rozan et al., Br. J. Nutr. 99: 1-8 (2008). The number of fructose or glucose residues (degree of polymerization "DP") of inulin amounted to 99.9% 5 s with an average DP, component, s 23.

TABLE 1:
The composition of the three feeds containing VFB, cellulose or in�Lin (percentage of ingredients by weight
The viscous fiber complex (VFC) (cognac/xanthan gum/alginate (70:13:17)) PGX granules®Insoluble fiber (cellulose)Soluble, non-viscous fiber (inulin)
The equation number (Research DietsD08012504D08012507D08012503
Casein20%20%20%
Methionine0,3%0,3%0,3%
Corn starch50%50%50%
Maltodextrin15%15%15%
Fiber*5% VFC (PGX®)5% cellulose5% inulin
Corn oil5%5%5%
The mixture of salt/minerals 3,5%3,5%3,5%
The mixture of vitamins1%1%1%
The choline bitartrate0,2%0,2%0,2%
Dye0,1%0,1%0,1%
* Fiber pellets VFC, sold under the name PolyGlycopleX®(PGX®) (InnovoBiologic Inc., Calgary, Alberta, Canada), cellulose (Research Diets, new Brunswick, new Jersey) and inulin (Raftiline®HP, Orafti, Muddy, Belgium), respectively.

Study design

Thirty (30) male rats ZDF/Crl-Polymorphismfa/faobtained from Charles river (Charles River (Kingston, NY) at the age of five weeks. Animals were placed individually in suspended mesh cages made of wire, which corresponded to the size recommended in the latest Guide for the Care and Use of Laboratory Animals DHEW (NIH). All studies were approved by Eurofins Institutional Animal Use and Care Committee (the Committee). In the room where animals were kept, regulated the temperature and humidity, was provided with a 12-hour cycle of day and night, clean and no pest. Animal� were acclimatized for one week after arrival, and the animals had access to food and water ad libitum.

After adaptation, rats randomly determined in one of three groups based on initial blood glucose and body weight. Each group of rats were given one type of food containing VFC (sold under the name PGX®), cellulose, or inulin (Raftiline®HP inulin obtained from chicory), all at 5% (V/V), as shown above in TABLE 1, during the time of 8 weeks. The basic procedures observations were made during the 8-week study, including measurement of weight food three times a week, weekly measurement of body weight and blood sampling for glucose and insulin. It should be noted that the analysis of glucose, not fasting began on 3 week at a time, as the analysis of fasting began in 1 week. In animals not fasting insulin was measured only at the last moment. Analysis of the state of not fasting was added to the study due to the observation of large effects VFC to stabilize glucose levels, despite the fact that the fiber was physically present in the gastrointestinal tract, probably due to the fact that Zucker rats ate continuously day and night. In animals in a state of fasting serum triglycerides were measured in the survey, whereas animals in the state of not fasting was only made final changes�Linux (IDEXX, North Grafton, MA).

For all studies the fences of blood samples for glucose and insulin were produced around the same time of day (mid morning). The study culminated in the two oral test for glucose tolerance intervals of one week and the autopsy.

Measurement

The following measurements were carried out during 8 nedelkova research:

Eating: Before and after the introduction of the experimental food weight of food was measured 3 times a week.

Body weight was measured once a week. The content of blood glucose and insulin Before and weekly intervals after administration of the experimental food blood sampling was performed by retro-orbital bleeding after an overnight fast. Fences blood samples for glucose and insulin was performed once a week at roughly the same time of day (mid morning). A small number were analyzed using a portable glucometer. After extraction of the sample for insulin analysis, 1 ml was subjected to coagulation; 0.5 ml of serum was extracted and analyzed for triglyceride content. Fences additional samples were produced by tail incision, when the animals had access to food. The content of glucose in the blood was measured by using a blood glucose meter Bayer Ascensia Elite Glucometer (Bayer Health Care, �arreton, New York). Insulin content was measured using ELISA (Ani Lytics, Gaithersburg, MD).

Oral test glucose tolerance (OGTTs) in 9 weeks and 10 weeks after the study ended with the two oral test glucose tolerance (OGTTs) in rats in a state of fasting and not fasting, not fasting OGTT was performed. The original sample of blood for insulin analysis and glucose measurements was taken for OGTTs fasting and not fasting. The original blood sample for final not fasting OGTT was also used for clinical biochemical blood analysis, as described below.

OGTT for animals in the state of fasting and not fasting induced by oral treatment with glucose (2 g/kg glucose, through coercive power). Fences of blood samples was performed after 30, 60, 90 and 120 minutes after glucose load and was analyzed for glucose and insulin. Upon completion of the second test on the glucose tolerance of rats killed by an overdose of isoflurane and relevant bodies have learned to conduct histopathological analysis.

Indicators of homeostatic model assessment (HOMA) were calculated during the study, mg of glucose x insulin (U/ml2). This is generally a reliable way of demonstrating changes in insulin resistance, where more� low rates NOMA represent a large reduction in peripheral insulin resistance. Indicators of a generalized index of insulin sensitivity (CISI) research regarding oral test glucose tolerance (OGTT) were also calculated using the following formula:

CISI=1000(Clucbase×Insbase)×(Glusmean×Insmean)

This indicator takes into account the CISI amplitude of glucose and the area under the curve with a higher index indicating improved insulin sensitivity.

The original sample of blood for insulin analysis and glucose measurements was taken for OGTTs fasting and not fasting. The original blood sample for final OGTT (not fasting) was also used for clinical biochemical analysis of blood, including: electrolytes, urea nitrogen blood (BUN), creatinine, alkaline phosphatase, aspartataminotransferaza (ALT), alanineaminotransferase (AST), bilirubin (direct + indirect) and total plasma cholesterol (a�Aliz was conducted using IDEXX, North Grafton, MA).

Serum triglycerides: animals in the state of fasting serum triglycerides were measured in the survey, whereas animals in the state of not fasting was done only to end the measurement (analysis was conducted using IDEXX, North Grafton, mA).

Clinical biochemical analysis of blood: the Initial blood sample for the final OGTT not on an empty stomach was used for clinical biochemical blood tests including electrolytes, urea nitrogen blood (BUN), creatinine, alkaline phosphatase, aspartataminotransferaza (ALT), alanineaminotransferase (AST), bilirubin (direct + indirect) and total plasma cholesterol (analysis was conducted using IDEXX, North Grafton, mA).

Analysis of tissues: One lobe of the liver, one kidney, and pancreas were fixed in 10% neutral buffer formalin (NBF). The pancreas was transferred into 70% ethanol after 24 hours. Tissue was processed and sealed in paraffin. Liver and kidney were sectionlevel with a diameter of about 5 microns and stained with hematoxylin and eosin (H&E). The pancreas has consistently sectionlevel twice with a diameter of about 5 microns, and the sections were stained using H&E or immunohistochemical staining using mouse antibodies against rat insulin (1:300 rabbit anti-Krysin�th insulin, Cell Signaling Technology, Danvers, MA).

Immunohistochemistry:

Immunohistochemistry was carried out as described below. Isotype control antibody (normal rabbit IgG, R&D Systems, Minneapolis, mn) was used to assess the overall level of non-specific staining and background staining. After dewaxing was carried out the restoration of the antigen when using Declere solution®(Cell Marque™ Corporation, Rocklin, CA) for 15 minutes at a temperature of 120°C, followed by hot Declere solution®for 5 minutes at room temperature. Endogenous peroxidase activity was suppressed by incubation in 3% hydrogen peroxide in deionized water for 10 minutes. The sections were treated for 20 minutes in 5% normal goat serum. Then the sections were treated with the first antibody for 60 minutes, with subsequent incubation for 30 minutes in biotinilated goat anti-rabbit antibody. Then the sections were treated in ABC Elite Reagent®(Vector, Burlingame, CA) for 30 minutes. In the end, the samples were treated in diaminobenzidine for 5 minutes followed by hematoxylineosin contrasting color.

After opening the additional lobe of the liver was subjected to immediate freezing was embedded in Oct and sectioned with a diameter of microns and stained with Sudan black for content analysis of lipids (free fatty acids and triglycerides).

All sections, stained using H&E, were assessed for morphological changes, usually related to the observed changes in ZDF, for example, increase tubular dilatation and an increase in tubular degeneration in the kidney, cellular degeneration of pancreatic islet and liver steatosis. These changes were classified semi-quantitatively on a scale from 0 to 5 depending on the definition of severity, where 5 is the most severe indicator.

The liver slices, stained with Sudan black, were evaluated and classified semi-quantitatively for the presence of vacuoles positive for Sudan black on a scale from 0 to 5, where 5 is the most severe indicator.

The percentage of islet area with cells positive for insulin was measured on sections of the pancreas, immunohistochemically stained using antiinsulin antibodies. The morphometric measurements were taken. Ten islets in the pancreas were identified manually by a veterinary pathologist. The area positive for insulin staining within these islets were labeled similarly, and the percentage of islet areas positive to insulin was calculated using the software image processing ImagePro®Plus.

Statistical methods:

Mogok�atna collected quantitative data were analyzed through bilateral variance analysis of repeated measurements (ANOVA). Significant effects entailed a posteriori comparison when using the criteria for multiple comparisons of Bonferroni, as described in Motulsky H. Intuitive Biostatistics, NY, University Press (1995).

Chemical analyses of insulin, cholesterol and blood measured only at the end of the study in rats in a state not on an empty stomach were analyzed by one-way ANOVA. Significant effects resulted in a posteriori comparisons using criteria multiple comparisons Dunnet (MCTS), as described in Motulsky H. Intuitive Biostatistics, NY, University Press (1995). Non-quantitative or discrete data (such as histological characteristics were analyzed by means of the criterion of the Kruskal-Wallis test, as described by Motulsky H. Intuitive Biostatistics, NY, University Press (1995). Significant effects resulted in a posteriori comparisons using MCT Dunnet.

Results

Body weight and food consumption

FIGURE 1A graphically depicts the effect of diet containing VFC, cellulose, or inulin on body weight (g) over time during the 8-week study in diabetic Zucker rats. As shown in FIGURE 1A, the increase in body weight versus time was reduced in rats receiving VFC, compared with animals treated with cellulose, or animals treated with inulin, starting from 1 week. At baseline vs� diabetic Zucker rats had similar mass bodies (about 160 g). In the next three weeks, rats treated food containing cellulose or inulin, has added an average of about 40 g compared with rats treated with food containing VFC. Significant differences between rats receiving VFC, compared with the diet containing cellulose or inulin was observed, ranging from 1 week to 8 week (the Symbol "***" indicates p<0,001, MCTS, Bonferroni). Significant differences were observed between rats receiving a diet containing inulin and cellulose.

FIGURE 1 graphically depicts the effect of diet containing VFC, cellulose, or inulin, the consumption of food (g/day) over time during the 8-week study in diabetic Zucker rats. As shown in FIGURE 1, the food intake was significantly reduced in rats receiving VFC, during the first three weeks (the symbol "*" indicates p<0.05 to 1 week; the symbol "***" indicates p<0,001 2 weeks; and the symbol "**" indicates p<0.01 after 3 weeks). Eating in a group VFC remained lower for the remainder of the Protocol, although after 4 weeks of the study, the more levels were not statistically different from the other two groups. Significant differences were observed between rats receiving diet containing inulin and cellulose.

Rats treated food containing VFC, Oba�but ate 20-23 g/day (made the correction for spillage; approximately 70-85 kcal/day). Rats treated food containing cellulose or inulin, usually ate 21-27 g/day (made the correction for spillage; approximately 75-100 kcal/day).

Thus, these results demonstrate that the increase in body mass with respect to time is usually observed in ZDF rat model was significantly reduced in animals treated with VFC.

Glycemic control: blood sugar and metabolism

FIGURES 2A-D graphically depict the effect of diet containing VFC, cellulose, or inulin, glucose in blood on an empty stomach (FIGURE 2A), serum fasting insulin (FIGURE 2B), glucose in blood on an empty stomach (FIGURE 2C) and indicators of homeostatic model (HOMA), fasting (FIGURE 2D) in ZDF rats over time during the 8 week study. As shown in FIGURE 2A, the values of blood glucose in rats in the fasting state were not significantly increased in any of the rats, there was a slight increase in glucose values regarding rats receiving VFC (the symbol "*" indicates p<0,05 after 3 and 6 weeks).

As shown in FIGURE 2B, the levels of serum insulin in rats in the fasting state were reduced in rats receiving VFC, during the period of the study, and serum levels of insulin were reduced to statistically significant levels ranging chere� five weeks (the symbol "***" indicates what p<0,001 4 weeks).

As shown in FIGURE 2C, the values of blood glucose in rats in a state not on an empty stomach were significantly reduced in rats receiving VFC, starting in about five weeks (the symbol "***" indicates p<0,0001 5 weeks) compared with rats treated with cellulose and inulin.

As shown in FIGURE 2D, rats treated with VFC, has significantly reduced NOMA, starting five weeks of the study (the symbol "*" indicates p<0,05), 5-7 weeks (p<0.05) and 8 weeks (the symbol "**" indicates P<0.01).

In General, under conditions of fasting (i.e. animals examined in the morning after about 16 hours without access to food), ZDF rats remained significantly lower concentrations of glucose in the blood compared with the concentrations of glucose in the blood observed in the conditions of feeding (not fasting) (compare FIGURE 2A with FIGURE 2C). As shown in FIGURE 1A, for all groups in the state of fasting values of glucose in the blood is usually observed in the range between 95 mg/DL and 145 mg/DL, which is considered marginally diabetic with minor differences observed between the groups receiving VFC, cellulose, or inulin.

As shown in FIGURE 2B, under conditions of fasting, the rats receiving the diet containing VFC, remained much more stable serum concentrations of insulin than rats, Paul�chausie food containing cellulose or inulin. As shown in FIGURE 2B, the serum levels of fasting insulin were reduced in ZDF rats treated with VFC, during the study period, a significant reduction was observed, starting in five weeks, and remained significant after 8 weeks (p<0,001 4 weeks, as indicated by the symbol "***"). Significant differences were observed between rats receiving diets containing inulin and cellulose.

Insulin resistance in this study in rats in the fasting state was assessed by calculating the homeostatic model assessment (HOMA). As shown in FIGURE 2D, the indicators NOMA has increased during the study for all groups, but significantly lower in relation to rats receiving a diet containing VFC than for rats treated with cellulose or inulin. Significant differences between VFC compared with cellulose or inulin were observed in 5, 6 and 7 weeks (p<0,05, as indicated by an asterisk ( * ) and 8 weeks (p<0.01, as indicated by the symbol "**"). Significant differences were observed between rats receiving the diet containing cellulose or inulin.

FIGURE 3A graphically depicts the performance of the generalized index of insulin sensitivity (CISI) regarding diabetic Zucker rats in the fasting state, receiving food containing VFC, cellulose, or inulin, during the 8-week studies�the study. As shown in FIGURE 3A, figures CISI, calculated on test OGTT on animals in the fasting state were significantly higher (p<0.01, indicated by the symbol "**") regarding animals treated with VFC, further demonstrating improved insulin sensitivity for this group VFC compared with groups treated with cellulose and inulin.

FIGURE 3B graphically depicts the performance of the generalized index of insulin sensitivity (CISI) regarding diabetic Zucker rats not fasting, fed, containing VFC, cellulose, or inulin, during the 8-week study. As shown in FIGURE 3B, the animals are not fasting, receiving VFC, showed significantly higher rate CISI (p<0.001 are indicated by the symbol "***"), hence, higher insulin sensitivity compared with groups treated with cellulose and inulin. The maximum glucose levels was observed 30 minutes after glucose tolerance test, group VFC had significantly lower maximum value than other two groups.

As shown in FIGURE 2C, under conditions not on an empty stomach (feeding) (i.e., the animals investigated in the morning with continuous access to food in the last 24 hours), rats treated with diet containing VFC, maintained lower levels of glucose in the shelter� compared with rats treated with food containing cellulose or inulin, during all weeks of the study. Study of glucose in the blood began during the third week of the study and continued until the eighth week. The study of glucose of the animals feeding condition was added to the study Protocol, provided that the values of glucose in the fasting state were very close to the normal range and are not bound by any particular theory, it is believed that many of the mechanistic actions VFC include its direct contact with food.

Despite the fact that under the conditions of feeding insulin was measured only at the last moment, was observed improved insulin sensitivity similar to insulin sensitivity observed in animals in the fasting state for measurement in the last time. As shown in FIGURE 2C, the response to glucose in the blood in a state of feeding was significantly reduced in animals treated with VFC (p<0,001, as indicated by the symbol "***"), compared with animals treated with inulin or cellulose. Significant differences were observed between rats receiving a diet containing inulin and cellulose.

FIGURE AP graphically depicts the performance of HOMA, calculated in relation to diabetic Zucker rats not fasting, fed, containing VFC, cellulose, or inu�Jn, for the last blood sampling 8-week study. As shown in FIGURE 3C, the index HOMA-IR was significantly lower in the group treated with VFC (p<0,001) compared with groups treated with cellulose and inulin. As noted above, lower rates NOMA represent a more significant reduction in peripheral insulin resistance.

Lipid profile

Serum triglycerides were measured in animals in the fasting state (measured throughout the study) and not on an empty stomach (measured only at the end of 8 weeks study). FIGURE 4 graphically depicts serum levels of triglycerides measured in diabetic Zucker rats in the fasting state, fed, containing VFC, cellulose, or inulin, over time during the 8 week study. As shown in FIGURE 4 regarding animals in the state of fasting, the animals receiving VFC, showed an early and significant lowering effect on triglycerides compared with groups treated with inulin and cellulose. After 2-3 weeks of serum triglycerides decreased in all groups, animals treated pulp had lower triglycerides compared with animals treated with inulin and VFC. As shown in TABLE 2 below, in animals in a state not on an empty stomach, as measured at the end of the study, sivaraman�e triglycerides were similar in animals that receiving VFC and cellulose, and in animals fed inulin was significantly lower triglyceride levels than the other two groups.

At the end of 8 weeks study, we measured the plasma cholesterol in the initial sample obtained from animals in a state of feeding before the last OGTT. As shown below in TABLE 2, the animals were hypercholesterinemia and VFC significantly reduced cholesterol levels by more than half compared with groups treated with cellulose and inulin.

Most vulnerable organs: histological evaluation of liver, pancreas and kidneys Although of the above data regarding glucose and insulin show improved insulin sensitivity and glycemic control in the treatment of VFC, was analyzed tissues to assess the impact of VFC to reduce the extent of damage to organs such as kidneys. The kidney is especially known for susceptibility to diabetic nephropathy, which probably is associated with hyperglycemia and polirovannom. For all measured the degree of tissue damage was assessed as an indicator of the ability of treatment VFC to delay the progression of diabetes and/or reduce the intensity of symptoms associated with diabetes.

