Use of yeast cell walls for treating and/or preventing hyperinsulinemia

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics. There are yeast cells walls used in preparing a pharmaceutical composition for treating and/or preventing hyperinsulinemia in a non-diabetic patient. The yeast cell walls have total content of glucans and mannans min. 34.0 wt % of total solids and content of glycogen min. 10.0 wt % of total solids.

EFFECT: use of the declared invention enables higher effectiveness of using the yeast cell walls, as well as effectiveness in preventing and treating hyperinsulinemia caused by insulin resistance, overweight, obesity or metabolic syndrome.

2 ex, 3 tbl, 4 dwg

 

The present invention relates to the use of yeast cell walls for treating and/or preventing hyperinsulinemia.

Insulin is a hypoglycemic hormone, secretory β-cells of the islets of Langerhans of the pancreas. Insulin can reduce glycemia, in particular through activation of glucose transport in the target tissue (muscle, liver and adipose tissue).

Type 2 diabetes is characterized by insulin resistance, which is converted during the first phase of hyperinsulinism. Some types of obesity is also caused by hyperinsulinism. For diabetes with insulin resistance may be prescribed medications that can reduce excess sugar in the blood without stimulating insulin secretion (e.g., Metformin).

In the document JP-A-61-167622 proposed agent for diabetes containing cell fraction of yeast, called in this document the cell wall, and obtained by hydrolysis of yeast with reduced bitterness for at least 2 hours at a temperature of from 50 to 70°C. and aqueous extraction of soluble components. Specified cellular fraction of yeast, in particular, has the content glucans about 14,8%, the content of mannans about 13.9 percent. The specified cell fraction also has the glycogen content of about 24.9 percent. Glycogen, not only is em a reserve polysaccharide, also present in the muscles and, in particular, in the liver. This glycogen is a reserve substance of yeast used yeast as a source of energy for their survival. Although he is one of the main components of cell fractions in this Japanese application, it is not part of the cell wall of yeast.

In the patent application WO 2005/021015 described the use of yeast cell walls for the prevention and treatment of hyperglycemia and stabilizing blood glucose levels where these yeast cell walls have a low content of glycogen and can be obtained using a simple method of enzymatic autolysis or hydrolysis. The total content of glucans and mannans these yeast cell walls is at least 34,0% mass/mass of dry matter.

β-glucan of the cell wall of the yeast are essentially polymers of glucose, glucose units are in the main chain are linked β-1,3 linkages, and whose branches are linked β-1,6 linkages, β-glucan yeast insoluble and have low viscosity.

β-glucan, purified from yeast cell wall, have immunostimulating properties. In particular, it is shown that β-glucans of yeast capable of binding to macrophages and activate them. β-glucans of yeast were investigated for their application as antibacter the social, antiviral and anticancer agents (D.S. Adams, Journal of Leukocyte Biology, 1997).

The essence of the present invention is the use of yeast cell walls as agent for regulating insulin.

One of the objects of the invention is, in particular, the use of yeast cell walls for treating and/or preventing hyperinsulinemia.

One of the objects of the present invention is the use of yeast cell walls to obtain a pharmaceutical composition intended for treating and/or preventing hyperinsulinemia, where the yeast cell wall are:

- total content glucans and mannans at least 34,0% dry matter content by weight, and

the glycogen content of less than 10.0% of the dry matter content by weight.

The term "yeast cell wall" refers to the insoluble fraction of yeast cells that are not enriched in chromium, obtained after autolysis or enzymatic hydrolysis, in particular, proteases, leading to solubilize at least 50% and preferably at least 60% mass/mass of dry matter of whole yeast cells and preservation of the structural polysaccharides of the cell wall, i.e. β-glucans and mannans, where these mannans are in the form of mannoproteins.

This autolysis or enzymatic hydrolysis of conduct that is they way to solubilisate a big part of the reserve sugars yeast cells, that is, glycogen and trehalose. Yeast cell wall is produced by the Department solubilizing fractions using autolysis or enzymatic hydrolysis, preferably for at least 18 hours. Preferred ways of autolysis of yeast suspension described in str-377 in the book "Yeast Technology" 2ndedition, 1991, G.Reed and T.W.Nogodawithana, published by Van Nostrand Reinhold, New York, ISBN 0-442-31892-8. Yeast cell wall, thus obtained, is then typically dried using the conventional method of drying such as spray drying or drying on heated rolls.

