Grape extract, food additive therefrom and such extract and additive production methods

FIELD: food industry.

SUBSTANCE: invention relates to a polyphenol grape extract, a compound for oral administration, a food product, beverage, a taste additive, a nutraceutical product, a revitalising composition and therapeutic remedy including the said grape extract. According to the invention, the grape extract contains nearly 5-15 wt % of monomers, nearly 5-20 wt % of dimers, nearly 3-10 wt % of trimers, nearly 2-10 wt % of tetramers and nearly 2-10 wt % of pentamers.

EFFECT: grape extract reduces blood pressure with patients suffering from prehypertonic condition or metabolic syndrome.

27 cl, 4 dwg, 8 tbl, 7 ex

 

This application has priority application U.S. Ser. No. 60/721721 dated September 28, 2005, the description of which is included here by reference in full.

The present invention relates to a novel grape extract, and methods of making such grape extract. New grape extract is used essentially to reduce blood pressure, for example, in patients with prehypertension condition or metabolic syndrome. The present invention also relates to a food additive containing grape extract according to the invention.

Grape seeds contain about 5-8 wt.% of flavonoids. Flavonoids constitute an important group of dietary polyphenolic compounds, which are widely represented in plants. It was identified more than 4,000 chemically unique flavonoids from plant sources such as fruits, vegetables, legumes, nuts, seeds, herbs, spices, flowers, as well as drinks such as tea, cocoa, beer, wine, and grape juice.

The term flavonoids in relation to grape seed means a monomer flavan-3-Ola, namely, (+)-catechin, (-)-epicatechin and (-)-epicatechin-3-gallate. Two or more chemically bonded monomer flavan-3-ol referred to as proanthocyanidins or oligomeric proanthocyanidins ("OPCs"), which include procyanidins and prodelphinidins. OPCs containing two monomial�and called dimers, three of monomer are called trimers, four of monomer are called tetramers, five of monomers called pentamer, etc. In structural terms, the oligomers have a chain length of 2 to 7 (from dimers to heptamers); whereas polymers are components with a chain length of more than 7. After a lengthy discussion there was a consensus on assessment methods with grape seed Committee (National Association of food) determining OPCs, as proanthocyanidins containing two or more monomer including polymers or condensed tannins. Thus, the oligomers of grape extracts include, for example, dimers and trimers, and, obviously, the polymers can have up to sixteen units.

Below is the typical structure of proanthocyanidin, showing lengthening units and terminal units. Extension units are, for example, epicatechin (2) and epigallocatechin (3) linking group, while the terminal unit is represented by a group epicatechin-gallate (4).

As polyphenolic compounds commercial use, such as grape extract, these compounds can be isolated from grapes in a more concentrated form.

The basic process by which is extracted, purified and concentrated polyphenol with�organisations from whole grapes, grape pomace and seeds described in U.S. patent 6544581 entered here by reference in full.

It was reported that in addition to antioxidant activity in animal studies flavonoids exhibit anti-cancer effects, reducing growth of new blood vessels and have anti-inflammatory, antimicrobial and antiallergic activity. It was also found that grape extract according to the invention can be applied to reduce blood pressure in patients with prehypertension condition and in patients with metabolic syndrome. It is also assumed that the grape extract according to the invention will be further to lower blood pressure to reduce oxidized LDL cholesterol in patients with metabolic syndrome. Elevated levels of LDL cholesterol is recognized as a risk factor for development of atherosclerosis. There is strong evidence that the modified LDL oxidation initiates the development of the pathological process. Consequently, the decrease in the concentration of oxidized LDL may reduce and/or prevent the development of atherosclerosis in patients with metabolic syndrome.