Kidney

All sections of diabetic kidney tissue, painted with H&E, were assessed for mortal�technological change, associated with morphological changes that are typically observed in ZDF, which includes the increase in tubular dilatation and an increase in tubular degeneration/regeneration. Some parameters of renal pathology showed differences between diabetic Zucker rats (ZDF) receiving the diet containing VFC and ZDF rats treated with food containing inulin or cellulose.

FIGURE 5A graphically depicts the effect of nutrition containing VFC, cellulose, or inulin, diabetic Zucker rats after eight weeks on renal tubular dilation on the basis of histological score ranging from 0 to 5, where 5 is the most severe indicator. As shown in FIGURE 5A, tubular dilatation was assessed as absent in ZDF rats treated with VFC. For comparison, tubular dilatation was present in ZDF rats treated with cellulose and inulin. The figures shown in FIGURE 5A, showed a significant treatment effect (p<0.001 are indicated by an asterisk ( * ) between groups treated with VFC and cellulose or inulin. Significant differences were observed in the volume of tubular dilation between animals treated with inulin and cellulose.

FIGURE 5B graphically depicts the effect of diet containing VFC, cellulose, or inulin, diabetic Zucker rats after eight weeks on renal tubular degeneration/reg�neratio on the basis of histological score ranging from 0 to 5, where 5 is the most severe indicator. As shown in FIGURE 5B, the rats receiving the diet containing VFC, showed the average tubular degeneration/regeneration of 0.1, which is 1 (the minimum weight) for one rat, and 0 (within normal limits) for the other 9 rats per group. For comparison, the average for rats receiving the diet containing cellulose or inulin, amounted to 1.0, as further shown in FIGURE 5B. therapeutic effect of VFC to reduce the severity of tubular degeneration/regeneration was significant (p<0.01, indicated by the symbol "**") with a significant difference observed between the groups receiving VFC in comparison with cellulose or inulin.

FIGURE 5C graphically depicts the effect of diet containing VFC, cellulose, or inulin, diabetic Zucker rats after 8 weeks on renal mesangial expansion on the basis of histological score ranging from 0 to 5, where 5 is the most severe indicator. As shown in FIGURE 5C, the estimation of glomerular mesangial expansion showed lower rates in the group receiving the diet containing VFC compared with the diet containing cellulose or inulin. Despite the fact that the indicators concerning mesangial expansion was reached statistical significance (p<0,05, the only pairs of treatment groups that are significantly different at the a posteriori study were group receiving diet containing VFC and inulin (p<0.05 are indicated by an asterisk ( * ), with a strong tendency to decrease compared to cellulose nutrition.

Pancreas

FIGURE 6 graphically depicts the percentage of the area of immunoreactive insulin pancreatic islet that is present in diabetic Zucker rats treated with diet containing VFC, cellulose, or inulin, at the end of the 8-week study, as determined by staining using anti-rat insulin antibodies. As shown in FIGURE 6, rats treated with diet containing VFC, retained a higher area of insulin immunoreactivity calculated as a percentage of total islet area (i.e. the larger the mass of pancreatic beta cells), as measured by insulin immunohistochemistry compared with rats receiving the diet containing cellulose or inulin. Analysis ANOVA showed a significant treatment effect (p<0,0001 indicated by the symbol "***"), whereas a posteriori study showed differences between the rats receiving the diet containing VFC and cellulose (p<0,001). The differences between the animals received a diet containing inulin and cellulose were observed. It is important to note that the data amount�inannie data showing lower concentrations of serum fasting insulin (FIGURE 2B) and greater insulin sensitivity (FIGURE 3B), indicate that diabetic Zucker rats treated with diet containing VFC, retain a much greater reserve capacity for insulin secretion compared with rats receiving the diet containing cellulose or inulin.

FIGURE 7A graphically depicts histological indicator regarding infiltrates of mononuclear inflammatory cells of the pancreatic islet present in diabetic Zucker rats treated with diet containing VFC, cellulose, or inulin, at the end of the 8-week study based on the index from 0 to 5, where 5 is the most severe indicator. As shown in FIGURE 7A, the difference in treatment effect on indicators regarding the presence of mononuclear infiltrates in the Insula was observed.

FIGURE 7B graphically depicts histological indicator regarding cellular degeneration of pancreatic islet that is present in diabetic Zucker rats treated with diet containing VFC, cellulose, or inulin, at the end of the 8-week study based on the histological score ranging from 0 to 5, where 5 is the most severe indicator. As shown in FIGURE 7B, the indicators regarding the degeneration of islet cells �tattvamasi in rats, treated with food containing VFC, and had a tendency to increase in rats fed with the diet containing cellulose or inulin; however, these differences did not reach statistical significance.

FIGURE 7C graphically depicts histological indicator concerning the amount of fibrosis pancreatic islet present in diabetic Zucker rats treated with diet containing VFC, cellulose, or inulin, at the end of the 8-week study based on the histological score ranging from 0 to 5, where 5 is the most severe indicator. As shown in FIGURE 7C, the figures regarding the volume of islet fibrosis tended to decrease in rats treated with diet containing VFC compared to the rats receiving the diet containing cellulose or inulin; however, these differences did not reach statistical significance. Indicators regarding the presence of hemorrhage or hemosiderin tended to lower in rats receiving the diet containing the VFC, but the results were not statistically significant (data not shown).

The liver

FIGURE 8A graphically depicts the effect of nutrition containing VFC, cellulose, or inulin, diabetic Zucker rats after eight weeks on hepatic steatosis as measured by staining with Sudan black, on the basis of histological p�ratio of from 0 to 5, where 5 is the most severe indicator. As shown in FIGURE 8A, the rats receiving the diet containing VFC, showed a slight fatty liver disease (measured by staining with Sudan black) than rats fed with the diet containing cellulose or inulin. According to the index from 0 (within normal limits) to 5 (severe), rats treated with diet containing VFC, on average, received a 3.4. That compares with a rate of 4.6 regarding rats receiving the diet containing cellulose, and 4.1 regarding rats receiving a diet containing inulin. Groups differed significantly between rats receiving a diet containing VFC, compared with the diet containing cellulose and inulin (p<0.01, as indicated by the symbol "**"). Significant differences between the rats received a diet containing inulin and cellulose were observed.

Rats receiving a diet containing VFC, also showed a small microvesicular the vacuolization of hepatocytes compared with rats receiving the diet containing cellulose or inulin. FIGURE 8B graphically depicts the effect of nutrition containing VFC, cellulose, or inulin, diabetic Zucker rats after 8 weeks on hepatic microvesicular the vacuolization on the basis of histological score ranging from 0 to 5, where 5 is the most severe indicator. As shown in FIGURE 8B, mikrobasic�polar vacuolization was assessed as severe in all rats, treated with food containing cellulose or inulin (average of 4.6). For comparison, rats treated with diet containing VFC, received an average of 3.2 (easy). MCT Dunnet showed significant difference between groups receiving a diet containing VFC and cellulose (p<0,001, as indicated by the symbol"**") but not between groups receiving a diet containing inulin and cellulose.

FIGURE 8C graphically depicts the effect of nutrition containing VFC, cellulose, or inulin, diabetic Zucker rats after eight weeks on hepatic macrovesicular the vacuolization on the basis of histological score ranging from 0 to 5, where 5 is the most severe indicator. As shown in FIGURE 8C, in all treatment groups microvesicular vacuolization of hepatocytes was generally less pronounced compared to microvesicular the vacuolization of hepatocytes, which is reflected in lower severity (compare FIGURE 8B to 8C). Despite the fact that rats receiving a diet containing inulin showed a declining trend of vacuoles compared with the rats receiving the diet containing cellulose, this difference was not statistically significant. There was observed significant difference between the groups receiving diet containing VFC, compared with the diet containing cellulose and inulin (< Of 0.001, as indicated by the symbol "***"). Significant differences between groups receiving a diet containing inulin and cellulose were observed. Cystic degeneration of hepatocytes and fibrosis also showed a tendency to indicators of lesser severity in rats receiving a diet containing VFC, but it did not reach statistical significance (data not shown).

As shown in TABLE 2 below, some indicators of clinical chemistry regarding hepatic damage showed significant therapeutic effects through VFC. Liver enzymes alanineaminotransferase (ALT) and aspartate aminotransferase (AST) are released into the blood by means of hepatocellular damage even with intact cell membranes. Rats Sprag-Dole without overt liver disease have levels of ALT from 22 to 48 IU/L (reference data IDEXX). The data shown in TABLE 2, showed the General therapeutic effects on the levels of ALT and AST. A posteriori study showed lower levels of ALT in blood of rats receiving a diet containing VFC, compared with rats receiving the diet containing cellulose or inulin (p<0,05) and significantly higher levels of ALT in blood of rats receiving a diet containing inulin, compared with rats receiving a diet containing pulp (p<0,05).

AST in the blood showed a similar profile of results as complement�flax shown in TABLE 2. Rats Sprag-Dole without overt liver disease have levels of AST from 33 to 53 IU/L (reference data IDEXX). Rats receiving a diet containing VFC, averaged about 170 IU/L, rats receiving the diet containing cellulose, averaged approximately 870 IU/L, whereas rats receiving a diet containing inulin, averaged 1010 IU/L. Overall treatment effect was statistically significant (p<0,0001), although the difference between groups receiving a diet containing inulin and cellulose was not significant, difference between groups receiving a diet containing VFC and cellulose, was significant (p<0,001, MCTS Dunnet).

Rats treated with diet containing VFC, were lower serum levels of alkaline phosphatase than in rats receiving the diet containing cellulose or inulin, as shown in TABLE 2. The normal range on this parameter in rats Sprag-douli in the absence of known liver disease or bone disease is 0-267 IU/L (reference data IDEXX). As shown in TABLE 2, the mean serum levels of alkaline phosphatase regarding rats receiving a diet containing VFC, were within the normal range, whereas averages about rats receiving the diet containing cellulose or inulin, go beyond normalisation. The decrease of alkaline phosphatase between the groups receiving diet containing VFC, compared with the diet containing cellulose or inulin, was significant (p<0,001). Lower serum levels of alkaline phosphatase using VFC have a protective effect on cholestasis, whereas the increase in ALT and AST indicates hepatocellular damage (S. D. Pratt et al., Harrison's Principles of Internal Medicine 15 th Edition, pp.1711-1715 (2001). On the contrary, globulin and bilirubin is cleared by the liver, but increase reflect impaired hepatic function.

Normal range of globulin in rats Sprag-douli is 2.8-4.5 g/DL (reference data IDEXX). As shown in TABLE 2, the concentration of globulin averaging 3.4 g/DL for rats receiving a diet containing VFC, and 4.0 and 3.9 g/DL for rats receiving the diet containing cellulose and inulin, respectively. The effect of fibre type was significant (p<0,001) with significant difference between groups treated with food containing VFC and cellulose (p<0,001, MCTS Dunnet), but not between groups receiving a diet containing inulin and cellulose. Similarly, rats treated with diet containing VFC, total bilirubin (direct and indirect) averaged 0.13 mg/DL, as shown in TABLE 2, whereas rats treated with diet containing cellulose and inulin, the average was 0.19 and 0.18 mg/DL, respectively. Range of rules�found values for rats Sprag-douli is 0-0. 4 mg/DL (reference data IDEXX). therapeutic effects were statistically significant (1W ANOVA, F(2,28)=4,93, p<0,05) with significant difference between the groups receiving diet containing VFC (p<0,05, MCTS Dunnet), but not between groups receiving a diet containing inulin and cellulose. Despite the fact that the levels of globulin and bilirubin were within normal limits for all groups, rats treated with diet containing VFC, showed significantly lower concentrations (p<0,001) of two analytes compared to the other groups, suggesting improved liver function.

The normal range for bilirubin in rats Sprag-douli is 0-0. 4 mg/DL (reference data IDEXX). Significant differences in bilirubin between treatment groups were observed. Albumin, which is synthesized by the liver was similar in all treatment groups.

TABLE 2:
Plasma chemistry of key analytes, taken at the conclusion of the study in diabetic rats Zucker in a state not on an empty stomach (original dimension final OGTT not fasting)
PowerVFC (PGX®)CelluloseInulin
Cholesterol (mg/DL) 179,6±6,4***383,7±23,2350,8±21,3
Aspartate aminotransferase (AST) (IU/L)Of 165.9±24,5***871,4±109,31010,1±169,1
Alanineaminotransferase (ALT) (IU/L)93,3±13,3*299,4±30,9472,7±77,7*
Bilirubin (mg/DL)0,1±0,0*0,2±0,00,2±0,0
Alkaline phosphatase (IU/L)134,2±7,7***327,7±46,8302,3±30,3
Globulin (g/l)3,4±0,1***4,0±0,13,9±0,1
Albumin (g/DL)3,1±0,12,9±0,12,8±0,1
Nitrogen blood urea (mg/DL)A 10.4±0,613,4±0,717,0±2,6
Triglycerides (mg/DL)276,4±24,6276,7±43,5 352,6±67,6
* significantly different from the cellulose group (p<0,05)
** significantly different from the cellulose group (p<0.01)
*** significantly different from the cellulose group (p<0,001)

Discussion of results:

This study on the model of diabetic Zucker rats demonstrates that meals containing VFC, significantly improves glycemic control, reduces kidney damage, preserves pancreatic beta-cells, improves insulin sensitivity and thus reduces the overall load of glucose in humans. Moreover, the decrease in the rate of gain of body weight by approximately 10% during the study was observed in rats receiving a diet containing VFC, compared to other diet enriched with fibers. This can partly be explained by the decrease in food consumption during the study, although decreased food consumption was significant only during the first three weeks of the study. As further shown in example 4 VFC also increases the secretion of GLP-1 and PYY, inducing saturation.

The number of VFC granules used in this study was 5% VFC added to rat food. As shown in FIGURES 1A and 1 B, the use of food day in Cass�tive rats receiving VFC, averaged about 22 g/day, therefore, 22 grams 1.1 g VFC. Suppose that the average weight of Zucker rats of approximately 300 g (see FIGURE 1A), the dose in this study per kg was approximately 3,66 g/d/kg. Assume that a person's weight is about 60 kg, then the dose will be equivalent to about 219,6 g/day for humans. When using the conversion dose based on surface area of the body in an amount constituting from 0.1 to 0.15 rat dose dose for humans, as described by Reagan-Shaw et al., FASEB Journal 22:659-661 (2007), can be translated into a range of doses for a person weighing 60 kg lag of about 22 grams to about 33 grams VFC per day or from about 366 mg/kg/day to about 550 mg/kg/day.

In this study, in diabetic Zucker rats in the fasting state (i.e. animals examined in the morning after about 16 hours without access to food) were no significantly elevated levels of glucose, probably due to compensation provided by the hyperinsulinemia observed as the disease is only beginning to emerge. The animals were without food for 16 hours before the study in all dietary groups, insulin levels were higher in the groups treated with cellulose and inulin, compared with the group receiving VFC, which in combination with �the providers NOMA and CISI indicates a greater peripheral insulin resistance in groups treated with inulin and cellulose compared with animals treated with VFC. Therefore, it appears that VFC does not need to be present in the intestine to improve insulin sensitivity. Not being bound to any particular theory, the improved insulin sensitivity observed in animals treated with VFC who were without food for 16 hours before the study can be related to increased proglucagon expression (S. P. Massimino et al., J. Nutr. 128: 1786-1793 (1998); R. A. Reimer et al., Endocrinology 137:3948-3956 (1996)); or could be due to increasing regulation of muscle GLUT-4 (Y. J. Song et al., Clin. Exp.Pharm. Physiol. 27:41-45 (2000)).

As the animals were without food for 16 hours before the study, were slightly hyperglycemic, starting three weeks of the study, the level of glucose in plasma was also measured in rats in a state not on an empty stomach (i.e., constant access to food prior to the study). It was determined that in animals was investigated in a state of fasting, the animals in groups treated with cellulose and inulin, were hyperglycemic, whereas animals in the group treated VFC, had glucose levels that were reduced almost to neadiabaticheskikh levels. Insulin levels in the state of not fasting were measured only at the end of the study, it was found that the insulin was significantly reduced � animals receiving VFC and indicators NOME and CISI also showed improved insulin sensitivity in animals fed VFC, compared with other groups.

Therefore, taking into account the results that serum insulin was significantly reduced in animals treated with VFC, in the state of fasting and not fasting, and glucose in the blood was significantly reduced in animals treated with VFC studied in the state not on an empty stomach, it is necessary that the treatment VFC diabetic Zucker rats was effective to delay early progression of diabetes.

In addition to improvements of glycemic control, it was determined that animals treated VFC, was also reduced organ damage compared with the animals treated with cellulose and inulin. Diabetic nephropathy is a clinically important complication of diabetes, especially thickening of the glomerular basement membrane and expansion mezangij and tubules, and tubular degeneration that occurs due to metabolic disorders and hemodynamic changes (H. R. Brady and V. M. Brenner: Pathogenesis of Glomerular Injury, in Harrison's Principles of Internal Medicine 15 th ed., E. Braunwald et al., pp.1572-1580 (2001)). Interestingly, in this study it was determined that significant damage to the organs occurs very quickly in young diabetic Zucker rats with early diabetes, despite relatively�individual mild degree of diabetes. It is important to note that animals treated VFC, was observed the presence of significantly higher density of pancreatic β-cells at the end of the 8-week trial compared with the groups treated with inulin or cellulose. These data indicate that diabetic Zucker rats treated with diet containing VFC, within eight weeks has kept a much greater reserve capacity for insulin secretion. It should be noted that the observed preservation of pancreatic β-cells regarding inhibitors of DPP IV, which increase the levels of insulin secretagogue GLP-1, and in some studies using inhibitors of DPP IV insulin is higher than the control in models of type II diabetes, especially postprandial insulin levels (A. Viljanen et al., J. Clin. Endocrinol. Metab. 94:50-55 (2009)).

In the model of diabetic Zucker rats used in this study, the levels of glucose, not fasting was sufficient to cause kidney damage. It was determined that in the group treated VFC, renal damage was less, especially concerning mesangial expansion. Enhanced glycemic control during normal feeding and subsequently reducing the glycation of tissues, probably served as the main factor in reducing renal damage. It is interesting to note that pistol�Gia showed that VFC has significantly protected the kidneys from damage polirovannom, indicating a reduction in the total load of glucose and, consequently, reduced glycation. The FDA is considering reduced glycation as a primary marker regarding anti-diabetic effects, and the mere reduction of blood glucose is no longer considered enough for approval of drugs.