Yeast cell walls according to the invention have a total content of glucans and mannans in the cell wall (systematically expressed in equivalent weight of glucose and mannose, respectively - see measurements below) at least 34,0% mass/mass of dry matter and the content of glycogen (systematically expressed in the equivalent mass of glucose - see measurements below) less than 10.0% mass/mass of dry matter.

Under "hyperinsulinemia" mean as basal hyperinsulinemia and the postprandial hyperinsulinemia. In one preferred form of implementation of the yeast cell walls according to the invention is used for warned is I and/or treatment of postprandial hyperinsulinemia. Alternative yeast cell walls according to the invention is used for prevention and/or treatment of basal hyperinsulinemia. In another form of implementation of the yeast cell walls according to the invention is used for prevention and/or treatment as basal hyperinsulinemia and postprandial hyperinsulinemia.

Basal hyperinsulinemia is characterized by a high basal level of insulin in the plasma. The basal level corresponds to the levels of insulin between meals, in particular when the fasting person. Physiological basal level is usually equal to or below 10 IU/l in slim healthy individual. Therefore, basal hyperinsulinemia may be characterized basal level between meals (in particular, during starvation) more than 10 IU/l

Postprandial hyperinsulinemia corresponds to excessive insulin response after a meal.

On postprandial hyperinsulinemia may indicate oral test glucose tolerance (OTG). Physiological response to the test OTG characterized by a peak of insulin secretion is usually between 30 and 60 IU/l in healthy slim individual. Therefore, postprandial hyperinsulinemia can be characterized by the response to OTG with peak insulin secretion more than 60 IU/L.

The level of insulin in the plasma of patients who NTA measure using conventional techniques known to experts in this field of technology. In particular, RIA test (radioimmunological analysis) can be performed on plasma samples obtained from the patient (e.g., RIA, Biosurce, Medgenix Diagnostics, Rungis, France).

The goal of treatment of hyperinsulinemia is to decrease basal insulin levels in plasma and/or restoration of physiological insulin response after a meal.

The purpose of the prevention of hyperinsulinemia, in particular, is to maintain the basal level of insulin in the plasma at physiological values and/or in maintaining the physiological response of insulin after a meal.

The invention is, in particular, is the treatment and/or prevention of postprandial hyperinsulinemia.

The object of the invention, in particular, is the use as defined above, where the cell wall belong to the yeast of the genus Saccharomyces cerevisiae.

These yeast preferably represent Baker's yeast. Baker's yeast are yeast belonging to the species Saccharomyces cerevisiae, produced essentially by aerobic propagation or cultivation, as described in the book "Yeast Technology", quoted above, and who have not applied for any purpose to their autolysis or enzymatic hydrolysis, in contrast to, for example, beer yeast, which is the tsya byproduct of beer production and therefore, served for the production of beer before their gathering for enzymatic autolysis or hydrolysis. These yeast were cultured essentially anaerobic conditions (since the production of beer is an anaerobic process).

The object of the present invention is the use as defined above, where the yeast cell walls have a total content of glucans and mannans at least 40.0% of the dry matter content by weight, preferably at least 45.0% in the dry matter content by weight.

It is useful to yeast cell walls according to the invention had a protein content (N x 6.25 from 17,0 to 35.0% of the dry matter content by weight, preferably of 18.0 to 26.0% of the dry matter content by weight.

A particular case of the invention is the use as defined above, where the yeast cell wall content of mannan less than 30% dry matter content by weight.

In one preferred form of the invention, the content of mannans of yeast cell walls is between 20 and 26% of the dry matter content by weight. This form of implementation corresponds to the yeast cell walls obtained by enzymatic autolysis or hydrolysis, separation solubilizing fractions and drying the insoluble fraction, such as you described is e in example 1.1 (the section under the heading: Getting yeast cell walls, containing the mannans). Specified product after oral tolerance test glucose (OTG) gives the possibility of obtaining reduced peak insulin compared with placebo in patients with type 2 diabetes (see example 2).

The object of the invention, in particular, is the use as defined above, where the yeast cell wall content of mannans less than 2% of the dry matter content by weight, in particular less than 1% of the dry matter content by weight, in particular less than 0.1% of the dry matter content by weight.

In another preferred form of the invention, the content of mannans of yeast cell wall is lower than 0.1% of the dry matter content by weight.

The mannans can be removed by hot alkali treatment. Hot alkaline treatment is placed in aqueous suspension of yeast cell walls according to the invention, obtained as described above, and heating the suspension in an alkaline medium at about 70°C and 100°C for a maximum time of three hours. Faction solubilizing this treatment, containing the greater part of mannoproteins and even all mannoprotein, is removed by centrifugation and washing. Residual not solubilizing fraction is collected and usually dried.