Regarding the impact of treatment on VFC serum and hepatic lipid profiles, plasma cholesterol was significantly reduced in the group treated with VFC. Effect on serum triglyceride levels were more variable. However, hepatic lipid levels (steatosis) and hepatic measurements, such as serum bilirubin, ALT and AST were significantly reduced in the group receiving VFC, indicating reduced liver damage in the group treated with VFC. Moreover, on the basis of histological analysis also determined that animals treated VFC, were reduced indicators of hepatocellular damage and reduced serum levels of alkaline phosphatase, which may indicate that animals treated VFC, were reduced cholestasi, and reduced hepatic steatosis, a common concomitant symptom of metabolic syndrome (A. Viljanen et al., J. Clin. Endocrinol. Metab. 94:50-55 (2009)).

Consequently, the efficiency required�of VFC demonstrates the ZDF in relation to glycemic control, reduction of kidney damage and preservation of pancreatic beta-cells. As demonstrated in this example, rats treated with VFC, renal damage was less, particularly mesangial expansion. Enhanced glycemic control and subsequent decrease glycation of tissues, probably served as the main factor in reducing renal damage. Therefore, the VFC can be used as a dietary Supplement to assist in reducing the intensity of the development and progression of early-stage metabolic syndrome, including the ability to slow the progression of organ damage induced by glucose, the accumulation of lipids in liver and the inhibition of the loss of pancreatic beta cells.

EXAMPLE 2

This example describes a study conducted in a rat model with obesity induced by diet with high sucrose content for the purpose of determining the effects of a dietary fiber composition containing a granulated mixture of viscous fiber (VFC granules) (also called complex fiber PolyGlycopleX (PGX®)) on pancreatic dysregulation, dyslipidemia and obesity.

Rationale: As described in example 1, a water-soluble complex fibers, granules VFC, also called PolyGlycopleX®(PGX®) (made from cognac mannan, xantana�Oh gum and alginate to create a highly viscous polysaccharide complex with a high water-holding and gel-forming properties), reduces body weight and increases insulin sensitivity in diabetic Zucker rats. However, the impact of VFC granules observed in diabetic Zucker rats on serum triglycerides (TAG), was variable. The variability of different fibers to reduce serum levels of TAG was observed in other studies and can establish a connection between the type of fiber and certain animal model (W. U. Jie et al., Biomed. Environ. Sci. 10: 27-37 (1997); A. Sandberg et al., Am. J. Clin. Nutr. 60:751-756 (1994); R. Wood et al., Metab. Clin. Exp. 56:58-67 (2007); and N. M. Delzenne et al., J. Nutr. 129: 14673-14708 (1999)). For example, research conducted by the Mao-Yu et al. showed that the decrease in TAG through unusable fibers depends on the severity of the increase in TAG and sustainability over time (Z. Mao-Yu et al., Biomed. Environ. Sci. 3:99-105 (1990)).

The research described in this example was conducted to determine the effects of granulated VFC (PGX®) increase in body weight, serum triglyceride (TAG) and hepatic steatosis in male rats Sprag-doli treated with sucrose, a model of obesity induced by diet (high sucrose content 65% in/in) famous, which leads to weight gain and sustainable increases in liver and serum TAG levels, especially in the chronic condition, with great precision simulates the type II diabetes in humans (A. M. Gadja et al., An. Lab News At 3: 1-7 (2007); M. Hafidi et al., Clin. Exp.Hyprten. 28:669-681 (2006); and R. Rozan et al., Br. J. Nutr. 98: 1-8 (2008)). The research described in this example was carried out for 43 weeks in order to capture a sufficient portion of the life cycle of rats and maximize sustained increase in serum TAG levels, which are a characteristic feature of this model.

Methods

Food for rats with increased fiber content:

Pellets of viscous fiber complex (VFC) (cognac/xanthan gum/alginate (70:13:17) (that is, the fiber mixture was processed by granulation to create complex sold under the name PGX®) were included in the main food rats (D11725: Research Diets, new Brunswick, new Jersey). Alternative nutrition, used in this study included other types of fibers, as shown in TABLE 3 below. Cellulose was chosen as the primary reference fiber which is insoluble and cannot be digested and considered an inert reference compound (J. W. Anderson et al., J. Nutr. 124: 78-83 (1994)).

TABLE 3:
The composition of the three feeds containing VFC or cellulose (percentage of ingredients by weight
Viscous Complex fiber (VFC) (cognac/xanthan gum/�lgint (70:13:17)) PGX granules ®Insoluble fiber (cellulose)
The equation number (Research DietsD08012504D08012507
Casein20%20%
Methionine0,3%0,3%
Corn starch50%50%
Maltodextrin15%15%
Fiber*5% VFC (PGX®)5% cellulose
Corn oil5%5%
The mixture of salt/minerals3,5%3,5%
The mixture of vitamins1%1%
The choline bitartrate0,2%0,2%
Dye0,1%0,1%
* Pellets fibers VFC, sold under the name PolyGlycopleX®(PGX®) (InnovoBiologic Inc., Calgary, Alberta, Canada).

Animal model: Male rats Sprag-Doly (SD) was chosen because rat receiving sucrose is considered a superior model of hypertriglyceridemia in the presence of normal genetic background (A. M. Gadja et al., An Lab News 13: 1-7 (2007)).

Study design: 30 male SD rats obtained from Charles river (Charles River (Kingston, NY) at the age of six weeks. Animals were placed individually in suspended mesh cages made of wire, which corresponded to the size recommended in the latest Guide for the Care and Use of Laboratory Animals DHEW (NIH). In the room where animals were kept, regulated the temperature and humidity, was provided with a 12-hour cycle of day and night, clean and no pest. Animals were acclimatized for four days prior to the survey.

Water: Filtered water tap water were provided ad libitum automatic water distribution system.

Food: After adaptation of rats in a random order determined in one of two groups in each group n=10, in the power of the two groups was added to the pulp at the rate of 5% (V/V) or 5% VFB (V/V) to 65% (W/V) sucrose. The food was almost isoenergetically, nutrition content of cellulose is to 3.90 kcal/� and meals with the contents of the VFC is at 3.98 kcal/g, the total content of dietary calories is approximately 3902. Rats received a diet with a high content of sucrose ad libitum with cellulose (initial body weight was 214,7±2.6 g) or VFB (initial weight was 220,8±3.5 g) for 43 weeks.

Measurement of study: Eating (daily), body weight (weekly) and weekly blood sampling for measurement of serum triacylglycerols (TAG) (analyzed by IDEXX, North Grafton, mn), blood glucose (by the meter Acensia Elite Glucometer) and serum insulin (Ani Lytics, Gaithersburg, MD) was conducted during the study. The study ended with a final blood test to measure glycation of hemoglobin and urea nitrogen blood. Then it was performed a limited autopsy, as indicated below. One lobe of the liver was subjected to instant frozen for analysis of lipid content when using histochemistry of the Sudan black. Then one lobe of the liver were fixed for staining with hematoxylin and eosin.

Statistical methods: the Increase in body weight was analyzed regarding statistically significant differences using repeated measures ANOVA and one-way ANOVA on differences in weight gain during the study. The alpha error rates regarding multiple comparisons controlled�was inovalis when using the Bonferroni. Indicators of histology were measured using a criterion of Kruskal-Wallis concerning nonparametric data.

Results:

FIGURE 9 graphically depicts the effect of granulated VFC (PGX®) or food containing cellulose, the increase in body weight and serum triacylglycerols (TAG) in rats Sprag-doli treated with sucrose, during the 43-week study (the symbol "*" indicates p<0.05; the symbol "**" indicates p<0.01; * * * " indicates p<0,001). The initial mass of bodies did not differ between the group receiving VFC (215±3 grams), and group treated cellulose (221±3 g). As shown in FIGURE 9, body weight increased over time in both groups due to a diet rich in sucrose, but weight gain was significantly attenuated in the group treated VFC, since the beginning of the study before 22 weeks compared with the group treated pulp (p<0,05). Repeated measurements showed a significant therapeutic effect on weight gain (p=0.04) rats receiving VFC showing reduced weight gain. Although final body weight did not differ significantly between groups (p=0,20; 660±26 g regarding the groups receiving VFC and cellulose, respectively), rats VFC retained 7% lower body weight at the conclusion of the study. Used�e food on rats, receiving VFC, was similar in rats treated with cellulose (data not shown).

As further shown in FIGURE 9, serum TAG levels were sustained at an early stage of the study, but increased over time in the group treated with the cellulose prior to the completion of the study in 43 weeks. For comparison, rats treated VFC, showed significantly lower serum TAG levels compared with the group treated pulp (p<0.01). The group receiving VFC, had the original TAG levels, which were slightly different from the original TAG levels in the group treated cellulose.

Rats treated with diet containing VFC, showed a lower degree of hepatic steatosis (measured by staining with Sudan black) than rats fed with the diet containing cellulose. Lipid content was determined by staining tissue sections of liver using Sudan black, slices were evaluated and classified semi-quantitatively for the presence of vacuoles positive for Sudan black according to the index scale from 0 to 5, where 5 is the most severe indicator. Indicators of severity was 3.9±0.3 for group, treated cellulose, and 2.7±0.4 for group receiving VFC that differed significantly. Also there is a steady tendency to decrease hepatocellular damage in cu�with, receiving VFC, compared with rats treated with cellulose, although the difference was not statistically significant ([p<0.07 for macrovesicular of vacuolization and p<0,11 for microvesicular of vacuolization, data not shown).

The levels of blood glucose and insulin were observed weekly during the entire study and not changed, it is assumed that for this animal model (A. M. Gadja et al., An. Lab News 13:1-7 (2007)).

Discussion:

As expected in this model of obesity induced by diet, rats Sprag-Doly (SD) treated with sucrose, with time rapidly increased the weight by about 18-25 weeks, at this time stabilized slower rate of weight gain. As shown in FIGURE 9, during the period of rapid weight gain, VFC granules significantly reduced the changes of body weight compared to cellulose with smaller declines observed during the phase of slower growth in the latter stage of the study (i.e. older rats). As further shown in FIGURE 9 plasma TAG exceeded only the initial level of older rats and VFC significantly blunted this increase TAG. In accordance with these data, hepatic steatosis was significantly reduced in animals treated with VFC, as measured by histomorphometry compared with animals treated with cellulose.

p> It is believed that weight reduction among subjects who consume indigestible fiber, is associated with one or more of the following conditions: reduced consumption of food, the reaction of the modified hormone responsible for the feeling of satiety, reduced absorption of nutrients, which is a complication slowing down my stomach and/or absorption of nutrients fiber (see N. C. Howarth et al., Nutr. Rev. 59:163-169 (2001); A. Sandberg et al. Am. J. Clin. Nutr. 60:751-756 (1994); G. Grunberger et at., Diabet. Metab. Res. Rev. 23:56-62 (2006); and J. R. Paxman et al., Nutr. Res. 51: 501-505 (2008)). It is interesting to note that in the present study showed a slight decrease in food consumption, therefore, this factor likely contributed to the observed weight loss in animals treated with VFC. Not being bound to any particular theory, it is possible to slower gastric emptying and reduced absorption of nutrients, food eaten, can be responsible for weight loss that may be caused by increased secretion of like protein (GLP-1) (N. N. Kok et al., J. Nutr. 128: 1099-1103(1998)).

Reduction of TAG in the liver or plasma has become the subject of many studies regarding dietary fiber, and the results vary widely (W. U. Jie et al., Biomed. Environ Sci. 10:27-37 (1997); A. Sandberg et al., Am. J. Clin. Nutr. 60: 751-756 (1994); R. Wood et al., Metab. Clin. Exp. 56: 58-67 (2007); and N. M. Delzenne et al., J. Nutr. 129: 14678-14708 (1999); P. Rozan et al., Br. J. Nutr. 98: 1-8 (2008)). He all the study�deposits show a marked decrease absorption TAG with some differences, observed between the types of fibers. For example, a study conducted Delzenne and Cook showed that oligofructose reduced hepatic steatosis by reducing lipogenesis in rats treated with fructose (N. M. Delzenne et al., J. Nutr. 129: 14678-14708 (1999)). Similarly, Kok and others suggest that the secretion of GLP-1 induced oligofructose fiber, also may be responsible for reduced lipogenesis and fat mobilization (N. N. Kok et al., J. Nutr. 128: 1099-1103 (1998)). Not being bound to any particular theory, it is likely that reduced lipogenesis and reduced absorption of fat played a role in the decline of TAG observed in animals treated with VFC in this study. Reduced absorption of nutrients explain the reduced weight gain observed in the absence of reduction of eating.

In conclusion, this study demonstrates that the pellets VFC significantly lower serum TAG in a rat model Sprag-doli (8D), treated with sucrose that existing medicines are not very effective lower. Reduced hepatic steatosis compared with decreased serum TAG, and such properties make pellets VFC useful dietary Supplement for the treatment of patients with hyperlipidemia and other aspects of metabolic syndrome, including weight loss.

EXAMPLE 3

This example describes a study of the Casa�individual redundancy weight and obesity in adults, demonstrating the impact of the dietary fiber composition containing granular viscous fiber complex (VFC granules) (also called complex fiber PolyGlycoplex (PGX®)) for short-term weight loss and associated risk factors.

Rationale: according To the latest data published by the world health organization, obesity has reached global epidemic proportions with more than 1 billion adults are overweight suffer from chronic disorders www.who.int estimated 3/15/08). Coronary heart disease and stroke, resistance to insulin (metabolic syndrome), type II diabetes, hypertension and cancer are widely known medical comorbidities of overweight (K. Fukioka Obesity Res 10 (Supp 12): 116S-123S (2002)). Moreover, recent epidemiological study confirmed that obesity in adults is associated with a significant reduction in life expectancy. This study showed that non-Smoking 40-year-old man and a woman lost an average of 7.1 and 5.8 years of life, respectively, due to obesity (A. Peeters et al., Ann. Intern. Med. 138:24-32 (2003)). Based on the above risk factors, there are many therapeutic effects regarding excessive weight/obesity, including surgical intervention, treatment drugs�prisoners who had been drugs and lifestyle changes, for example diet and exercise.

Important dietary strategy of any program of weight control should include the use of a significant amount of food with high content of fibers, especially of food or food additives containing viscous soluble fiber (K. M. Queenan et al., Nutr. J. (2007)). It is estimated that the average American uses about 2.4 grams of viscous soluble fiber per day - half from 5 to 10 grams of a viscous soluble fiber, is recommended for daily use (T. A. Shamliyan et al., J. Family Practice 55: 761-69(2006)).

Because of the difficulty of receiving the perfect quantity of soluble fiber using one power there is a clear need in the concentrations of soluble fiber that can be used as components of the food, or be used as additives to ensure consistent, high consumption of soluble fiber. Granulated VFC, also known as PGX® (PolyGlycopleX®), represents a novel high viscosity polysaccharide complex, which is produced through reaction of glucomannan, xanthan gum, and alginate using a process called EnviroSimplex®. The obtained polysaccharide complex (α-D-glucurono-α-D-manno-β-D-manno-β-D-glucan), (α-L-hourone-β-D-mannuronate), β-D-gluco-β~-D,. α-D-glucurono-α-D-manno-β-D-manno-β-D-gluco), (α-L-hourone-β-D-mannarino), β-D-gluco-β~-D-mannan is a new unit, as demonstrated in the structural analysis described in examples 5 and 6, and has the highest viscosity and water-holding capacity of any currently known fiber.

This example describes a study conducted to study the effectiveness of VFC granules and a slight change of lifestyle on weight loss, body mass index (BM), as well as cardiometabolic risk factors, including cholesterol, cholesterol of low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides, fasting insulin, fasting glucose and 2-hour test for glucose tolerance during a 14-week time period in adults who are overweight or obese. Ways:

Participants: a Total of 29 adults, leading a sedentary lifestyle (23 women; 6 men), aged 20 to 65 years, with a range of body mass index (BMI) constituting from about 25 kg/m236 kg/m2were invited to participate by placing a series of ads in local Newspapers. Subjects provided written consent before participation in this program. Analysis of data was conducted in accordance with standards outlined in the Helsinki �declaratii from 1975.

Anthropometric and other measurements: participants were measured twice per week height (cm) weight (kg) and waist and hip (cm) when using standard medical measuring tape. Waist and hips were carried out in the relevant anatomical localizations of about 2.2 cm above the navel and around the hips in the greater trochanter of the subjects wearing a disposable paper gown. The percentage of fat was determined using bioelectrical impedance analysis (RJL Systems, Michigan, USA) at baseline (before the start of the study and every two weeks after the start of the study. Computerized analysis of impedance data was used to determine the body mass index (BMI) and percent fat content.

Nutrition and supplements: Each volunteer received General instructions from the doctor regarding a healthy diet, weight loss and exercise. Moreover, advice on diet and exercise was conducted for the group every two weeks for 14 weeks. Special attention in these lectures was given not counting calories, but primarily focused on the control of portion size and how to maintain a diet low in fat, low glycemic index. General recommendations were also included in the programme, concentrating on the variety, type and time�Yeni exercises (for example, strength training and cardiovascular aerobic exercise), which increase total weight loss. Moreover, the subjects provided a granular viscous fiber complex (VFC) (pellets cognac/xantana/alginate (70:13:17), also called complex fiber PolyGlycopleX (PGX®)), which can be added to drinking or products (e.g., low-fat yogurt).

Five grams of the granules VFC must be consumed, drinking 500 ml of water 5-10 minutes before each meal, two to three times a day for 14 weeks, for a total daily consumption of 10 to 15 grams of granulated VFC/day.

Blood sampling and laboratory biochemical analysis: All laboratory measurements were performed by an independent lab in British Columbia, Canada. At baseline (before the start of the study) subjects were asked to abstain from food for ten hours before the procedure, blood sampling, which included the following tests: total cholesterol, triglycerides, HDL, LDL, glucose, insulin and 2-hour insulin. Oral test for glucose tolerance in the amount of 75 grams, was also conducted in accordance with the criteria and procedures established by the laboratory. Only people with aberrant risk factors have passed repeated the study using the above laboratory parameters after 14 weeks.

Statistics�technical analysis: a Computerized statistical analysis was performed using paired t-tests to assess several types of variables includes height, weight, BMI, % fat and various laboratory values before and after treatment. Significant results were obtained from those variables, which gave a value of p<0,05.

Results:

Weight loss and other anthropometric parameters: during the 14 weeks of using VFC has been a significant weight reduction in the group (-5,79±3.55 kg), waist size (-12,07±5,56 cm) % fat (-2,43±2,39%) and BMI (-2,26±1.24 kg/m2). The full results are shown below in TABLES 4 and 5.

TABLE 4:
Group 1: Men and women together
ResearchSample size0 week Average value and SD14 week Mean value and SDChange and SD% change
"Waist29103,58b±12,7891,51b±12,95-12,07±5,56b-11,65
*Hip29116,30b±7,67106, 83b�7,44 -9,47±4,15b-8,14
*% fat2940,30±8,2837,87±8,88-2,43±2,39-6,02
*p<0.05 from 0 weeks
a = the weight is specified in kilograms (kg)
b = waist and hips are specified in centimeters (cm)

31,27c±8,17
TABLE 5:
VM for all groups taken together
ResearchSample size0 week Average value and SD14 week Mean value and SDChange and SD% change
*Man635,03c±Of 4.0932,47c±3,78-2,56c±1,22-7,31
*Woman2333,45c±EUR 7.57-2,18c±1,26-6,52
*All29Equal to 33.78c±6,9631,52c±7,43-2,26c±1,24-6,70
*p<0.05 from 0 weeks
c=BMI in kg/m2

In this way both sexes individually showed a significant reduction in the studied variables weight loss, as shown below in TABLE 6 and TABLE 7. As shown below in TABLE 7, on average, men lost 8,30±2,79 kg over a 14-week study (average weight loss is 7,43%). As shown in TABLE 6, the women lost on average of 5.14±3,49 kg over a 14-week study (average weight loss is 6%).

TABLE 6:
Group 1: Women (n=23)
Research0 week Average value and SD14 week Mean value and SDChange and SD % change
*Weight84,29a±7,85Equal to 79.15a±8,77-5,14a±3,49-6,00
Waist98,98b±8,9987,55b±10,57-11,43b±5,71-12,00
*Hip115,19b±6,73105,92b±7,34-9,27b±4,29-8,00
*% fat43,88±4,5241,33b±6,15-2,55b±2,63-6,00
* p<0.05 from 0 weeks
a = the weight is specified in kilograms (kg)
b = waist and hips are specified in centimeters (cm)
TABLE 7:
Group 2: Men (n=6)
Research0 week Average value and SD14 week Mean value and SDChange and SD% change
*Weight111,81a±9,18103,51a±13,05-8,30a±2,79-7,43
Waist121,13b±9,65106,63b±10,23-14,50b±4,59-12,00
*Hip120,57b±7,62110,36b±7,39-10,21b±3,63-8,00
*% fat26,58±3,0124,62b±2,97-1,97b±1,15-7,00
p<0.05 from 0 weeks
a = the weight is specified in kilograms (kg)
b = waist and hips are specified in centimeters (cm)

The levels of lipids, compared with baseline values obtained before the study began after 14 weeks of use VFC among subjects was observed, the mean lowering of total cholesterol values, constituting John 19: 26% (n=17; p<0.05 from 0 weeks), and the average decrease in values of cholesterol low-density lipoprotein, which 25,51% (n=16; p<0.05 from 0 weeks). As shown in TABLE 8, there was a tendency to decrease triglyceride and increase in the values of cholesterol of high density lipoproteins in this study, however, the observed differences were not statistically significant.

Insulin and fasting glucose: After 14 weeks of use VFC subjects involved in this study occurred an average reduction in fasting glucose, comprising of 6.96% (n=20; p<0.05 from 0 weeks), the average deviation of 2-hour glucose tolerance gap of 12,05% (n=21; p<0.05 from 0 weeks), and the mean decrease in levels of fasting insulin, which 27,26% (n=17; p<0.05 from 0 weeks), compared with baseline measurements made prior to the start of the study.

TABLE 8:
Summary of laboratory data obtained during the 14-week trial with VFC (PGX®)
ResearchSample size0 week Average value and SD14 week Mean value and SDChange and SD% change
"Total cholesterol (mmol/l)175,69±1,074,60±0,82-1,09±0,63-19,26
**Triglycerides (mmol/l)171,92±0,981,52±0,56-0,40±0,89-20,97
**HDL mmol/l)171,48±0,531,53±0,770,05±0,673,33
*LDL (mmol/l)163,40±0,962,53±0,64-0,87±0,56-25,51
Fasting glucose level mmol/l)205,75±0,785,34±0,49-0,40±0,65-6,96
*2 hour glucose mmol/l)216,09±2,105,35±1,81-0,73±1,43-12,05
"Fasting insulin (pmol/l)1789,41±44,8465,04±33,21-24,37±36,29-27,26
"2-hour insulin (pmol/l)17433,53±270,32355,76±332,44-77,76±196,51-17,94
*p<0.05 with 0 weeks; **NS (minor) from baseline

Analysis of efficiency in the use of a questionnaire to be samozabvennuyu: In the questionnaire, due samozabvennuyu that participants completed at the end of the study, 97.7% of users VFC noted that they had a positive reaction to the product and control appetite and hunger.

Side effects of test drug: Use�ie VFC mostly well tolerated by participants with minor gastrointestinal symptoms (GI), containing the majority of all reported complaints. Sixty-eight percent noted that moderate GI symptoms (e.g., gas, bloating, constipation, loose stools) were within approximately three weeks from the receipt of VFC. At thirty-two percent of participants were moderate GI side effects in the course of the program, but they were not heavy enough to stop using. Recent controlled study on tolerance VFC (PGX®) was conducted in France, which is also confirmed by these recent data (I. G. Carabin et al., Nutrition J. 8: 9 (2008)).

Discussion: the Study concerning weight loss, controlled from a medical point of view described in this example demonstrates that the use of VFC granules along with General changes in diet and physical activity over a 14-week time period, benefits in modifying cardiometabolic risk factors in subjects with overweight and obesity. In General, there was a significant decrease in weight in the group (-5,79±3.55 kg), waist (-12,07±5,56 cm) and percent fat (-2,43±2,39%) from baseline. Moreover, data from the most recent physical changes were compared with significant reduction in levels of LDL fasting (-25,51%), fasting glucose (-6,96%) and fasting insulin (-27,26%) within a relatively short interval of 14 weeks.

It is interesting to note that men on average lost more weight (-8,30±2,79 kg) than women (-5,14±3,49 kg) for 14-week period of time. This change can be attributed to basic sex differences observed in energy expenditure at rest. Dr. Robert Ferraro and others have shown that the daily consumption of the people leading a sedentary lifestyle, about 5 - 10% is lower in women compared with men after statistical adjustments regarding age, activity and compositional analysis of body composition. (R. Ferraro et al, J. Clin. Invest. 90:780-784 (1992)).

The results obtained through VFC in reducing body weight (-5,79 kg), comparable with the results of those people who took orlistat, a drug against obesity (Xenical®, Alii®). Orlistat is a lipase inhibitor that reduces fat absorption (J. B. Dixon et al., Aust. Fam. Physician 35:576-79 (2006)). In a controlled study of 391 individuals with mild to moderate degree of overweight who took the drug orlistat at a dose of 60 mg three times a day during the 16-week period, lost 3,05 kg, compared to 1.90 kg in the placebo group (J. W. Anderson et al., Ann. Pharmacother. 40:1717-23 (2006).

The use of VFC has also led to a reduction of other risk factors associated with mild-to-moderate obesity. � overall there was a significant decrease in total cholesterol levels (-19,26%; -1,09 mmol/l) and levels of LDL cholesterol (-25,51%; -0,87 mmol/l) compared to baseline (p<0,05) after 14 weeks of treatment VFC. The resulting reduction in lipid values using VFC compared with the use of early development latinovich drugs like lovastatin (Mevacor™). For example, one study noted that within one month of starting treatment lovastatinom, total cholesterol and LDL cholesterol decreased by 19% and 27%, respectively, in people with elevated cholesterol (W. B. Kannel et al., Am. J. Cardiol. 66: 1B-10B(1990)).

Moreover, as described in examples 1 and 2, the use of VFC not only lowers the levels of blood lipids, but also can be used to reduce the intensity of the development and progression of early stages of metabolic syndrome. The increase in visceral adiposity, serum glucose and insulin levels along with hypertension and dyslipidemia are a group of clinical conditions that are collectively known as metabolic syndrome (E. J. Gallagher et al., Endocrinol. Metab. Clin. North Am. 37: 559-79 (2008)). The study showed that people who have metabolic syndrome by 50% at a higher risk of undergoing primary coronary events (D. E. Moller et al., Annu. Rev. Med. 56: 45-62 (2005)). In this regard, any weight loss, fasting insulin and glucose yield significant health benefits for those people, which�s suffer from this disease.

In this 14-week study using VFC has led to lower levels of fasting insulin with 89,41±44,84 pmol/l to 65,04±33,21 pmol/l (p<0,05). The reduction of fasting insulin reflects the improvement in insulin sensitivity and may in part be due to increased activity of GLP-1 and reduced the postprandial hyperglycemia along with improvements in insulin sensitivity that accompanies weight loss (see G. Reaven et al., Recent Prog. Horm. Res. 59:207-23 (2004)).

These data are consistent with the results obtained in the study of diabetic Zucker rats described in example 1, and suggest that therapeutic use of the VFC with the change in lifestyle brings practical benefits to people suffering from obesity and certain cardiometabolic risk factors. Unlike other types of standard medical interventions available for the treatment of obesity and elevated cholesterol levels, the use of VFC is associated with minimal side effects. This preferential safety profile along with therapeutic efficacy suggests that VFC should be considered as a first line treatment for people with overweight/obesity, elevated cholesterol and/or insulin resistance.

EXAMPLE 4

This example describes a study regarding zdorovyaki people of normal weight, showing increased levels of PYY in plasma and increased short-chain fatty acids (SCFA) in fecal mass after the addition of viscous fiber complex (VFC) compared to control subjects treated with skimmed milk.

Rationale:

Numerous dietary fibers have been shown to have numerous health benefits, including increased secretion of hormones of the gastrointestinal tract that is responsible for the feeling of satiety and improve bowel function (R. A. Reimer et al. Endocrinology 137: 3948-3956 (1996); Reimer and Russell, Obesity 16: 40-46 (2008); P. D. Cani et al., Br. J. Nutr. 92:521-526 (2004); T. C. Adam and R. S. Westererp-Plantenga, Br. J. Nutr. 93:845-851 (2005)). Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are anorexigenic peptides involved in the reduction of eating, while ghrelin, the only known hypothalamic orexigenic peptide, is associated with hunger (Wren and Bloom, Gastroenterology 132: 2116-2130 (2007)).

Although the mechanisms regulating this use of dietary fiber, are completely incomprehensible, it is believed that the production of short-chain fatty acids (SCFA) mediates some of the effects. SCFA and mainly acetate, butyrate and propionate are produced in the colon by anaerobic fermentation of fermentable dietary fiber, and are associated with the stimulation of hormones responsible for the feeling of satiety, and modulation of cyberotic�CSOs cholesterol.

The purpose of this study was to examine the levels of hormones gastrointestinal tract, which is responsible for the feeling of satiety, GLP-1, PYY and ghrelin, as well as the concentration of SCFA in faecal mass in healthy subjects after consumption of VFC (PGX®) or control (skim milk powder) for 21 days.

Ways:

Subjects: the Participants were a healthy, non-Smoking men and women aged 18 to 55 years with a BMI between 18,5 and 28.4 kg/m2(i.e. normal weight).

Study design: Randomized, double-blind, placebo-controlled study was conducted, as follows:

Participants in a random order determined in two groups:

Group 1 (n=27) had used the subject product, the granules of viscous fiber complex (VFC) (cognac/xanthan gum/alginate (70:13:17)) (that is, the fiber mixture was processed by granulation to create complex sold under the name PGX®supplied Inovobiologic Inc., Calgary, AB, Canada).

Group 2 (n=27) had used control product (skimmed milk, which was similar in color and texture on the tested product).

The control and the test product were pre-mixed with 10 g selling Breakfast ready produced FEATURE Pharma, France, and is Packed with 135 ml of a commercially available yogurt without additives. Participants laugh�Ali yogurt and pre-mixed product before swallowing.

Within the first seven days of the study participants consumed 2.5 g of the product (test or control) twice a day, as part of the two main meals. Within the last 14 days of the study participants consumed 5 g of the product (test or control) twice a day. For the period of the duration of the study participants were instructed regarding the abstinence from foods that are enriched with fibers, and restrictions on the use of dietary fiber to about 10 g per day. With the exception of pre-mixed product and yoghurt all other products were purchased and prepared by the participants as ordinary food.

Evaluation: the Evaluation of participants was carried out in four separate visit. Screening (visit 0, visit screening, "V0") included a physical examination.

Blood samples: a Sample of fasting blood was taken at visit 1, day 0 of the study (baseline). Visit 2=seven days of the study, after one week of use 5 g of the product. Visit 3=day 21 of the study, after two weeks of use of 10 g of the product. During each visit, blood sampling was performed in a tube treated with EDTA with the addition of diprolene A (0.034 mg/ml of blood; MP Biomedicals, Illkirch, France) and centrifuged at 3000 rpm for 12 minutes at 4°C. Plasma granulative a temperature of -80°C until analysis.

Collection of stool: stool Samples were obtained from subjects at baseline (V1, O), after one week of use 5 g/l product (V2, day 8 ± 1) and after two weeks of use of 10 g/l product (V3, day 22 ± 2). Subjects collected one sample of feces for 48 hours before each scheduled visit. Approximately 5 g of the sample was sent for analysis on dry ice.

Analysis of blood plasma:

GLP-1 Active GLP-1 was quantified using ELISA kit from LINCO research (Millipore, PT.Charles, Mo). According to the manufacturer, the sensitivity analysis is 152 PM for sample volume of 100 µl. The coefficient of variation for internal analysis is 8% and the coefficient of variation for a series of tests is 13% at 4pm (Millipore, PT.Charles, Mo).

PYY and ghrelin: PYY and ghrelin were quantified using ELISA kits from Phoenix Pharmaceuticals, Inc. (Burlingham, CA). The sensitivity analysis regarding PYY was 0.06 ng/ml and 0.13 ng/ml on ghrelin. The coefficient of variation for internal analysis amounted to < 5% for the two tests and the coefficient of variation for a series of tests amounted to < 14% and < 9% on PYY and ghrelin, respectively.

Insulin: Insulin was measured using ELISA kit from Milliport (PT.Charles, Mo). The sensitivity analysis is 2 mked/ml with coefficient of variation for the internal�analysis < 7% and the coefficient of variation for a series of tests < 11,4%.

Statistical analysis: the Results are presented as the mean ± standard error of average size (SEM). The levels of the three peptides were analyzed by repeated measurements ANOVA with the Bonferroni correction [two-factor analysis with time (V1, V2, V3) and power as parameters]. The Association between the two parameters were calculated using the Pearson correlation coefficient. The homeostatic model assessment regarding insulin resistance was calculated using the formula [HOMA-IR=fasting insulin (mked/ml) X fasting glucose (mmol/l)/22,5]. Data were analyzed using the software SPSS v of 16.0 (SPSS Inc., Chicago, Il).

Fecal analysis: measurement of SCFA were carried out according to van Nuenen and others, Ecology of microorganisms in health and disease 15:137-144 (2003). Briefly, stool samples were prosencephalon and a mixture of formic acid (20%), methanol and 2-ethyl butyric acid (internal standard, 2 mg/ml in methanol) was added into a clean supernatant.0.5 ml of sample was injected into GC column (Stabilwax-DA, length 15 m, internal diameter of 0.53 mm, phase thickness 0.1 mm; Varian Chrompack, Bergen op Zoom, the Netherlands) in gas chromatography (Chrompack CP9001 when using an automatic sampler. L - and D-lactate were determined farm�Advisory by clean supernatant through vials Cobas Mira plus (Roche, Almere, The Netherlands). The pH was measured using a microelectrode. Dry matter was measured by drying the final sample to dryness at 110°C for at least 2 days.

Statistical analysis. The results were presented as mean ± standard error of the mean (SEM). SCFA during three visits were analyzed by re-conducting the analysis of variance (ANOVA) visits (V1, V2, V3) as intersubjective factors and treatment, and intergroup factor. The correlation between SCFA and other estimated parameters (hormones saturation, glucose, insulin and index of insulin resistance HOMA-IR) was determined using correlation analysis Pearson. The level of statistical significance was accepted at P≤0.05.

Results:

54 subjects (25 men and 29 women) participated in the study and completed all 4 visits (V0-V3). None of the actors refused to participate in the study, and the product was a good move. The average age of the control group treated with the control drug (11 men, 16 women), was equal to 30.9±10.8 BPM, and initial BMI is 22.8±2,4. In the group receiving the study drug (VFC), the average age was of 32.3±10.3 a, and initial BMI is 22.7±2,1. There were no differences in baseline clinical and biochemical characteristics between the groups.

The values of body weight, level g�uCoz, insulin and index of insulin resistance HOMA-IR during V1, V2 and V3 are presented in TABLE 9 below.

TABLE 9
Body weight and biochemical parameters of the participants who received the control drug or VFC
The control group (skimmed milk powder)The study group (VFC)
V1 (0 day)V2 (7 day)V3 (21 day)V1 (0 day)V2 (7 day)V3 (21 day)
Body weight (kg)64,60±1,57N/a64,60±1,5268,20±1,71N/a68,43±1,67
Glucose (mmol/l)4,60±0,064,60±0,074,62±0,104,67±0,094,60±0,084,60±0,08
Insulin (mked/ml) Of 5.32±0,854,52±0,335,19±0,335,52±0,564,52±0,494,61±0,47
The index of insulin resistance HOMA-IR1,11±0,200,93±0,071,07±0,071,15±0,110,96±0,110,94±0,10

Values represent mean ± standard error of the mean (SEM) (n=27/group). N/a - not measured. When gender was included as a covariate in repeated measurements ANOVA, the difference between visits was a significant index of insulin resistance HOMA-IR (p=0,024) for the studied group.

As shown above in TABLE 9, there were no significant changes in body weight between V1 and V3 in the control and study groups. The concentration of glucose in fasting plasma did not differ over time or between groups. Although there was a decrease in the level of fasting insulin by 14% between V1 and V3 in the study group (i.e., PGX), this difference was not statistically different from the control group. Absolute and relative changes in the values of the index of insulin resistance HOMA-IR were equal -0,04 or -3.6% in the control group and -0,21 or -18,3% in the study group. Percentage reduction in the index� of insulin resistance HOMA-IR was significantly greater in the treatment group, than the control (P=0.03). Analysis of variance of repeated measurements showed P=0,067 for the effect of the visit. When gender was included as a covariate in the analysis of repeated measurements, the effect of the visit was statistically significant (P=0,024). In a separate analysis in men revealed a greater reduction in the value of an index of insulin resistance HOMA-IR than in women (P=0.042) between V1 and V3. The decrease in the index of insulin resistance HOMA-IR was the same for control and study group participants-men (-0,36±0.20 and -0,31±0,18 respectively). However, in women the results of the index of insulin resistance HOMA-IR were increased in the control group (+0,18±0.17) and decreased in the study group (-0,08±0,19).