Preferably, the mannans remove the hot alkaline treatment with the consequences of the overall acid treatment for complete removal of mannans.

These treatments are described in detail in the examples section.

Upon receipt of these products from the original yeast cell walls, subjected to hot alkaline treatment and may be acid-treated as described above (see also example 1.1, section titled: Getting yeast cell walls, free of mannans), and in which the mannans completely or almost completely removed (glucans represent much of the total content glucans and mannans), perhaps after the test OTG, get reduced peak insulin compared with placebo and compared with yeast cell walls, in which the mannans are not removed, patients suffering from type 2 diabetes or overweight (see example 2).

The object of the present invention is the use as defined above, where the yeast cell walls have a total content of glucans and mannans, equal to or less than 90% of the dry matter content by weight, in particular equal to or less than 80% of the dry matter content by weight, in particular equal to or less than 70% of the dry matter content by weight.

The object of the present invention more specifically is the use as defined above, where the yeast cell walls have a total content of glucans and mannans from 45 to 90% dry matter content by weight, in particular from 52% to 80% dry matter content by weight, preferably from 65% to 77% of the dry matter content by weight and more preferably from 72% to 77% of the dry matter content by weight. Therefore, according to one particularly preferred form of implementation, the content of glucans and mannans is between 72% and 77% of the dry matter content by weight.

In particular, in one specific form of implementation, the object of the present invention is the use as defined above, where the content of glucans and mannans is between 72% and 77% of the dry matter content by weight, and the content of mannans is less than 2% of the dry matter content by weight, in particular less than 1% of the dry matter content by weight, in particular less than 0.1%.

Alternative content glucans and mannans may be equal to or less than 55% of the dry matter content by weight, provided that it remains higher than 45% of the dry matter content by weight.

Therefore, according to one preferred form of implementation, the content of glucans and mannans is between 52% and 57% of the dry matter content by weight.

In particular, in one specific form of implementation of the object of the present invention is the use as defined above, where the content of glucans and mannans is between 52% and 57% of the dry matter content by weight, and the content of Ananov is less than 2% of the dry matter content by weight, in particular less than 1% of the dry matter content by weight, in particular less than 0.1%.

The object of the present invention is the use as defined above, where the yeast cell wall content of glycogen in less than 8,0% of the dry matter content by weight, preferably less than 5.0% of the dry matter content by weight, preferably less than 3.0% of the dry matter content by weight, preferably less than 1.0% of the dry matter content by weight, and more preferably less than 0.1% of the dry matter content by weight.

To remove glycogen completely or almost completely from yeast cell walls, yeast cell walls according to the invention can be subjected to hot alkaline treatment, such as described above, and preferably hot alkaline treatment with subsequent acid treatment.

At the above-mentioned processing may be getting a yeast cell walls according to the invention, having the total content of glucans and mannans from 45% to 90% dry matter content by weight, preferably from 52% to 80% dry matter content by weight, preferably from 65% to 77% of the dry matter content by weight and more preferably from 72 to 77% of the dry matter content by weight. When the specified processing is possible, in particular, obtaining a yeast cell walls is to, which also contain less than 1.0% of the dry matter content by weight of glycogen, preferably less than 0.1% of the dry matter content by weight of glycogen.

The pharmaceutical composition according to the invention can be introduced in various forms or presentations.

The pharmaceutical composition according to the invention contains at least one active substance, presents a yeast cell walls, and a pharmaceutically acceptable carrier.

The pharmaceutical composition according to the invention may contain one or more than one other active substance selected among hypoglycemic agents or insulin-sensitizing agents, and, in particular, among hypoglycemic sulfonamides, biguanide, Metformin and derivatives thiazolidinedione, inhibitors of α-glucosidase.

The composition may also contain one or more than one vitamin in particular selected from among vitamin a, vitamin C, vitamin D, vitamin E, vitamin K, vitamin B1 (thiamine), B2 (Riboflavin), B3 (Niacin), B5 (Pantothenic acid), B6, B8 (Biotin), B9 (folic acid), B12 (cobalamin), and/or one or more than one food mineral, in particular selected among calcium, phosphorus, potassium, sodium, magnesium and iron.

The object of the present invention are also pharmaceutical compositions, such as the definition is but higher contains chrome.

The pharmaceutical composition may contain one or more pharmaceutically acceptable excipients.