There was no significant differences in the levels of like peptide (GLP-1) fasting during visits or between groups (data not shown).

FIGURE 10A shows graphically the effect of controls compared to VFC on the levels of fasting peptide YY in healthy adults among all participants (n=54) while V1 (day 0), V2 (day 14) and V3 (21 days). The values are mean ± standard error of the mean (SEM). As shown in FIGURE 10A, a repeated measures analysis showed a statistically significant effect of visit (P=0.004) for levels of peptide YY on an empty stomach. When the results shown in FIGURE 10A, were stratified by BMI, participants with a BMI<23 display�whether a significant difference in the levels of peptide YY, as the effect of the visit (P=0.03) and treatment (P=0.037), as shown in FIGURE 10 V. analysis of Variance showed significantly higher levels of peptide YY in the study group compared with the control group at the end of the study (P=0.043). It should be noted that elevated levels of peptide YY are favorable, as this anorexigenic hormone, which is associated with decreased food consumption.

As shown in FIGURE 10C, a repeated measure ANOVA showed a significant effect of visit (P<0,001) for the General level of ghrelin on an empty stomach and treatment (p=0.037). As shown in FIGURE 10C, a decline to 89,7±20,0 and 97.7±26.6 mcmol/l was observed in the control group and the treatment group receiving VFC, respectively.

Peptide YY is negatively correlated with the level of glucose in V2 (r=-0,27, P=0.046). There were also significant negative correlations between ghrelin and insulin in V1 and V2 (r=-0,28, P=0,038 and r=-0,31, P=0,022, respectively) and between ghrelin and the index of insulin resistance HOMA-IR in V1 and V2 (r=-0,27, P=0,052 and r=-0,28, P=0,041, respectively).

Fecal SCFA and lactate:

As shown below in TABLE 10, the concentration of acetate were significantly higher in the group with VFC (PGX®) compared to control (P=0.01). There were no differences in the concentrations of acetate between the groups in V1 (baseline; p=0,286) or V2 (p=0,096), but concentrations were significantly higher with VFC (PGX ®) compared with the control group at V3 (p=0.018). There were no significant differences in treatment between groups in concentrations of propionate, butyrate, valerate, caproate or lactate. Repeated measures analysis showed a significant effect of treatment (P=0.03) on SCFA, which was defined as a higher level of SCFA in V3 of the subjects who received VFC (PGX®) compared to control (P=0,06). There was a significant effect of visit on the pH of feces (0,02) with a decrease in both groups between V1 and V3.

Correlation with the hormone of satiety, insulin and glucose

The results of the analysis in plasma levels of ghrelin, peptide YY, GLP-1, insulin, glucose and index of insulin resistance HOMA-IR are shown above in TABLE 9. As shown below in TABLE 10, there was a significant negative correlation between ghrelin and propionate on an empty stomach in V3 (r=-0,29; P=0.03). The change in the concentration of propionate between baseline and the last visit was calculated as V3-V1 and is named Delta propionate. Delta propionate was negatively associated with Delta insulin (r=-0,26; P=0.05) and Delta index of insulin resistance HOMA-IR (r=-0,25; P=0,07).

TABLE 10:
Fecal concentrations of short-chain fatty acids (SCFA) and �of actate entities after adding VFC (PGX ®) or the control product.
ControlVFC (POX"&)β-values
V1V2V3V1V2V3VisitVisit × treatment Treatment
SCFA (mmol/g of feces)
Total61,1±4,459,2±5,053,5±5,2t66,8±4,463,5±3,666,9±4,70,780,030,48
Acetate35,8±2,4 33,2±2,530,3±2,7*39,5±2,438,7±2,039,9±2,80,510,010,40
Butyrate10,0±1,111,1±1,59,5±1,212,4±1,310,7±0,911,6±1,00,770,260,31
Propionate11,4±1,210,8±1,110,2±0,410,9±0,810,7±0,811,5±1,00,890,850,55
The valerate3,1±0,33,7±0,43,0±0,43,4±0,33,1±0,33,3±0,30,660,980,20
The caproate0,58±0,100,41±0,110,55±0,090,41±0,080,50±0,120,330,940,60
Lactate (mmol/g of feces)0,62±0,090,74±0,090,46±0,070,52±0,090,48±0,090,46±0,070,050,220,12
PHAbout 6,82±0,096,71±0,166,43±0,256,69±0,096,68±0,096,40±0,080,020,770,88
The results represent the mean value ± standard error of the mean (n=27 / group). The symbol * represents a statistically significant difference between control and VFC (PGX®during 3 visits (V3). The symbolconstitutes a trend (p=0,06) for the difference between the control and VFC (PGX®) during 3 visits (V3).

Discussion

Analysis of the p�of ptid YY plasma

The results of the study described in this example demonstrate that the use of VFC increases levels of peptide YY on an empty stomach compared to the control product, and they are statistically significant in patients with BMI<23. The concentration of peptide YY in the plasma, as a rule, reduced in people with overweight and obesity (R. L. Batterham et al., Nature 418: 650-654 (2002)), and that the impaired secretion of peptide YY may contribute to the development of obesity and/or inhibit weight loss.

While participants of the control group noted a moderate decrease in the level of peptide YY during the three-week study, participants who consumed VFC, managed to retain, and those with BMI<23, actually increase their levels of peptide YY. It has recently been shown that microbial fermentation of prebiotics in healthy adults is associated with increased production of GLP-1 and peptide YY (P. D. Cani et al., Am. J. Clin. Nutr. (2009)). It has been shown that in rodents, short-chain fatty acids (SCFA), which are by-products of microbial fermentation of dietary fiber, directly stimulate the secretion of peptide YY (V. Dumoulin et al., Endocrinology 139:3780-3786 (1998)). It was shown that the cognac glucomannan, one of the original VFC products, increases the concentration of fecal acetate, propionate and butyrate in humans (H. L Chen et al., J. Am. Coll. Nutr. 27:102-108 (2008)). It has also been demonstrated that the viscosity of the ox�con independently affects the consumption of food, and this effect may be mediated by changes in the release of the hormone of satiety.

The level of fasting ghrelin was suppressed between 1 visit (day 0) and 3 visit (21 days) in the group consuming the study product containing VFC, and in the group consuming the control product. Since ghrelin stimulates food intake and promotes the development of obesity (A. M. Wren et al., J. Clin. Endochnol. Metab. 86:5992-5995 (2001); M. Tschop et al., Nature 407: 908-913 (2000)), compounds that weaken the progressive increase production of ghrelin to food intake, are attractive. While reducing the level of ghrelin on 8 mmol/l greater in the group VFC compared with the control group was not significantly different in this study, others demonstrated a decrease in the level of ghrelin and fasting is associated with food by eating dietary fiber (see, e.g., Parnell and Reimer, 2009). While the mechanisms by which dietary compounds inhibit ghrelin, still insufficiently known, it has been hypothesized that the absorption rate of nutrients and the osmolarity of the lumen of the intestine may play a role (Overduin et al., Endocrinology 746:845-850 (2005)). Further, in this regard it should be noted that VFC has a viscosity of from 3 to 5 times higher than any currently known polysaccharides, and therefore, probably changes the absorption of nutrients in �Chechnya.

In this study no difference in the level of GLP-1 on an empty stomach between the two groups for three weeks. This lack of change in the level of GLP-1 was observed with other dietary fibers (T. C. Adam and R. S. Westererp-Plantenga, Br. J. Nutr. 93:845-851 (2005); K. S. Juntunen et al., Am. J. Clin. Nutr. 78: 957-964 (2003)).

Although the concentration of glucose and insulin in healthy subjects who participated in this study was within the normal range, 14% reduction of insulin during the study in the study group and 5.3 fold greater reduction of the values of the index of insulin resistance HOMA-IR in the study group compared with the control group may be a symptom of a commensurate improvement in insulin sensitivity, which is consistent with the results obtained in examples 1 and 3. Thus, this study demonstrates that VFC (PGX®increases the level of fasting peptide YY, peptide gastrointestinal tract involved in the reduction of food intake among healthy participants.

Analysis of SCFA in the feces

As described above, fermentable dietary fiber can reduce power consumption and increase the secretion of anorexigenic hormones of the gastrointestinal tract. The generation of SCFA from microbial fermentation of dietary fibers in the distal intestine is thought to play a role in this regulation. Recently Cani et al., Am J. Clin Nutr 90:1236-123 (2009) demonstrated a significant correlation between the hydrogen breathing (a measure of microbial fermentation in the gut) and the level of GLP-1 in plasma, a potent insulinotropic hormone, which also reduces food intake. The present study is based on this data, showing a significant increase in the faecal concentration of the total amount of SCFA, acetate in particular, in individuals who consumed 10 g/day of a new functional fiber complex PGX®.

Acetate, propionate and butyrate are the major SCFA produced in the distal bowel. Receptors free fatty acids (FFAR), interact with SCFA in the gut, were recently identified as the receptor free fatty acids-2 (FFAR2) (also known as GPR43 - G-protein coupled receptor-43) and receptors free fatty acids-3 (FFAR3) (also known as GPR41 - G-protein coupled receptor-41). Cm. Ichimura A. et al., Prostaglandins &Other Lipid Mediators 89:82-88 (2009). FFAR2 is expressed in enteroendocrine cells which secrete peptide YY, which corresponds to the data that SCFA stimulate the release of peptide YY (Ichimura et al., 2009). It was shown that in vitro acetate and propionate inhibit lipolysis in 3T3-L1 adipocytes through the activation of FFAR2 and reduce the level of free fatty acids (FFA) in plasma in vivo in mice. Cm. Ge H et al., Endocrinology 149: 4519-4526 (2008). Increased FFA level was associated with insulin resistance and dyslipidemia. There is also evidence that taking propionate increases inside the number �aptina in mice FFAR3 (Ichimura et al., 2009). Given that leptin acts centrally to reduce food intake, it is possible that SCFA produced during microbial fermentation of dietary fiber, partly regulate the metabolism of the body using FFAR2 and FFAR3.

The results of this study demonstrate a significant increase of acetate and total SCFA by the end of the third week of receiving VFC (PGX®). Although there were no changes in body weight in our subjects during the three weeks of supplementation, there is the possibility that the consumption of fiber PGX®in the last study dose (10 g/day) may result in decrease of body fat, as has been shown with other soluble fibers such as oligofructose, within three months. (Parnell, J. A. et al., Am J Clin Nutr 89:1751-1759 (2009). A negative correlation between propionate and ghrelin fits in the overall reduction of food intake associated with dietary fiber, in particular, with high viscosity, such as VFC (PGX®). A negative correlation between insulin and index of insulin resistance HOMA-IR corresponds to the functional ability of these fibers to improve metabolic health and reduce insulin resistance.

In conclusion, we note that the results of this example show increased fecal acetate in individuals consuming moderate doses of viscous and soluble �elocon, VFC (PGX®) during a 3-week period of time. SCFA, propionate, negatively correlated with ghrelin, insulin and index of insulin resistance HOMA-IR fasting. As far as we know, this is the first report showing an increase in the concentration of fecal SCFA after taking VFC (PGX®), which implies that its fermentation in the colon can cause a cascade of physiological effects, potentially mediated through FFAR2 and FFAR3.

EXAMPLE 5

This example describes the analysis of the primary structure of granular viscous fiber complex (VFC) (cognac/xanthan gum/alginate (70:13:17) (i.e., the fiber blend was processed by granulation with the formation of complex commercial name PGX®).

Rationale:

Polysaccharides are natural polymers composed of Sugars (monosaccharides) linked through glycosidic hydroxyl group. They can be branched or linear, having a very high molecular weight from a few thousand daltons to more than two million. The primary structure of granulated VFC (70% cognac mannan, 17% xanthan gum, 13% of sodium alginate) was determined using the analysis of methylation, hydrolysis and chromatography and hydrolysis and NMR spectroscopy.

Brandy glucomannan partially acetylated (1,4)-β-D-glucomannan derived from the club�her Amorphophallus konjac or Konjac root (Bewley et at., 1985, Biochemistry of Storage Carbohydrates in Green Plants, Academic Press, New York, pp.289-304).

Xanthan gum is a microbial polysaccharide produced by Xanthomonas campestris. It has unique rheological and gelling properties. The structure of xantana built on a cellulose basis of β-(1,4)-linked glucose residues that have side trisaccharide chain mannose-glucuronic acid-mannose associated with each second residue of glucose in the main chain. Some terminal mannose residues are pirovinogradnoi group, and some of the internal mannose residues - acetyl groups (Andrew T. R., ACS Symposium Series No. 45 (1977)).

Sodium alginate is the sodium salt of the polysaccharide obtained from brown algae (e.g., Laminaria hyperborea, Fucus vesiculosus, Ascophyllum nodosum). The chemical structure consists of blocks of (1,4) linked-β-D-polymannuronic acid (poly M), (1,4) linked-α-L-prigorodovoy acid (poly G) and alternating blocks of the two uronic acids (poly-MG). Grasdalen, H., et al., Carbohydr Res 89: 179-191 (1981). Alginates form strong gels with divalent cations of metals, to describe this form of gel is used the model of "packing for the transportation of eggs." Cm. Grant, G. T., et al., FEBS Lett 32:195-198 (1973).

Methods

All the polysaccharides used in this Example were provided by InovoBiologic Inc (calgary, Alberta, Canada). Separate Polish�Itami were: brandy glucomannan (batch No. 2538 and 2681); xanthan gum (batch No. 2504 and 2505); sodium alginate (batch No. 2455, 2638 and 2639). Granulated VFC (PGX, batch No. 900495 and 2029070523) were made by mixing 70% of cognac-mannan, 17% xanthan gum and 13% of sodium alginate) with the addition of 30% to 60% (%by weight) of water to the VFB and the subsequent drying of the added water by the application of heat. Samples of the same ternary mixture (raw VFB) were taken before treatment (e.g., granulation); these are referred to as a three-component mixture No. 1 (TM1, batch No. 900285, 900416 and 1112050809).

1. Analysis of methylation

Rationale: Analysis using the method of gas chromatography and mass spectrometry partially metilirovannah acetates alditol was used to identify monosaccharide components of polysaccharides and provisions of the bonds (H. Bjorndal et al., Carbohydrate Research 5: 433-40 (1967)). Thus, the methylation analysis may reveal new and unexpected sugar, as well as the provisions of the bonds that were created in the process of adding three polysaccharides (cognac mannan, xanthan gum and alginate) together and processing them using thermal treatment and the granulation process. However, analysis of methylation does not show, as sugars are linked to each other (α or β). It is known that the methylation analysis is not satisfactory for the analysis of uronic acids (e.g., sodium alginate), to�which is not subject to methylation and resistant to hydrolysis (Percival et al., Chemistry and Enzymology of Marine Algal Polysaccharides, Academic Press 101 (1967)). Since sodium alginate is composed entirely of uronic acids (mannuronate and guluronate acids) for analysis of VFC was necessary to use additional methods, which include hydrolysis and analysis of neutral Sugars and uronic acids by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and 1H NMR spectroscopy, as described below.

Methods:

Analyzed the examples shown below in TABLE 11, which includes every single component VFC (cognac mannan, sodium alginate, xanthan gum), granular VFB (hereinafter referred to as "triple blend # 1" or "TM1") and granulated VFC (hereinafter referred to as PGX®). Were taken weighed quantity of each polysaccharide and their ternary mixture, a few drops of dimethyl sulfoxide was added to 450 µg of each sample. The samples were fully methylated using sodium hydroxide (NaOH/methyl iodide (Mel)), the samples were shaken and then treated with ultrasound for a total of four times within two hours. The samples were purified by extraction with chloroform, then gidrolizirovanny 2M trifluoroacetic acid (TFA) for two hours at 120°C and recovering boron-sodium deuteride (NaBD4) in 2M NH4OH for two hour�in at room temperature. Borat, resulting in the decay of boron-deuteride, were removed by triple add a mixture of methanol and glacial acetic acid (90:10) followed by lyophilization. Then the samples were azetilirovanie with acetic anhydride (1 hour at 100°C). Acetylated samples were purified by extraction with chloroform.

The results of the analysis of methylation:

TABLE 11:
Retention time (in minutes) of partially methylated acetate alditol in relation to sugars and linkages in lots of different samples determined by gas chromatography and mass spectrometry (CL = traces n. d. = not detected)
SamplePart numberTerm price mannose or glucose2-linked mannose4-linked mannose4-linked glucose3,4-linked hexose
Brandy mannan253812,52n. d.13,7813,86n. d.
Sodium alginate2455n. d.n. d.13,78n. d.n. d.
Xanthan gum250412,5413,68n. d.13,8514,65
Form of pellets VFB (TM1)900285n. d.traces (13,65)13,7713,8514,65
Form of pellets VFB (TM1)1112050 80912,47traces (13.62)13,7413,83traces (14,63)
Granulated VFC (PGX®)2029070 52312,51traces (13,66)13,7813,8614,65
Granulated VFC (PGX®)2029070 523 12,48n. d.13,7413,83traces (14,62)
"TM1"": a three-component blend # 1

TABLE 11 gives a summary of the results observed in the reconstructed ion chromatograms (not shown) of the analysis of contacts made on partially methylated the acetates alditol (PMAAs), obtained from seven samples. As shown in table 11, the sample of sodium alginate gave only a weak signal for 4-linked glucose. The comparison of the signals observed in each sample of polysaccharides, shows that components found in the powder cognac mannan, consistent with the stated structure, namely glucose and mannose linked through the 4 position with a short terminal side chains. A sample of xanthan gum, gave a weak signal on 2-linked mannose in addition to strong signals on terminal mannose and/or terminal glucose, and 4-linked glucose. The signal extracted from the sorbent at 14,65 minute, showed the nature of fragmentation, the corresponding 3,4-linked branched hexose. All signals of xanthan gum, consistent with the stated structure. Signals for approximately 14,65 minute of xanthan gum, and all samples and VFB VFC consistent with 3,4-associated point RA�branching hexose, observed in the sample of xanthan gum.

Thus, the overall profile of the transmitted signals in the samples contains components corresponding to the brandy mannan, and contains components that can be classified as xanthan gum (the branch point). These results methylation are consistent with the following conclusions. First, the form of pellets and VFB (TM1) and granulated VFC (PGX®have brandy mannan (4-linked mannose) and xanthan gum (3,4-linked branched glucose). Other methylated sugar in the spectrum can come from any of these biopolymers. Second, there are no other common biopolymers (for example, there is no evidence of the presence of galactomannan, carrageenan, etc.), 6-linked glucose (starch), etc. third, these results provide no evidence that formed the new sugar-like structures (for example, no masses corresponding to other sugars, as in granulated VFC, and in the form of pellets VFB, which would have similar mass spectra). Fourthly, this analysis is not able to identify a component of sodium alginate due to the fact that the source of uronic acid is not methylisourea (Percival et al., Chemistry and Enzymology of Marine Algal Polysaccharides, Academic Press 101 (1967)). Analysis of sodium alginate is used for hydrolysis and chromate�graphy and hydrolysis and NMR spectroscopy, as described below.

2. Analysis of the hydrolysis method and gas chromatography and mass spectrometry (GC-MS)

Rationale:

And xanthan gum, and sodium alginate contain uronic acids, namely glucuronic (xanthan gum) and mannuronate and guluronate (sodium alginate). These structural features are difficult to determine due to the extreme hydrolytic resistance of uronic acids in polysaccharides induced electron carboxyl group, making it very difficult to achieve in the first phase, acid-catalyzed hydrolysis, namely protonation of the glycosidic oxygen atom (Percival et al., Chemistry and Enzymology of Marine Algal Polysaccharides, Academic Press 104 (1967)). This leads to the high stability of these polysaccharides to attack. Methods of hydrolysis of sodium alginate in the prior art describes the processing of 90% H2SO4within a few hours followed by boiling for 24 hours after dilution (Fischer et al., Hoppe-Seyler's Z Physiol Chem 302:186 (1955)). Lately, however, discovered and used in a strong volatile acid, trifluoroacetic acid (TFA), which hydrolyzes a very stable connection of polyuronides with the added advantage of volatility to facilitate removal (L. Hough et al., Carbohydrate Research 21:9 (1972)).

This example describes a new analytical method that was developed for hydraulic�Lisa VFB and VFC (also known as "VFB/C") and characteristics of all products of hydrolysis (glucose, mannose, glucuronic acid, mannuronate acid and guluronic acid) using chromatography and complementary use of NMR.

Methods:

The analysis method GC-MS: Partially methylated acetates alditol (PMAAs) were separated and identified using gas chromatography and mass spectrometry (GC-MS). GC separation was carried out using a DB5 column, inject the sample directly into the column at 45°C and the temperature program of 1 min at 40°C, then 25°C/min to 100°C, then 8°C/min to 290°C and, finally, by holding at 290°C for 5 minutes. MS identification was performed with an ionization voltage of 70 eV in the scan mode in the mass range 50-620 daltons with a single resolution.

Partial hydrolysis Conditions: Hydrolysis: Conditions were developed for the hydrolysis VFB/C trifluoroacetic acid (TFA), which hydrolyzes polysaccharides as fully as possible, without attacking sugar to such an extent that the results could be masked by undesirable breakdown products.

TFA hydrolysis was carried out at 30-mg samples are presented in TABLE 11, which were placed in a sealed tube with 2M TFA and heated to 100°C for 1 h, 2 h, 4 h, 8 h, 24 h and 72 h. the Samples were removed from heat in a timely manner, TFA was evaporated in the freeze dryer, and the sample was investigated using thin-layer chromatography (TLC) (solvent�:butanol:ethanol:water, 5:3:2) on plates of silica gel (TLC silica gel 60°F Merck). Spots were visualized using sulfuric acid (5%) in methanol. It was found that the best conditions for hydrolysis of polysaccharides in VFB/C as much as possible before components Sugars, no attack of Sugars to such an extent that the results could be masked by undesirable degradation products, were 2M TFA incubation for 72 h at 100°C, filtered, freeze-dried x2. The results are shown in TABLE 12.

The results of the analysis hydrolysis:

TABLE 12:
The results of the TFA hydrolysis
SamplePart numberThe hydrolysis products of TFA
Brandy mannan2538A mixture of glucose and mannose
Sodium alginate2638The mixture mannuronate and guluronate acids
Xanthan gum2504Glucose, mannose, glucuronic acid
VFC granules (cognac/xanthan gum/alginate (70:13:17) PGX®Part number 2029070523Glucose, mannose, uronic acid

Chromatography:

After the discovery of the conditions of hydrolysis, which releases the sugar components of the three polysaccharides, was developed chromatographic method capable of providing both neutral sugars (glucose, mannose) of uronic acids (glucuronic acid, mannuronate acid and guluronic acid).

Acid Dionex chromatography is a chromatographic method that is widely used for sugars and related compounds. This determination method is much more sensitive compared to many other methods that have been used in the past, such as the refractive index.

Methods:

Equipment: Dionex ICS-3000 ion exchange chromatography system with dual pump, an electrochemical detector, a data processing system Chromeleon.

Materials: Water (deionized and filtered), sodium hydroxide (50% solution, electrochemical qualifications WEEK), sodium acetate, anhydrous (299,5%).

td align="justify"> The injection volume
TABLE 13:
Chromatographic conditions:
The deviceSystem liquid chromatog�aafia Dionex, equipped with a pulsed amperometric detector
ColumnDionex CarboPac PA1 (250×4 mm) Dionex CarboPac PA1 protective column (50×4 mm)
EluentA: water B: 500 mM sodium acetate in 10 mM NaOH In: 100 mM NaOH
GradientTime%B%
0015,5
20015,5
21500
32500
32,50100
420100
42,5015,5
52015,5
Flow rate1 ml/min
10 µl
Column temperature30°C
Recording time52 min

Specimen preparation: (concentration of ~0.02 mg/ml)

Sample solutions were prepared at a concentration of approximately 0.02 mg/ml of initial concentration in the D2O 30 mg/ml (NMR samples). Aliquots (15 µl) of the hydrolysate and standard solution dissolved in deionized water (30 mg/ml) and was diluted to 0,0225 mg/ml with deionized water for analysis. Were also prepared with standard solutions of each of the expected components of the three polysaccharides: glucose and mannose (from cognac glucomannan and xanthan gum), glucuronic acid (xanthan gum) and mannuronate and guluronate acid (from sodium alginate).

Samples were injected in a Dionex CarboPac PAl (250×4 mm) column with a protective column (50×4 mm) at 30°C. the Column was suirable with gradient of solvent formed from A: deionized water; B: 50 mmol sodium acetate (anhydrous, ≥ 99.5% pure) in 100 mmol NaOH (electrochemical qualifications WEEK) and b: 100 mmol NaOH at a flow rate of 1 ml min-1, as shown in TABLE 13.

The results of chromatographic analysis:

TABLE 14 shows the cut�the objectives Dionex ion chromatography of hydrolysates of different samples of fibers.

TABLE 14:
The results of Dionex ion chromatography of hydrolysates
SampleRetention time (min)/height (number plates - nC)/ACC. area (%)
Standards:
glucose13,98 min/175,56nC99,95%
mannose15,22 min/56,23nC99,61%
glucuronic acid25,58 min/214,36nC94,79%
Manureva acidOf 25.75 min/327,64nC97,95%
gourova acid*25,13*
Test samples (hydrolysates)
brandy mannan (Batch No. 2538)13,98 min/45,26nC41,05% (glucose)
15,22 min/57,94nC57,27% (mannose)
xanthan gum (Batch No. 2504)13,98 min/6,49nC46,99% (glucose)
15,22 min/3,99nC29,93% (mannose)
25,58 min/2,7nC4,87% (glucuronic acid)
sodium alginate (Batch No. 2638)13,95 min/0,428nC3,89% (glucose)
15,23 min/0,275nC1,90% (mannose)
25,13 min/7,06nC14,95% (gourova acid)
Of 25.75 min/35,03nC75,37% (Manureva acid)
form of pellets13,98 min/15,79nC40,94% (glucose)
VFB(TM1)15,20 min/18,76nC52,42% (mannose)
Part number 90041625,58 min/1,44nC0,83% (glucuronic acid)
Of 25.75 min/3,09nC1,96% (Manureva acid)
granular13,98 min/15,70nC40,62% (glucose)
VFC15,20 min/18,76nC52,50% (mannose)
(PGX®)25,58 min/1,42nC0,82% (glucuronic acid)
Part number 900495Of 25.75 min/3,15nC2,03% (Manureva acid)
*the hydrolysis of sodium alginate (provided that the second largest peak accounted for guluronate acid)

TABLE 15:
Commercial biopolymers and their monosaccharide components
Name of commercial biopolymerThe composition of Sugars
Starchglucose
Carrageenangalactose
Sodium alginateManureva acid, gourova acid
Gum plodotvornogo tree/guar gumgalactose, mannose
Brandy glucomannanglucose, mannose
Phytelephas mannanmannose
Xanthan gumglucose, mannose, glucuronic acid
Arabinogalactan larcharabinose, galactose
Cellulose ethersglucose
Acacia gum (gum Arabic, etc.)complex mixture
VFC (PGX®)glucose, mannose, glucuronic acid, Manureva acid

As shown in TABLE 14, the results of the analysis of GC-MS show that the components of sugar and sugar acids were well separated within a 35-minute recording time. As shown in TABLE 14, TFA hydrolysis VFB/C gives a unique profile, which traces Dionex clearly observed four monosaccharides, namely glucose, mannose, glucuronic acid and Manureva acid. These results correspond to the composition of the VFB/C, including brandy glucomannan (mannose, glucose), xanthan gum (glucose, mannose, glucuronic acid) and sodium alginate (Manureva acid and guluronate acid).

TABLE 15 shows the monosaccharide components of various commercial biopolymers, showing that VFC (PGX®) has a unique profile monosaccharide components. Thus, these results demonstrate that TFA hydrolysis and GC-MS separation can be used to distinguish the VFC from other combinations of monosaccharides.

Thus, GC-MS analysis of partially methylated acetates alditol cognac glucomannan and xanthan gum showed the presence of characteristic Sugars and relationships expected from their known primary structures. Brandy glucomannan gave GC peaks, sootvetstvuyushie-linked glucose 4-the associated mannose and terminal glucose and/or mannose (mainly from the side chains). Xanthan gum gave strong peaks corresponding terminal mannose and/or glucose (from side chains) and 4-linked glucose (main chain), plus a peak for 3,4-linked hexose (glucose) and a weak peak for 2-linked mannose (both of the side chains). Almost all these peaks were also detected in GC-MS analysis of partially methylated acetates alditol TM1 and granulated VFC (PGX®as shown in TABLE 11, showing that they both contain brandy glucomannan and xanthan gum. Trace peak elution positions 2-linked mannose from the component of xanthan gum, was too weak to give it a massive range, but the signals in the retention time by 12.47 and 14,65 minutes correspond to terminal mannose and 3,4-linked hexose (glucose) xanthan gum, respectively. It is important that these analyses did not reveal any unexpected additional Sugars or linkages of Sugars in TM1 or granulated VFC (PGX®) that would come from other components of biopolymers or from any new Sugars or linkages of Sugars, which could be formed during processing. As expected, GC-MS analysis of partially methylated acetates alditol TM1 and granulated VFC was not able to detect �omponent sodium alginate.

Whereas high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), TABLE 14 shows the measured retention time of the standards, components expected components of hydrolysis TM1 and granulated VFC along with the results of the chromatograms obtained from hydrolysates TM1 and granulated VFC. As shown in TABLE 14, four of the possible components (glucose, mannose, glucuronic acid and Manureva acid) were detected in the hydrolysates TM1 and granulated VFC. The fifth component, gourova acid, was not detected in this analysis, which is likely due to the relatively low content of sodium alginate in the mixture. It was not revealed unexpected components of hydrolysis. These results are consistent with the results obtained by GC-MS analysis of partially methylated acetates alditol, supporting the conclusion that TM1 and granulated VFC contain chemically unmodified brandy glucomannan and xanthan gum. Furthermore, the definition of mannuronate acid in the hydrolysates suggests the additional presence of chemically unaltered sodium alginate.

3. The nuclear magnetic resonance spectroscopy of intact or partially hydrolyzed polymers, Monomeric standards

Rationale:

Spectroscopy nuclear �Ananova resonance (NMR) is a valuable tool for the analysis of organic molecules both spectra contain a wealth of information about their primary structure through the arrangement of protons (hydrogen atoms) in the molecule. Thus, at a basic level the value of NMR spectra 1 H is to provide an appropriate level of primary structural information that can identify different characteristics, using well established rules of chemical shifts and integrals of standard and unknown compounds in the required mixture. Carbohydrates have a number of characteristic features on MRI, making it useful for analysis. The two main features of the NMR spectra of carbohydrates are (I) the so-called "anamaria" resonances, which are protsvetanie with C1 in the sugar ring and typically occur at lower fields than the second feature; (II) a "shell ring" of protons associated with the remaining sugar ring. For example, for glucose alpha and beta anomeric resonances correspond to the level of 5.2 and 4.6 ppm, respectively, and the shell ring is located between a 3.1 and 3.9 million shares, respectively.

Interestingly, uronic acids have NMR spectra that are somewhat different from the typical spectrum of hexos described above, and their resonances rather overlap each other in a slightly stronger field than for glucose and mannose (from 3.9 to 5.6 ppm to�her). Santi et al., 12th Int. Electronic Conf on Synthetic Organic Chemistry (ECSOC-12): 1-30 (2008). Thus, the NMR spectra of hydrolysates of the polysaccharides (e.g., VFB/C) can be used to determine the fingerprint of the structural units (glucose, mannose, uronic acids, etc.) in polymers.

In the study described in this example, the NMR spectra were used for identification of complex mixtures, which are obtained by the hydrolysis of various polysaccharides and VFB. It should be noted that all polysaccharides can be investigated using NMR-level polymers because of problems with physical characteristics such as viscosity.

Methods:

Samples of each of the polysaccharide and ternary mixtures were partially hydrolysed with 2M TFA at 100°C for four hours and 24 hours. Filtered samples of hydrolysates (30 mg) was dissolved in D2O (1 ml) and subjected to freeze-dried before re-dissolving in D2O and by placing in vials for NMR spectroscopy. The standard solutions of the alleged two of the three monosaccharides and uronic acids were prepared in a similar manner.

Were obtained NMR spectra of hydrolyzed and standard solutions at 298,1 To using Bruker 400 MHz Advance III with broadband automatically configurable multi-sensor and the device under variable temperature control� software Bruker Topsin. For most samples was performed 16 scans, in addition to guluronic acid, which received 256 scans.

The results of NMR analysis:

1H NMR spectra of standard monosaccharides and uronic acids were well laid out, and their characteristic chemical shifts were found in both hydrolysates of xanthan gum, and sodium alginate, as well as in the hydrolysate VFC (PGX®) (data not shown). Chemical shifts observed for glucose and mannose from xanthan gum, were laid on the anomeric resonances (4,6-5,2 M. D.) and the resonances of the rings Sugars (3-4 M. D.). Chemical shifts observed for mannuronate and guluronate acids from sodium alginate, were closer to each other (between 3.6 to 5.2 m. etc). Resonances uronic acids detected in the hydrolysate of granulated VFC (PGX®) were a distinct combination of those that were detected in the hydrolysates of xanthan gum, and sodium alginate, which also confirms the presence of chemically unaltered sodium alginate in granulated VFC (PGX®).

Thus, the NMR spectra of pure standards, components of hydrolysates and hydrolysates VFC has demonstrated that VFC (PGX®consists of polysaccharides with unmodified primary structural features monosaccharide components with unchanged glycoside bonds.

General conclusions:

®) almost unchanged in comparison with pre-mixed, unprocessed, granular VFB (TM1). As described in this example, using the classical method of methylation has been shown that components of cognac glucomannan and xanthan gum contained the expected fragments and context, and that there were no unexplained additional structural components, which could be obtained by blending or processing components VFB/C.

Since sodium alginate, one of the components VFB/C, resistant to methylation, further methods were used to complete the structural analysis, including partial hydrolysis, chromatography and NMR to obtain further evidence about the unchanging nature of the primary chemical structure VFB/C.

Thus, the studies described in this example, support a conclusion that the granulated VFC (PGX®chemically does not change its primary features in the pelletizing process.

EXAMPLE 6

This example describes the analysis of rheological and macromolecular properties of granular viscous fiber complex (VFC) (cognac glucomannan/xanthan gum/alginate (70:13:17)) granules (i.e., the mixture of fibers was subjected to granulation to �education complex, commercial name (PGX®). The results described in this example show that the interaction occurs between the components of granulated VFC-level polymer to create networks and zones connection with the formation of a new polysaccharide with the following nomenclature: α-D-glucurono-α-D-manno-β-D-manno-β-D-gluco), (α-L-hourone-β-D-mannarino), β-D-gluco-β-D-mannan.

Rationale:

The studies described in this example were conducted to study the question of whether triple granulated mixture VFB/C, including brandy mannan, xanthan gum and sodium alginate, the network and the connection zone with the participation of all three components, which affects the rheological properties of the solution that are unique to processed/granulated VFC compared with untreated/non-granular VFB or its individual components brandy mannan, xanthan gum or sodium alginate. The presence of non-covalent macromolecular interactions between the three polysaccharides in granulated VFC (PGX®) in solution was investigated using the methods described below. Since the pair interaction between brandy glucomannan and xanthan gum was expected from the results of the study described in example 5, a further analysis was done specifically for any research participation �retey polysaccharide, sodium alginate, in the possible formation of triple bonds.

1. Measurement of rheological properties

In the first study of the rheological curves were obtained for several concentrations of rough/granular VFC (ternary mixture No. 1, referred to as "TM1") and granulated VFC (PGX®), and they were compared with rheological curves for each individual component solutions VFB/C at the same concentrations to identify synergies in the rheological behavior of aqueous solutions of ternary mixtures.

TABLE 16:
The specimens used in the rheological study
SamplePart number
Granulated VFC (PGX®)2029070523
Granulated VFC (PGX®)900495
Form of pellets VFB (TM1)1112050809
Form of pellets VFB (TM1)900416
Sodium alginate2638
Sodium alginate 2639
Xanthan gum2504
Xanthan gum2505
Brandy glucomannan2538
Brandy glucomannan2681

Sample preparation:

Separate polysaccharides and ternary mixtures were studied in a solution of deionized distilled water. Exact sample were dissolved at concentrations of 0.1 g, 0.2 g and 0.5 g in 100 g of water (with 0.1%, 0.2% and 0.5%, respectively) at 25°C and gidratirovana for two hours with stirring, in which the linkage of water was placed on a magnetic stirrer and vortex created prior to adding the samples, which were weighed to four decimal places, then slowly poured in the center of the vortex. After two hours, the solutions were mixed using a high speed mixer (stirrer IKA (15 thousand revolutions per minute) for 1 minute, to ensure that all particles of the material were fully mixed. Then the samples were further mixed for 1 hour before they were considered suitable for analysis.