In one preferred form of implementation of the pharmaceutical composition according to the invention suitable for administration through the mouth.

The pharmaceutical composition may in particular be in the form of tablets, capsules, pills, powder, granules, or suspensions.

The pharmaceutical composition according to the invention may in particular be in the form of acceptable dose, appropriate amount of dry matter of the yeast cell walls according to the invention is less than 10 g, preferably from 8 to 9,

In one preferred form of the implementation object of the present invention is the use as defined above, where the yeast cell walls have a dry matter content equal to or greater than 90%, preferably equal to or greater than 94%, and more preferably equal to or greater than 96% mass/mass.

When the content of dry matter equal to or greater than 90%, preferably equal to or greater than 94%, and more preferably equal to more than 96%, obtaining a better preservation of yeast cell walls, in particular the best bacteriological stability and better stability against harmful reactions, enzymatic or not fermentative the CSOs origin.

The object of the present invention is the use of yeast cell walls, as defined above, for the prevention and/or treatment of hyperinsulinemia, either basal or postprandial hyperinsulinemia, regardless of the origin of hyperinsulinemia.

The object of the invention, in particular, is the use of yeast cell walls, as defined above, for the prevention and/or treatment of hyperinsulinemia due to insulin resistance, overweight, obesity or metabolic syndrome, any type of a patient suffering from hyperinsulinemia this origin.

However, although the invention is meant for the prevention and treatment of any type of hyperinsulinemia, any type of patient, in one preferred form of implementation of the yeast cell walls according to the invention is used for obtaining a medicinal product intended for the prevention and/or treatment of hyperinsulinemia in patients who are not diabetic. In this case, the object of the present invention, in particular, is the use of yeast cell walls, as defined above, for the prevention and/or treatment of hyperinsulinemia due to insulin resistance, overweight, obesity or metabolic syndrome in people who are not diabetics.

Alter ative yeast cell walls according to the invention can also be applied to obtain medicines intended for prevention and/or treatment of hyperinsulinemia in patients with type 2 diabetes. The object of the present invention, therefore, is, in particular, the use of yeast cell walls, as defined above, for the prevention and/or treatment of hyperinsulinemia due to insulin resistance, overweight, obesity or metabolic syndrome in a person with type 2 diabetes.

Under "type 2 diabetes" also imply non-insulin-dependent diabetes INST. Type 2 diabetes is characterized by a fasting glucose of more than 1.26 g/l

When fasting glucose is between 1.10 g/l and 1.26 g/l, use the term anomaly of glycemia.

In particular, an object of the present invention is the use of yeast cell walls, as defined above, for the prevention and/or treatment of hyperinsulinemia during the first stages of type 2 diabetes.

By "insulin resistance" refers to the absence of insulin-dependent response of tissues to insulin action. Then the pancreas continues to secrete insulin levels which in the plasma is too high.

Under the "overweight" or "obese" refers to the excess weight. Adults overweight characterised by a body mass index (BMI) between 25 and 30 kg/m2and obesity is the body mass index (BMI)over 30 kg/m 2. The body mass index in humans, determine the following formula:

The terms "overweight or obese people who are not diabetics" means excess weight or obesity, as defined above, in humans, glycemia are properly regulated.

In particular, the level of glucose in the plasma of fasting in people who are not diabetics, is less than 1.26 g/DL (7 mmol/l), in particular less than 1.10 g/DL (6.1 mmol/l).

The term "metabolic syndrome" refers to a group of risk factors of cardiovascular disorders, stroke, and type 2 diabetes.

The diagnosis of metabolic syndrome is based on the evaluation of several parameters, including the waist size, cholesterol levels and triglycerides, fasting glucose, insulinemia and blood pressure.

In particular, according to NCEP-ATPIII (National educational program on cholesterol - III Report of the expert group on the treatment of adults), metabolic syndrome is diagnosed if there are three or more than three of the following risk factors:

- waist size of more than 88 cm in women and over 102 cm in men,

- HDL cholesterol less than 1 mmol/l in men and 1.2 mmol/l in women,

- the level of triglycerides equal to or greater than 1.7 mmol/l,

- fasting glucose equal to or is more of 6.1 mmol/l,

- blood pressure greater than 130 mm Hg/85 mm Hg.

Postprandial hyperinsulinemia may, in particular, play a role in the development of metabolic syndrome.

The present invention also relates to a method for treating and/or preventing hyperinsulinemia in a patient, comprising the administration to a patient the pharmaceutical composition according to the invention.

A method of treating and/or preventing hyperinsulinemia may, in particular, to provide a method of treating and/or preventing hyperinsulinemia due to insulin resistance, overweight, obesity or metabolic syndrome. In one preferred form of implementation of the patient is a patient who is not a diabetic. Alternative, the patient may be a patient with type 2 diabetes.

The invention applies in particular to the specified method, wherein the pharmaceutical composition is administered to the patient via the oral route.

Various methods according to the invention can, in particular, to include the introduction to the patient a pharmaceutical composition in a daily dose corresponding to 1 to 10 g, preferably 8-9 g of yeast cell walls according to the invention, where the daily dose indicated it is possible to enter in a single dose or during a meal, such as Breakfast, or a few is a partial doses, that is distributed during the day.

The methods according to the invention may also include at least one stage for validation basal level of insulin the patient and/or insulin response following the meal, or test OTG, after a single or continuous introduction of pharmaceutical compositions.

The methods according to the invention can also include phase measurement for measuring the basal level of insulin the patient and/or to monitor the response of insulin after a meal or test OTG before one-time or continuous introduction of the above pharmaceutical compositions.

The present invention also relates to a method of reducing or stabilizing the basal level of insulin in the plasma of patients suffering basal hyperinsulinemia, comprising the administration to a patient an effective amount of the pharmaceutical composition according to the invention.

The way to reduce or stabilize the basal level of insulin in the plasma can, in particular, to treat a patient suffering from basal hyperinsulinemia due to insulin resistance, overweight, obesity or metabolic syndrome. In one preferred form of implementation of the patient is a patient who is not a diabetic. Alternative, the patient may also present with the OI patient with type 2 diabetes.

The present invention also relates to a method for reducing peak of insulin secretion after a meal in a patient suffering from postprandial hyperinsulinemia, comprising the administration to a patient an effective amount of the pharmaceutical composition according to the invention.

The way to reduce peak of insulin secretion after a meal can, in particular, to treat a patient suffering postprandial hyperinsulinemia due to insulin resistance, overweight, obesity or metabolic syndrome. In one preferred form of implementation of the patient is a patient who is not a diabetic. Alternative, the patient may be a patient with type 2 diabetes.

DESCRIPTION of GRAPHIC MATERIALS

- Figure 1: Kinetics of postprandial insulinemia in patients with overweight (n=12).

The results of the 3 days of the study are given as mean ± SOS. Insulinemia in the controls, which took milk, shown as a solid line with squares, the people who took the yeast cell wall without mannans (beta-glucan), dashed line with diamonds, and the people who took the yeast cell wall with mannans, in the form of a dotted line with triangles. The level of insulin (IU/l) are shown as a function of time in minutes (min).

- 2. The trend in postprandial blood insulinemia, shown as the area under the curve (ACC) in patients with overweight (n=12).

The results of the 3-day studies are presented as mean ± SOS. The area under the curve obtained from the control of the people who took the milk is shown in the black bar, the people who took the yeast cell wall without mannans (beta-glucan), in the form of densely shaded column, and the people who took the yeast cell wall with mannans, in the form of loosely shaded column. The area for insulinemia (IU/l time) are shown for the period 0-300 minutes (total), the period of 0-120 minutes and the period 120-300 minutes.

- Figure 3: Kinetics of postprandial insulinemia in people with type 2 diabetes (n=11).

The results of the 3-day studies are presented as mean ± SOS. Insulinemia in the controls, which took milk, shown as a solid line with squares, the people who took the yeast cell wall without mannans (beta-glucan), dashed line with diamonds, and the people who took the yeast cell wall with mannans, in the form of a dotted line with triangles. The level of insulin (IU/l) are shown as a function of time in minutes (min).

- Figure 4: Trend in postprandial blood insulinemia, shown as the area under the curve (PPC) is patients with type 2 diabetes (n=11).

The results of the 3-day studies are presented as mean ± SOS. The area under the curve obtained in the controls, which took milk is shown in the black bar, the people who took the yeast cell wall without mannans (beta-glucan), in the form of densely shaded column, and the people who took the yeast cell wall with mannans, in the form of loosely shaded column. The area for insulinemia (IU/l time) are shown for the period 0-300 minutes (total), the period of 0-120 minutes and the period 120-300 minutes.