Measurement of the rheological curve:

The rheological properties of the solution was measured using the rheometer [Bohlin Gemini, using C14 DIN 53019 concentric cylindrical inner and outer�ical measurement system at 25,0±0,1°C. The equilibrium shear rate was measured for a number of constantly applied shear stresses shear, increasing from 0.1 PA to 10 PA. Rheological properties were initially characterized using rheological curves of viscosity against shear rate.

Results:

FIGURES 13A, 13B and 13C illustrate the rheological curves cognac glucomannan, xanthan gum and sodium alginate, respectively, at 0.1%, 0.2% and 0.5% in mass percent, measured at 25°C. the Rheological curves of solutions of certain polysaccharides, shown in FIGURES 13A-C show that xanthan gum is the most powerful thickener (FIGURE 13 b), then the brandy glucomannan (FIGURE 13A) and finally sodium alginate (FIGURE 13C). The small difference in rheological properties was observed between the solutions of different batches of samples of individual polysaccharides. A solution of xanthan gum had the most extensive areas of pseudoplasticity many decades of shear rate, where the logarithmic plots were linear.

FIGURES 11A-C illustrate a comparison of the rheological curves of rough/granular VFB (TM1) and granulated VFC (PGX®) at 0.5% (mass percent) (FIGURE 11A), and 0.2% (mass percent) (FIGURE 11) and 0.1% (mass percent) (FIGURE 11C).

The data shown in FIGURES 11�, were further considered by bringing each rheological flow to the exponential relationship between viscosity η and the shear rate D is as follows:

η=KDn-1

Where K is the consistency coefficient (represents the total thickness), and η is the turnover rate (indicating the deviation from the Newtonian viscosity) obtained from the angular coefficients respectively logarithmic graph of viscosity against shear rate, which is linear to power-law fluids. The value To reflect the total consistency, and η indicates the deviation from the Newtonian viscosity (η=1). A Newtonian fluid has a value of η equal to 1 and, decreasing below 1 η, the viscosity of the fluid decreases when more shift.

As shown in FIGURES 11A-WITH all the samples of rough/granular VFB (TM1) and granulated VFC (PGX®) gave very similar rheological curves for each concentration, suggesting that the low activity of water in the preparation or previous exposure pre-cooked mixture affect the properties of mixtures. It should be noted that the treatment took place at a much lower overall water activity than during heat treatment dilute solution. The rheological curves of mixtures VFB/C were closer to the curve of xanthan gum; they are consistent with a power law� and showed significant pseudoplastic, but the magnitude of viscosity and pseudo-plasticity at each concentration were higher than that of xanthan gum, individually. This is clearly shown by the differences in the power law K and η values between solutions of mixtures of VFB/C and xanthan gums, as shown in FIGURE 12A.

FIGURE 12A illustrates a comparison of the power-law for K rough/granular VFB (TM1) and granulated VFC (PGX®) and xanthan gum. As shown in FIGURE 12A, the untreated samples/granular VFB (TM1) and granulated VFC (PGX®) gave very similar values of K for each concentration, and the value of K increased with increasing concentration. It should be noted that higher values of K correspond to higher viscosity and lower values of η consistent with a greater degree of pseudoplasticity in the range of concentrations.

FIGURE 12B illustrates a comparison of the power-law for η rough/granular VFB (TM1) and granulated VFC (PGX®) and xanthan gum. As shown in FIGURE 12B, the values of η were also the same for all samples VFC at each concentration, but indicated the possible presence of a minimum value or maximum η when the degree of reduction of shear viscosity in the range from 0.30 to 0.35%.

Based on the proportions that were used DL� create granulated VFC (70% cognac glucomannan, 17% of xanthan gum and 13% of sodium alginate), one could expect that the rheological properties of the mixture will be broadly similar 100% brandy the glucomannan, suggesting that polysaccharides no interactions. However, given that the cognac glucomannan is the predominant polysaccharide in the ternary mixtures, and xanthan gum and sodium alginate are impurity components, rheological properties of solutions of rough/granular VFB (TM1) and granulated VFC (PGX®) provide a clear indication of the interaction between the polysaccharides in these mixtures.

Conclusions:

The results of the comparison of the rheological curves of granulated VFC (PGX®), shown in FIGURE 11, with the rheological curves of the individual components shown in FIGURES 13A-13C show that there is an interaction between the polysaccharides in granulated VFC, which has led to a greater increase in the viscosities and degrees of pseudoplasticity than would be expected for a particular triple of the composition present in the raw/non-granular VFB (TM1) or granulated VFC (PGX®). In General, the rheological properties of samples of granulated VFC (PGX®) were closest to xanthan gum, but surprisingly, the viscosity of the granulated VFC (PGX®) were higher than the xanthan gum. This is shown in FI�URACH 12A and 12B, that emphasize higher values of K, and below about 0.45% concentration lower values for η granulated VFC (PGX®) compared to xanthan gum. Given that the content of xanthan gum in the raw/non-granular VFB (TM1) and granulated VFC (PGX®) is only 17%, and the remaining 83% are less strong thickeners brandy mannan and sodium alginate, these results are clear evidence of the interaction that occurred between the polysaccharides in the samples of granulated VFC (PGX®).

2. Study of the effect of concentration of sodium alginate and/or heat treatment

Were conducted additional experiments to determine the effect of different concentrations of sodium alginate and the effect of thermal treatment on the rheological and macromolecular properties VFB/C as follows.

Methods:

Were prepared with a mixture of brandy glucomannan, xanthan gum and sodium alginate. The mixture contained brandy glucomannan (km) and xanthan gum (XG) at a constant ratio (KM:XG=4,12:1) and different amounts of sodium alginate (from A0 to A33) (0%, 2%, 5%, 8%, 11%, 13%, 17%, 21%, 24%, 27%, 30% and 33%). All the samples were first prepared in the form of dry fiber mixtures of the two (cognac glucomannan and xanthan gum) or three (cognac glucomannan, Xan�anew gum and sodium alginate) components. Each sample (mixture) was weighed to four decimal places (dry), were thoroughly mixed using a hand shaker and maintained at -19°C until required time. Aqueous solutions of each mixture were made in a concentration of 0.5% by adding 5.0 g of each of the samples (mixes) to 1 kg of deionized water, stirring using a magnetic stirrer (i.e., the whirlwind was first created in deionized water and the samples then slowly poured into the center of the vortex) and were left to rehydrate to a homogeneous condition within four hours. Aqueous solutions of the mixtures were stored at 5°C until sample preparation.

Heat treatment

Then were taken in 20 ml aliquots of each solution and processed as follows: (I) incubated at room temperature (22°C) (without heating) or (II) heated to 90°C in a drying oven with thermostatic control (samples were in sealed containers to avoid losses due to evaporation, and periodically shaken to ensure a more complete hydration) or one hour (AN to AN) or four hours (AN to AN).

Rheological curves at 25°C of aqueous solutions of mixtures of brandy glucomannan, xanthan gum and sodium alginate containing brandy glucomannan (KM) and xanthan gum (XG) at a constant ratio (KM:XG=4,12:1) and variable amounts� of sodium alginate (0%, 2%, 5%, 8%, 11%, 13%, 17%, 21%, 24%, 27%, 30% and 33%) was measured in a concentration of 0.5%. As described above, the solutions were either not heated or heated in one hour or heated for four hours.

Results:

Rheological curves (measured at 25°C) for non-heated two-component (A=0) and ternary mixtures are shown in FIGURE 14A. Rheological curves (measured at 25°C) for two-component (A=0) and ternary mixtures that were heated for one hour are shown in FIGURE 14C. Rheological curves (measured at 25°C) for binary and ternary mixtures that were heated for four hours, shown in FIGURE 14C.

As shown in FIGURE 14A, for unheated mixtures, the viscosity decreased with increasing content of sodium alginate that would be expected if two or more strong thickener (KM and XG) would be replaced with a weaker a thickener sodium alginate.

As shown in FIGURE 14C, for mixtures that were heated for one hour ternary mixture with higher content of sodium alginate retain their viscosity is higher compared to the mixtures with the same ratio of alginate, which is not heated (shown in FIGURE 14A). As shown in FIGURE 14C, for mixtures that were heated for four hours, the viscosity were similar to those observed after heating for one hour. These results show that the heating of a ternary mixture containing sodium alginate, led to the triple interaction between the polysaccharides.

FIGURES 15A and 15B illustrate the dependence of K and η from the proportion of sodium alginate in the mixture for 0.5% aqueous solutions of mixtures of brandy glucomannan, xanthan gum and sodium alginate with a constant ratio KM:XG (4,12:1) and different amounts of alginate (from 0 to 33%). FIGURE 15A illustrates the power-law dependence of K values for unheated and heated triple mixtures from the content of alginate. Rheological curves for solutions of the ternary mixtures that have not been exposed to heat treatment, consistent with a power law and mainly showed a decrease in viscosity with increasing content of sodium alginate. As shown in FIGURE 15A for unheated solutions, power dependence of the K values showed a small initial increase with increasing the content of sodium alginate, but MIM was followed by a significant decline. As it turned out, the maximum value of K consistent with the content of sodium alginate from 3 to 5%. As shown in FIGURE 15B, the value of η was increased (relative to Newtonian viscosity) with increasing content of sodium alginate in the mixture. This indicates a transition from a high viscosity and pseudoplasticity binary mixture of brandy g�of komandan and xanthan gums (sodium alginate) to a much lower viscosity and less pseudoplasticity ternary mixture with 33% content of sodium alginate. These results show that sodium alginate acts as a mild thickener.

One would expect lower values of K and increase of η greater than about 5% of sodium alginate, as shown in FIGURE 15A and FIGURE 15, if the two more powerful thickener (cognac glucomannan and xanthan gum) would be replaced by a weaker, less pseudoplastic thickener (sodium alginate). As shown in FIGURE 15A, similar data were obtained for the solutions, which was heated for one hour, indicating that the initial drop in value due To heat treatment when the concentration of sodium alginate approximately less than 5%, but 11% and above reduction in the K value was much less steep than in solutions that are not heated. The maximum value of K was heat-treated solutions with a higher content of sodium alginate from 8% to 11% in the ternary mixture. As shown in FIGURE 15B, the value of η thermally treated solutions remained low and did not change throughout the range of the content of sodium alginate. For a limited number of solutions, and heated for four hours, K and η were similar to that obtained for a similar solution, which was heated for only one hour (data not shown).

Summary of results:

Overall, these results show that those�chemical processing triple solution significantly increases the overall level of macromolecular interactions. In contrast to the situation during the treatment, when the rheological properties before and after treatment remain the same, these samples were subjected to heat treatment in dilute solutions and consisted of recently mixed components. Since the value for solutions To powder mixtures containing from 0% to about 5% of sodium alginate, actually decreased after heat treatment, this higher level of interaction was unlikely to be mediated by increased interaction between brandy glucomannan and xanthan gum.

Overall, these data show that after heat treatment of a mixture of sodium alginate or restored and strengthened interaction between brandy glucomannan and xanthan gum, or was himself involved in the interaction with the other two polysaccharides in solution. Thus, these results suggest that sodium alginate may be added to glucomannan and xanthan gum in an amount of more than 8% to 20% in combination with heat treatment without significant damage to the rheology of binary mixtures.

3, Sedimentation in the analytical ultracentrifuge

Rationale:

Unexpectedly high viscosity VFC (cognac/xanthan gum/Aly-inat (70:13:17)) granules (i.e., the fiber blend was processed by granulation with the formation of complex commercial name PGX®) also known as PolyGlycopleX ®(α-D-glucurono-α-D-manno-β-D-manno-β-D-gluco), (α-L-hourone-β-D-mannarino), β-D-gluco-β-D-mannan (PGX®) led us to study the hydrodynamic properties of mixtures of brandy glucomannan, xantana and alginate, as evidenced by their rate of sedimentation in the analytical centrifuge, in order to find the interaction at the molecular level, which may provide the molecular basis of these macroscopic observations. This study used the technique of sedimentation velocity in the analytical centrifuge as a sensor to study the properties of mixtures in which the glucomannan was the dominant component, supplemented with xanthan gum and alginate.

Methods:

Polysaccharides

All the polysaccharides used in the study were put InovoBiologic Inc, (calgary, Alberta, Canada), namely: brandy glucomannan, party No. 2538; xanthan gum, party No. 2504, and sodium alginate, batch No. 2455/2639. Polysaccharides were studied both individually and in the composition of ternary mixtures containing granulated VFC (referred to in this study as "PGX®") and the form of pellets VFB (referred to in this study as "TM1"). The samples were dissolved in deionized distilled water, and then cialiseregalis in solutions with ionic strength is 0.0001 M To 0.001 M, 0.01 M, 0.1 M and 0.2 M in phosphate-chloride buffer p�and pH ~6.8. Ion power > 0.05 M were supplemented by the addition of NaCl.

Analytical ultracentrifugation

Technique deposition velocity in the analytical ultracentrifuge was used as a detector for the study of interaction. A method without the use of the solutions has the advantage over other methods, since it does not need columns, membrane materials, and other media for separation or immobilization, which could disrupt or interfere with interaction phenomena (S. E. Harding Analytical Ultracentrifugation Techniques and Methods, pp.231-252, Cambridge: Royal Society of Chemistry (2005)). Used ultracentrifuge Beckman XL-I equipped with refractive optics Rayleigh. Data were obtained using the CCD camera. The initial scan was performed at a low rotor speed of 3000 rpm for monitoring the presence of particles with very high molecular weight (not detected) to adjust the rotation speed of up to 45,000 rpm. The sedimentation coefficients s were adjusted to standard conditions of density and viscosity of water at 20.0°C to obtain S20,w. Scanning was performed with a two-minute interval during the operation time of ~12 hours. Data were analyzed from the point of view of the distribution of the sedimentation coefficient distribution g(s) compared to s (see, e.g., S. E. Harding, Carbhydrate Research 34: 811-826 (2005)), using the "method of least squares g(s)" algorithm SEDFIT (Dam &Schuck, Methods in Enzymology 384:185 (2003)), based on finite element analysis method Claverie et al., Biopolymers 14: 1685-1700 (1975). Analysis of changes in the distribution of sedimentation coefficient was used to establish the presence of interaction. The total load concentration 2.0 mg/ml or a 0.5% (0.5 g in 100 g of water) was used to monitor and mixtures.

Results and discussion

The integrity of the reagents

Brandy glucomannan, xanthan gum and alginate were first characterized using the analytical ultracentrifuge to determine their molecular integrity.

FIGURE 16 illustrates the apparent distribution of sedimentation coefficients g*(s) versus sedimentation coefficient (s) for glucomannan (FIGURE 16A), sodium alginate (FIGURE 16B) and xantana (FIGURE 16C) at loading concentration of 2 mg/ml and 1=0,0. The speed of rotation of the rotor at 45,000 rpm, temperature = 20,0°C. the ordinate axis represents the number of stripes per unit of Svedberg (S), and the abscissa unit of Svedberg.

FIGURE 17 illustrates the apparent distribution of sedimentation coefficients for rough/granular VFB (TM1) at ionic forces 0-0.2 M (FIGURE 17A); TM1 when ionic forces of 0-0. 01 M (FIGURE 17B); granulated VFC (PGX®) when ionic forces of 0-0. 01 M (FIGURES� 17C); and granulated VFC (PGX®) when ionic forces of 0-0. 02 M (FIGURE 17D). The speed of rotation of the rotor at 45,000 rpm, temperature = 20,0°C.

FIGURE 18 illustrates the effect of ionic strength (expressed in units of molar concentration M) on the amount of material with a sedimentation coefficient of the > 3,5 S for rough/granular VFB (TM1) (FIGURE 18A); or granulated VFC (PGX®) (FIGURE 18). To facilitate a logarithmic scale, a value of 1=0,00 presented, with 1=0,00001 M.

Unimodal areas were seen in all cases of apparent distribution of sedimentation coefficients (FIGURES 16A, b, C). In these circumstances, brandy glucomannan has a visible weighted average sedimentation coefficient s20,w~1.6 S, alginate ~1.3 S and xanthan gum is ~3,5 S, where 1S=10-13sec.

The formation of the complex and the effect of addition of electrolyte

Plots of the distribution of sedimentation coefficients were then generated for the following ternary mixtures: rough/granular/unheated VFB (TM1) (FIGURE 17A, b) and granulated/heat treated VFC (PGX®) (FIGURE 17C, D) in the same total loading concentration used for the control (2 mg/ml), up to a maximum of 10S. As our criterion of interaction, we evaluated the amount of material with the apparent sedimentation coefficient greater than the highest�th of the control specimens deposited - xanthan gum: sedimentation of material > 3,58 was considered as a criterion of interaction products.

TABLE 17 shows the concentration of deposited material > 3.5 S. the Concentration of the load on the ultracentrifuge cell in each case was equal to 2.0 mg/ml.

TABLE 17:
The concentration of deposited materials > 3.5 S
Sample03.58 (band)
Glucomannan0
Alginate0
Xanthan gum0,1±0,1
TC1(rough/ granular VFB)3,4±0,1
PGX®(granulated VFC)0,8±0,1

TABLE 17 shows a clear increase in the concentration of deposited material for mixtures of TM1 and granulated VFC compared with the individual components, although there is still a significant portion of the unreacted material, especially at low rates of sedimentation (~2S). FIGURE 18 and TABLE 18 also show the influence of increasing ionic strength on appeared�of substances with higher sedimentation.

TABLE 18 shows the results of the influence of ionic strength on TM1 (rough/granular VFB). The concentration of the load on the ultracentrifuge cell in each case was equal to 2.0 mg/ml.

TABLE 18:
The influence of ionic strength on TM1 (non-granular VFB)
The ionic strength (M)c > 3.5 S (strip)
0,03,4±0,1
0,00013,2±0,1
0,0013,4±0,1
0,010
0,050
0,10
0,20

TABLE 19 shows the results of the influence of ionic strength on PGX®(granulated VFC). The concentration of the load on the ultracentrifuge cell in each case was equal to 2.0 mg/ml.

TABLE 19:
The influence of ionic strength on PGX®(granulated VFB)
The ionic strength (AL)c>3.5 S (strip)
0,00,8±0,1
0,00012,8±0,1
0,0012,7±0,1
0,010
0,050
0,10
0,20

It is seen that for both granular and non-granular mixtures a significant amount of material with a higher sedimentation was observed at ionic strength 0.01 M, above which the appearance of such material was suppressed (FIGURES 18A, B). FIGURE 18A illustrates the influence of ionic strength (expressed in units of molar concentration M) to the amount of material with a sedimentation coefficient of the > 3,58 rough/granular VFB (TM1). FIGURE 18B illustrates the effect of ionic strength (expressed in units of molar concentration M) to the amount of material with a sedimentation coefficient of the > 3,58 for granulated VFC (PGX®).

The distribution of sedimentation coefficients of ternary mixtures

The distribution of sedimentation coefficients DL� mixtures containing a fixed ratio of glucomannan: xanthan gum, and alginate concentration (from 0% to 33%). Mixtures were either not heated (0), or heated for one hour (H1) or four hours (H4).

The distribution of sedimentation coefficients were determined from samples in deionized distilled water at a total concentration of the load of 5 mg/ml (0.5%). Results for unheated samples are shown in FIGURE 19A. Results for heated samples are shown in FIGURE 19B. As shown in FIGURES 19A and b, in the absence of alginate are no significant interactions between the products of a binary mixture of glucomannan: xanthan gum with a predominant content of glucomannan was not observed for unheated samples (JSC) and heated for one hour (AN) or four hours (AN) with the distribution of sedimentation coefficient similar to that of a control sample glucomannan (see Abdelhameed et al., Carbohydrate Polymers, 2010). However, the situation changes in the presence of alginate. As shown in FIGURE 19A, unheated ternary mixture showed some interaction with the alginate content 13%, 17%, 21% and 24%, based on the emergence of substances with a higher sedimentation coefficient, but no significant effect of alginate was observed when its concentration is above 27%. For the heated samples, as shown on P�SUNKE 19B, it was noted the formation of complexes with alginate concentration of about 8% in accordance with the rheological measurements. It should be noted that some samples with a high content of alginate, which is heated within one hour, such as AN (21% of the mixture of alginate heated within one hour), AN, AN, AN and all the samples that were heated for four hours and contained alginate, formed gels after heat treatment process and could not be analyzed using the method of estimation of sedimentation rate. This implies the presence of interaction in the original solutions of sufficient strength to translate them into a state of gel. In contrast, molecular interactions in unheated solutions were insufficient for the formation of such a phenomenon of gelation.

Conclusions

A mixture of glucomannan, xantana and alginate demonstrate the presence of reaction products, which can be removed by adding moderate amounts of electrolyte. These observations are consistent with the interaction in ternary mixtures, which can be suppressed by adding a supporting electrolyte with higher ionic strength of 0.01 M. the Interaction is not stoichiometric, since a significant portion of the material deposited at low sedimentation coefficients (<3,5 S when issued,� above).

4. Comparative treatment VFC and sodium alginate with calcium chloride

Rationale:

Granulated VFC (PGX®) forms in water solutions with high viscosity, but does not form a viscous gel. The above results are consistent with the formation of the complex interactions of the three components (cognac glucomannan, xanthan gum, and alginate) polymers. To determine whether alginate to be separated from the VFC, an experiment was conducted to check the possibility of separation of alginate from VFC in a solution of calcium ions. Alginates are known for having mediated calcium deposition and gelling characteristics (K. Clare, "Algin," in R. L. Whistler and J. N. BeMiller, Eds., Industrial Gums, Academic Press 116 (1993); A. Haug et al., Acta Chem. Scand. 19:341-351 (1965)). Pure solutions of sodium alginate react strongly and immediately to the addition of calcium ions with the formation of any precipitates or gels depending on the mode of addition of calcium (Clare et al. (1993); Haug et al. (1965)). Thus, in a typical reaction of gelation insoluble calcium salt, such as anhydrous calcium diphosphate, is added to the solution of sodium alginate followed by the addition of slowly decaying acids, such as glucono deltatech that causes the slow release of ions of Ca++with the formation of a homogeneous gel. If, however, calcium ions Ca++adding�tsya quickly as in the case of calcium chloride, then there is an instantaneous precipitation. Prigorodnoye segments of alginate molecules known fact that most firmly bonded with ions of Ca++(Kohn et al., Acta Chemica Scandinavica 22:3098-3102 (1968)), but if these segments become less available due to the interaction with one or two other polysaccharides, precipitate calcium alginate may be limited.

Methods:

Aliquots of solutions of raw VFB (TM1) and processed/granulated VFC (PGX®) in deionized distilled water at 0.5% (0.5 g in 100 g of water) were diluted to 0.1, 0.05 and 0.01% in mass percent. For each concentration in the solution was added 5 ml of 10% CaCl2 solution.2H2O and thoroughly mixed before the formation of the ion concentration of Ca2+0,5%. Exactly the same addition of ions of Ca2+was made to (1) parallel sets of control solutions of pure sodium alginate containing the same concentration of alginate and the solution of raw VFB (TM1) and processed/granulated VFC (PGX®); and (2) solutions of binary mixtures of either cognac glucomannan or xanthan gum with sodium alginate containing the same concentration of sodium alginate (measured in g per 100 g of water) and the same relative proportions of the two polysaccharides as 0.5% solutions raw VFB (TM1) and processed/Grand�graded VFC (PGX ®). The solutions were allowed to stand for 30 minutes before visual inspection for the presence/absence of sediment. Results:

The results shown below in TABLE 20.

TABLE 20:
The concentration of samples and results
% VFC (granular PGX®) (part number 900495)The planting of calcium (y/N)Alginate % (batch No. 2638)The planting of calcium (y/N)
0,5N0,075D
0,1N0,015D
0,05N0,0075D
0,01N0,001N

As shown in results, shown in TABLE 20, shows that in the presence of 0.5% ions of Ca++that will be precipitating calcium alginate to the level of not less than 0,0075%, no signs of planting in solutions of granulated VFC(PGX ®) to an equivalent level of alginate. This conclusion is consistent with what components VFB/C interact in solution, forming zones of connections and the network (i.e. at secondary and tertiary level of polysaccharide structure), which then protect the individual components from the existence of abilities that they could demonstrate in a pure form. Precipitation of calcium alginate were formed in the respective solutions of sodium alginate, in addition to the most diluted solution, which contained an insufficient amount of alginate to be deposited is 0.5% of the ions of Ca++.

Conclusion:

This study demonstrates the behavior of alginate in granulated VFC (PGX®), no planting or gel formation during rapid injection of calcium ions after the addition of calcium chloride. In a parallel control experiment, the calcium chloride is added to pure solutions of sodium alginate in decreasing concentrations, which resulted in an instant precipitate calcium alginate, even at very low concentration of alginate. These results show that Prigorodnoye segments of a macromolecule (alginate), which are usually firmly bonded with ions of Ca++in solution, were less available (or unavailable) for such interaction in solutions VFB/C, where in addition to sodium alginate was present brandy gluco�Annan and xanthan gum. It may be associated with these segments in the macromolecule, which are less accessible or inaccessible to ions of Ca++due to alternative interactions with one or two other polysaccharides. Precipitation of calcium alginate was observed after the addition of ions of Ca++to binary solutions or cognac glucomannan or xanthan gum with sodium alginate, which suggest that the interaction between sodium alginate requires the presence of two other polysaccharides.

Discussion overall results

The results of the initial structural analysis VFB/C described in example 5 show that after the granulation of the primary structure of the components of the polysaccharides present in granulated VFC, remain constant and that there are covalent interactions, either before (TM1), or after processing, including heat treatment (granulated VFC). However, the results of the analysis of macromolecular associations described in this example demonstrate that noncovalent interactions occur, leading to a new polysaccharide complex, which is formed in VFB/C at the macromolecular level. Rheological studies clearly show that the viscosity of solutions as rough/granular VFB (TM1) and granulated VFC (PGX®) is much higher than would expected�ü from a combination of the thickening properties of the individual polysaccharides in the mixture. Overall rheological characteristics of VFC in the solution closer to the individual solution of xanthan gum, but the viscosity VFC even higher than that of xanthan gum. Considering that the VFC design studied in this sample (70% KM, 17% of xantana, 13% of sodium alginate), contains only 17% strongest of thickeners, xanthan gums, and 83% weaker thickeners, cognac mannan (70%) and sodium alginate (13%), it was possible to expect that its rheological properties in water are similar to those of cognac mannan. However, it was found that the rheological properties of the solution VFC were actually closer to that of xanthan gum, and its viscosity was higher than that of xanthan gum.

Further study of the rheology and sedimentation of solutions of the ternary mixtures containing variable amounts of alginate prepared in the laboratory, confirmed the interaction of three components, and heat treatment solutions it has strengthened, especially when the content of sodium alginate in the mixture was greater than 5%. In addition, experiments with the addition of ions of Ca++showed that to prevent the precipitation of calcium alginate requires the presence of two other polysaccharides.

These results show that in the solution of sodium alginate interacts with brandy glucomannan and xanthan gum, creating the network and the connection zone, with about�education complex polysaccharide following items: α-D-glucurono-α-D-manno-β-D-manno-β-O-gluco), (α-L-hourone-β-D-mannarino), β-D-gluco-β-D-mannan.

As described in examples 1-4, it was found that the appointment of granulated VFC (PGX®) is useful for preventing, treating or alleviating one or more symptoms associated with metabolic disorders or disorder, such as metabolic syndrome, diabetes type I, diabetes type II, a disease of the pancreas or hyperlipidemia in a subject in need.

While the illustrative variants have been illustrated and described, it should be borne in mind that various changes may be made without deviation from the essence and scope of the present invention.

1. Nutritional therapy for preventing, treating or alleviating one or more symptoms associated with a metabolic disorder or disorder of metabolism, containing a composition of a polysaccharide dietary fiber high viscosity including viscous fiber mixture or a complex consisting of from 48% to 90% (in mass percent) of glucomannan, from 5% to 20% (percent by weight) of xanthan gum, and from 5% to 30% (percent by weight) alginate, and at least one macro element selected from the group, consisting of protein, carbohydrate and fat, where nutritional therapies have been developed to allow the dose of the composition of polysaccharide dietary fiber high �Ascoli from 20 g/day to 35 g/day for a period of time, effective to prevent, treat or alleviate one or more symptoms associated with metabolic disorders or disorder.

2. Health food in accordance with claim 1, wherein the dietary fiber composition comprises from 60% to 80% (in mass percent) of glucomannan, from 10% to 20% (percent by weight) of xanthan gum, and from 10% to 20% (in mass percent) of alginate.

3. Health food in accordance with claim 1, wherein the metabolic disorder or disorder is selected from the group consisting of metabolic syndrome, type I diabetes, type II diabetes, diseases of the pancreas and hyperlipidemia.

4. The method for production of therapeutic food according to claim 1, comprising the step of adding an effective amount of the composition of dietary fiber, containing glucomannan, xanthan gum and alginate, medical nutrition product, where the product is a medical food designed to provide the dose of the composition of polysaccharide dietary fiber high viscosity of 20 g/day to 35 g/day

5. Method in accordance with claim 4, characterized in that the product for therapeutic feeding is made for preventing, treating or alleviating one or more symptoms associated with a metabolic disorder or disorder of metabolism.

6. Method in accordance with claim 5, otlichuy�Xia, the dietary fiber composition comprises from 60% to 80% (in mass percent) of glucomannan, from 10% to 20% (percent by weight) of xanthan gum, and from 10% to 20% (in mass percent) of alginate.

7. The composition is a polysaccharide dietary fiber high viscosity for preventing, treating or alleviating one or more symptoms associated with a metabolic disorder or disorder of metabolism, containing a mixture of fibres or their complex comprising from 48% to 90% (in mass percent) of glucomannan, from 5% to 20% (percent by weight) of xanthan gum, and from 5% to 30% (in mass percent) of alginate, characterized by the fact that it's given to someone who needs it, from 20 g/day to 35 g/day for a time period effective for preventing, treating or alleviating one or more symptoms associated with a metabolic disorder or disorder of metabolism in the subject.

8. The composition in accordance with claim 7, wherein said metabolic disorder or disorder is selected from the group consisting of metabolic syndrome, type I diabetes, type II diabetes, diseases of the pancreas and hyperlipidemia.

9. The composition in accordance with claim 7, characterized in that the subject who is in need, suffering from or at risk of development of resistance to insulin or is suffering from or under�Ergen risk induced by glucose damage to the organs.

10. The composition in accordance with claim 7, characterized in that it is administered at least once daily during a period of at least two weeks.

11. The composition in accordance with claim 7, characterized in that it is a composition as a medical nutrition product.

12. The composition is a polysaccharide dietary fiber high viscosity to alleviate at least one symptom associated with the progression of insulin resistance in a subject suffering from or at risk of developing diabetes type II, containing a mixture of fibres or their complex comprising from 48% to 90% (in mass percent) of glucomannan, from 5% to 20% (percent by weight) of xanthan gum, and from 5% to 30% (in mass percent) of alginate, characterized by the fact that it's given to someone who needs it, at a dose of 20 g/day to 35 g/day for at least two weeks.

13. The composition in accordance with claim 12, characterized in that it comprises from 60% to 80% (in mass percent) of glucomannan, from 10% to 20% (percent by weight) of xanthan gum, and from 10% to 20% (in mass percent) of alginate.

14. The composition in accordance with claim 12, characterized in that it is administered as a medical nutrition product.



 

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27 cl, 23 tbl, 371 ex

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30 cl, 15 dwg, 8 tbl, 16 ex

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11 cl, 3 tbl

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15 cl, 2 ex

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11 cl, 18 dwg, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds of general formula I, or their racemic mixture, or their individual optic isomers, or pharmaceutically acceptable salts possessing the properties of TGR bile acid receptor agonist. The invention also refers to methods for preparing the compounds. In general formula I , X represents amino group R'R"N, wherein the substitutes R' and R" can be optionally identical, or represents hydrogen, C1-C6alkyl, C3-C6cycloalkyl; substituted C1-C6alkyl, wherein the substitute is specified in phenyl or phenoxy, each of which can be substituted by halogen in turn, C1-C3alkyl, C1-C3alkoxy, phenyloxy, C3-C6cycloalkyl, 5-6-merous heteroaryl with 1 nitrogen atom; aryl specified in phenyl optionally substituted by fluorine, C1-C3alkyl, C1-C3 alkoxy; 5-6-merous heteroaryl with nitrogen atom as heteroatom; C2-C4alkenyl, acyl specified in C1-C6alkylcarbonyl or C3-C6cycloalkylcarbonyl; or substituted oxygroup, which represents hydroxy group, wherein hydrogen is substituted by C1-C6alkyl optionally substituted by hydroxy, di(C1-C3alkyl)amino, phenyl, which can be substituted by halogen in turn, C1-C3alkyl, C1-C3alkoxy; C2-C4alkenyl; and 5-6-merous heterocyclyl with nitrogen atom, or sulphur atom, or oxygen atom as heteroatom; R1a and R1b represents hydrogen, C1-C3alkyl, or R1a and R1b together form methylene chain -(CH2)n-, wherein n=2-5; R1c and R1d represents hydrogen, C1-C3alkyl; R2 represents acyl group specified in C1-C6alkylcarbonyl, wherein alkyl can be substituted by phenyl or phenoxy, each of which can be substituted by halogen in turn, C1-C3alkyl, C1-C3alkoxy; C3-C6cycloalkylcarbonyl; phenylcarbonyl, which can be substituted by halogen, C1-C3alkyl, C1-C3alkoxygroup, oxygroup, C1-C3alkylene dioxygroup; 5-6-merous heteroarylcarbonyl with nitrogen atom, or oxygen atom, or sulphur atom as heteroatom, optionally substituted by carboxy, halogen or C1-C3alkoxycarbonyl, substituted aminocarbonyl group, wherein the substitute can be specified in C1-C6alkyl optionally substituted by C1-C3alkoxycarbonyl, halogen, 5-6-merous heteroaryl together with nitrogen atom, or oxygen atom or nitrogen atom as heteroatom; C3-C6cycloalkyl; phenyl optionally substituted by halogen, C1-C3alkyl, C1-C3alkoxy, C1-C3alkoxycarbonyl, C1-C3alkylenedioxygroup; 5-6-merous heteroarym with nitrogen atom, or oxygen atom or nitrogen atom as heteroatom optionally substituted by carboxy, C1-C3alkoxycarbonyl; aminocarbonyl group substituted by C1-C3alkyl; sulphonyl group specified in alkylsuphonyl optionally substituted by hydroxyl group, cyano group, phenyl, which is optionally substituted by C1-C3alkyl, halogen, C1-C3alkoxy group; henylsulphonyl oprtionally substituted by C1-C3alkyl, halogen, C1-C3alkoxy group, cyano group, C1-C3alkylene dioxygroup, or 5-6-merous heteroarylsulphonyl with nitrogen atom, or sulphur atom, or oxygen atom as heteroatom optionally substituted by halogen, C1-C3alkyl, C1-C3alkoxy group; R3 represents hydrogen.

EFFECT: compounds can be used for preparing the pharmaceutical composition applicable in treating or preventing metabolic diseases, such as diabetes, obesity, diabetic obesity, metabolic syndrome, hypercholesterolemia, dislipidemia.

14 cl, 17 dwg, 8 tbl, 16 ex

FIELD: medicine.

SUBSTANCE: invention represents a method for preparing a drug substance of polyprenylphosphates and beta-sitosterol consisting in pre-mixing polyprenylphosphates and sorbitol in a mortar, a size of which fits the total volume of the mixed substances. Between a wall and/or a bottom of the mortar and the introduced polyprenylphosphates, there is a layer of sorbitol; the blended mixture of the polyprenylphosphates in sorbitol is homogenised. Sorbitol, a dry mixture of polyprenylphosphates in sorbitol in a ratio of 1:8 and beta-sitosterol are added into a homogeniser, and pulsed homogenisation is performed for 10 minutes. The homogenisation is performed at a rotation rate of 500-700 rpm for 4 minutes, 1000-1200 rpm for 2 minutes and then 500-600 rpm for 4 minutes; the rotation rate variation requires a pause of 20 minutes. Each pause of the process is followed by the intensive agitation of a mixture cup; the prepared powder is sieved at a mesh size of 20 mcm; if a sieving weight makes less than 0.1% of the weight of the loaded ingredients; the homogenisation process is terminated.

EFFECT: preparing the drug substance with the maximum effective concentration of the active substances uniformly weight-distributed and higher bioavailability.

3 cl, 2 ex, 1 tbl, 7 dwg

FIELD: medicine.

SUBSTANCE: group of inventions relate to field of pharmaceutics, in particular, to pharmaceutical composition for treatment of phenylketonuria, which contains effective quantity of version of Anabaena variabilis (AvPAL) phenylalanine-ammonia-lyase, where claimed version additionally contains polyethylenglycol, as well as pharmaceutically acceptable carrier, which contains stabiliser, where stabiliser represents L-phenylalanine, trans-cinnamic acid or benzoic acid. Also claimed are: method of phenylketonuria treatment and method of reducing phenylalanine concentration in subject's blood.

EFFECT: group of inventions ensure application of prokaryotic PAL, which has higher phenylalanine-converting activity and/or lower immunogenicity in comparison with PAL of wild type.

56 cl, 19 ex, 11 tbl, 19 dwg

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