EXAMPLES

Example 1: getting a yeast cell walls according to the invention

1. Material and methods

Obtaining a yeast cell walls containing the mannans

Water yeast milk (i.e. suspension of yeast cells in water) Saccharomyces cerevisiae having a dry matter content of between 12 and 18% mass/mass, is subjected to hydrolysis using endogenous enzymes of yeast cells, not necessarily with the addition of proteases, exogenous to yeast cells, such as, for example, papain. The hydrolysis is carried out at 50°C for 24 hours to solubilisate at least 60% mass/mass of dry matter of the yeast cell.

In practice, and usually autolysis and enzymatic hydrolysis according to the invention is carried out at a temperature between 45°C and 55°C for 18-36 hours without the use of any f is rment, which can solubilisate glucan or mannoprotein.

Solubilizing fraction is separated from the insoluble fraction in successive stages by centrifugation and washing with water.

The insoluble fraction is dried on heated rolls to a dry matter content of 95% mass/mass. The formed agglomerates are removed by sieving, and get a yeast cell walls according to the invention.

Obtaining a yeast cell walls that do not contain mannans

Yeast cell wall, obtained as described above, placed in a water slurry and treated in an alkaline medium at a temperature between 70°C and 100°C for a period of not more than three hours. In particular, the suspension in an alkaline aqueous solution of sodium hydroxide heated to 85°C for two hours.

Faction solubilizing this treatment, is removed, and the remaining not solubilizing fraction is collected, washed, and usually dried. Solubilizing fraction containing mannoprotein, as well as glycogen, is removed by centrifugation and washing.

Yeast cell walls obtained after the hot alkaline treatment, is preferably subjected to acid treatment to remove any traces of mannoprotein. The insoluble fraction is then acidified with phosphoric or acetic acid, for the eat incubated at 80°C for one to two hours. Solubilizing fraction containing the remains of mannoproteins, glycogen, and some lipids are removed by centrifugation and washing.

Measurement of glycogen content

To the sample 20 mg of dried yeast cell walls, that is, having a dry matter content of at least 90% mass/mass, add 0.5 ml of 0.25 M PA2CO3, and the mixture is kept at 95°C for 4 hours.

The mixture is then brought to a pH of 5.2 by adding 0.3 ml of 1 M acetic acid and 1.2 ml of 0.2 M sodium acetate and mixing ingredients. Add distilled water to a total volume of 2 ml

0.5 ml of the suspension thus obtained, incubated for 15 hours in the presence of excess amyloglucosidase Aspergillus niger, such as commercially available from the firm ROCHE under catalog No. 102857 at 55°C.

After centrifugation released glucose analyzed by enzymatic analysis.

Enzymatic analysis of glucose, in particular, described in "Methods of Biochemical Analysis and Food Analysis - using Single reagents", published by BOEHRINGER MANNHEIM GmbH Biochemica © 1989, p.50-55, and preferably it is done using "Test-Combination D-Glucose/-Fructose" catalog No. 139106 from subsidiaries ROCHE, BOEHRINGER MANNHEIM GmbH/R-BIOPHARM GmbH, Darmstadt, Germany.

Amount (mg) of glucose, analyzed, thus, corresponds to the amount of glycogen present in about what the style, expressed in equivalent mass of glucose.

The measurement of the total content of glucans and mannans

A sample of 20 mg of dried yeast cell walls, that is, having a dry matter content of at least 90% mass/mass, is subjected to acid hydrolysis by mixing with 20 ml of 2 N. Hcl, and the mixture is kept in a sealed test tube with screw cap for 4 hours at 103°C in a furnace with shaking every 15 minutes

Then the acidic solution, thus obtained, neutralize, and determine the relative amount of glucose and mannose by enzymatic neutralized in solution.

This enzymatic analysis of glucose and mannose are also described on p.50-55 quoted above guidelines, and preferably it is done using "Test-Combination" catalog No. 139106.

Calculate the difference between the amount of glucose (expressed in mg), identified using this method, and the amount of glucose (also expressed in mg)defined for these yeast cell walls using the above-described method of measurement of glycogen content.

This difference (in mg) between two analytically defined quantities of glucose corresponds to the total number of glucans present in the sample, where this quantity is expressed in equivalent mass of glucose.

Quantity (in mg) of analytically determined the military mannose corresponds to the total number of mannans, present in the sample, where this quantity is expressed in equivalent weight of mannose.

2. Results

Yeast cell wall, thus obtained, have a dry matter content of 95% mass/mass.

The composition of yeast cell walls containing the mannans, and yeast cell walls, free of mannans (received hot alkaline treatment), are shown in table 1.

Table 1:
The composition of yeast cell walls according to the invention
Yeast cell wall, containing the mannansYeast cell wall that does not contain the mannans
The analysis in g per 100 g of productThe absorption of energy in kcal/gThe analysis in g per 100 g of productThe absorption of energy in kcal/g
Carbohydrates:to 49.92,04655,12,259
Glucan28,2 55,1
The mannans21,70
Proteins23,61,2982,20,121
Lipids11,81,10921,62,03

For both types of yeast cell wall content of glycogen was less than 10% of the dry matter content by weight.

Example 2: Effect of yeast cell walls according to the invention on insulin levels of people with excess weight, non-diabetics, and people with non-insulin dependent diabetes

In the rest of the document yeast cell wall, containing the mannans, called "yeast cell wall", and yeast cell wall that does not contain the mannans, called "beta-glucan".

1. Materials and methods

Subjects, characteristics and sample size

The study involved 24 volunteer 2 types: 12 healthy volunteers with overweight and 12 patients with type 2 diabetes (men or women).

For volunteers with overweight inclusion criteria is the of n: body mass index between 25 and 30 kg/m 2(inclusive), aged between 30 and 65 years (inclusive), normal fasting glucose: <7 mmol/l, HbAlc: <6%, total cholesterol: ≤7.0 mmol/l, triglycerides: ≤4.0 mmol/l

For subjects with type 2 diabetes inclusion criteria were: age between 30 and 65 years (inclusive), treatment with Metformin and/or hypoglycemic sulfonamides and/or glinide and/or glitazone for at least 3 months, HbAlc: <10%, total cholesterol: ≤7.0 mmol/l, triglycerides: ≤4.0 mmol/l

The main exclusion criteria were: type 1 diabetes, type 2 diabetes, treated with insulin or acarbose, insulinopenia, chronic renal or liver failure, history of chronic gastrointestinal disease, endocrine pathology, treatment with possible interference in the metabolism of carbohydrates and diet, intolerance to cow's milk, claustrophobia (calorimetric measurement in terms of Canopy®), pregnant women, people who consume large quantities of products from the list of prohibited foods.

Only one person with diabetes has been insulinopenia during the first induced hyperglycemia, and therefore it was excluded from the test because they did not meet certain inclusion criteria. 23 other subject completed the study.

Oral TEC is on glucose tolerance (OTG)

The test was performed within 3 days of hospitalization, separated by intervals of from 7 to 14 days. Each subject gave each of the 3 OTH, which were carried out in random order.

In tests OCTG used 8 g of yeast cell walls and 9 g of beta-glucans. It should be noted that the consumption of yeast cell walls at the scheduled dose of 8 g does not exceed the consumption levels observed for Baker's yeast and nutritional yeast.

The products were dissolved in 167 ml of 30% solution13C-glucose containing 50 g13C-glucose. For the test food tolerance placebo to 167 ml13C-glucose was added 28 ml of whole milk and 50 ml of semi-skimmed milk to obtain the equivalent energy consumption of 8 g of yeast cell walls and 9 g of beta-glucans.

Metabolic day was divided into two parts: the basal period (T-120-t0), during which we measured basal values (fasting), then the postprandial period of 4 h 45 min (T15-T300) after the test OTG.

During the test OTTH the patient was given 50 g13C-glucose at t0. Depending on shuffle conducted a possible addition to the solution just before receiving or yeast cell walls, or beta-glucans, or milk. This solution should be ingested in a period of less than 10 minutes. The patient remained in the supine position for which the subsequent 5 hours, avoiding sleep. Within days of the study the subject was allowed to drink water (300 ml).

Determined basal levels of insulin in plasma (between T-120 and t0) and in the postprandial period from T15 to T300. Samples of venous blood was immediately centrifuged, and plasma was collected for analysis of insulin. Analysis of insulin in plasma was performed using radioimmunological assay (Medgenix Diagnostics®, Rungis, France).

Statistical processing

The results are presented as mean per group ± standard error of the mean (SOS). The results obtained in 3 conditions (yeast cell wall beta-glucan and placebo (milk)were compared in pairs using the nonparametric test of Wilcoxon signed on the matching series. To implement these statistical tests used the software Statview (Statview, Abacus Concepts, Berkeley, CA).

2. Results

Watched a good digestive tolerance of both the tested yeast products.

The trend in insulinemia through 3 tests OTG in subjects with overweight

Increasing insulinemia in response to oral glucose was significantly lower in the presence of beta-glucans than with placebo.

Peak insulin was significantly lower in the presence of beta-glucans than with placebo (63,0±7,7 IU/l vs to 91.6±13,4 IU/l if the consumption of milk (p<0,01) (figure 1). The area under the curve insulinemia the presence of beta-glucans was significantly lower than with placebo (p<0,05) (figure 2 and table 2).

For yeast cell walls was observed a trend towards lower peak insulin and area under the curve compared with placebo.

Table 2
Peak insulin IU/l and the area under the curve of insulinemia IU/l* time in subjects with overweight. The results are expressed as mean ± SOS (n=12). p<x = significant difference compared to placebo (milk).
Insulinemia if overweightMilkYeast cell wallBeta glucan
Peak insulinto 91.6±13,484,2±13,263,0±7,7 p<0,01
The area under the curve 0-300 min8715±11308570±14446736±591 p<0,05
The area under the curve 0-120 min6499±9036214±9604816±504 p<0,05
The area under the curve 120 min2216±3352356±548 1920±158

The trend in insulinemia through 3 tests OTG in subjects with type 2 diabetes

In subjects with type 2 diabetes reduced the insulin response was also observed in the presence of beta-glucans and in the presence of yeast cell walls.

Peak insulin was significantly reduced in the presence of beta-glucans, and yeast cell walls (respectively 44,2±6,0 IU/l and 43.8±4,7 IU/l vs 56,1±8,8 IU/l for milk, p<0,05) (figure 3 and table 3).

The area under the curve was significantly reduced in the presence of yeast cell wall beta-glucans compared with placebo (figure 4 and table 3).

Table 3
Peak insulin IU/l and the area under the curve of insulinemia IU/l* time in subjects with type 2 diabetes. The results are expressed as mean ± SOS (n=12). p<x = significant difference compared to placebo (milk).
Insulinemia if overweightMilkYeast cell wallBeta glucan
Peak insulin56,1±8,843,8±4,7 p<0,0544,2±6,0 p<0,05
Set the ü under the curve 0-300 min 9049±10707947±821 p=0.058248±878 p<0,05
The area under the curve 0-120 min4325±6193644±3813708±404
The area under the curve 120 min4725±587430±5024540±498

The results of insulinemia in both groups, subjects who are not diabetics, overweight and subjects with type 2 diabetes, thus, show a significant decrease in insulin response after administration of the yeast cell wall, free of mannans. The same applies to the reception of yeast cell walls containing the mannans, in subjects with type 2 diabetes.

1. The use of yeast cell walls to obtain a pharmaceutical composition intended for the treatment and/or prevention of hyperinsulinemia in patients who are not diabetic, where the yeast cell walls have
- total content glucans and mannans at least 34,0% dry matter content by weight and
the glycogen content of less than 10.0% of the dry matter content by weight.

2. The use according to claim 1, where the yeast cell wall belong to the yeast of the genus Saccharomyces cerevisiae.

3. The application is about to claim 1, where the yeast cell walls have a total content of glucans and mannans at least 40.0% of the dry matter content by weight.

4. The use according to claim 1, where the yeast cell walls have a total content of glucans and mannans at least 45,0% dry matter content by weight.

5. The use according to claim 1, where the yeast cell wall content of mannans less than 30% dry matter content by weight.

6. The use according to claim 1, where the yeast cell wall content of mannans less than 2% of the dry matter content by weight.

7. The use according to claim 1, where the yeast cell wall content of mannans less than 1% of the dry matter content by weight.

8. The use according to claim 1, where the yeast cell walls have a total content of glucans and mannans equal or less than 90% of the dry matter content by weight.

9. The use according to claim 1, where the yeast cell walls have a total content of glucans and mannans equal or less than 80% of the dry matter content by weight.

10. The use according to claim 1, where the yeast cell walls have a total content of glucans and mannans from 45% to 90% dry matter content by weight.

11. The use according to claim 1, where the yeast cell walls have a total content of glucans and mannans from 52% to 80% dry matter content by weight is E.

12. The use according to claim 1, where the yeast cell wall content of glycogen in less than 8,0% of the dry matter content by weight.

13. The use according to claim 1, where the yeast cell walls have the glycogen content of less than 5.0% of the dry matter content by weight.

14. The use according to claim 1, where the yeast cell walls have a dry matter content greater or equal to 90%.

15. The use according to claim 1, where the yeast cell walls have a dry matter content greater or equal to 94%.

16. The use according to claim 1, where hyperinsulinemia due to overweight, obesity or metabolic syndrome.



 

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