Patients with prehypertension condition classified as patients whose systolic pressure is between 120 to 139 mm Hg.CT., or diastole�die pressure is in the range from 81 to 89 mm Hg.PT. This classification is based on the Seventh report of the joint national Committee on prevention, detection, evaluation and treatment of high blood pressure Seventh Report of the Joint National Committe on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC7), p. 87, NIH Publication No. 04-5230. Patients with prehypertension condition is usually not treated using medicines, instead they offer to lead a healthy lifestyle. These recommendations include maintaining a normal body weight; maintaining a physically active lifestyle; following a healthy eating plan that emphasizes fruits, vegetables and dairy products with low fat content; selection and preparation of products with a minimum content of sodium; and moderate consumption of alcoholic beverages or eliminating them. Maintaining a healthy lifestyle, as a rule, is effective as a first step to prevent and control abnormal blood pressure.

"Metabolic syndrome", also called "Syndrome X, Syndrome of insulin resistance" or "Deadly Quartet", characterized by an increase in risk factors for cardiovascular disease, stroke and/or diabetes type II. The cause of the metabolic syndrome may be the overproduction of cortisol, a stress hormone that causes NAC�came internal abdominal fat and insulin resistance. At present for patients with metabolic syndrome drug therapy is not carried out. Risk factors that characterize the metabolic syndrome include increased mass of adipose tissue in the abdomen (abdominal obesity), insulin resistance increases the risk of developing diabetes, hyperinsulemia, high levels of fat in the blood, increase in blood pressure and increased level of lipid content in serum. In the National Educational Program on cholesterol The National Cholesterol Education Adult Treatment Panel (ATP III) patients with metabolic syndrome defined as patients with at least three of the following risk factors:

Risk factorDetermining the level
Abdominal obesity is recognized when the waist circumference is*
Men> 102 cm (>40 inches)
Women> 88 cm (>35 in)
Triglycerides≥ 150 mg/DL
HDL-cholesterol
Men�s < 40 mg/DL
Women< 50 mg/DL
Blood pressure≥ 130 ≥ 85 mm Hg. article
Glucose level≥ 110 mg/DL
*Overweight and obesity are associated with insulin resistance and metabolic syndrome. The presence of abdominal obesity, however, is much more associated with risk factors of metabolic syndrome than with estimated body mass index. Therefore, to determine this component of the assessment of the metabolic syndrome, as the body weight, it is recommended that a simple measurement of waist circumference.
Some male patients may have multiple risk factors for developing metabolic syndrome if your waist circumference is the minimum excess, for example, from 94 to 102 cm (37 to 39 inches). Such patients may be a significant genetic predisposition to insulin resistance. They should also change the way of life, similarly to men with a significant increase in waist size.
The American diabetes Association has recently established a norm ≥ 100 mg/DL, above which the patient's diagnose�is prediabetes state (reduced level of glucose) or diabetes. The new rule is applied to determine the lower boundary of an elevated glucose as one criterion of metabolic syndrome.

Conditions associated with metabolic syndrome include diabetes mellitus type II, dis-lipoproteinemia, myocardial infarction, stroke, and other atherosclerotic disease, as well as the risk factors of these diseases, including, typically, insulin resistance, abdominal obesity, which is the accumulation of abdominal fat, elevated lipid and glucose levels in serum, increased diastolic and/or systolic blood pressure and hypertension.

There is a need in grape extract and food additive containing grape extract, which can be used as adjunctive therapy, or improved health, such as lowering blood pressure in patients with prehypertension state or in patients with metabolic syndrome.

In the drawings:

Fig.1 shows the relationship between initial blood pressure and systolic pressure in patients with metabolic syndrome treated grape extract according to the invention.

Fig.2 shows the relationship between initial blood pressure�tion and reduction of diastolic pressure in patients with metabolic syndrome treated grape extract according to the invention.

Fig.3 shows changes in the concentration of oxidized LDL in patients with metabolic syndrome treated grape extract according to the invention.

Fig.4 shows the relationship between the changed concentration of oxidized LDL and the initial concentration of oxidized LDL in patients receiving 300 mg of grape extract according to the invention.

The present invention relates to a grape extract that is effective in the treatment prehypertensive status and metabolic syndrome and/or conditions, including metabolic syndrome. Generally, grape extract according to the invention comprises, by weight, of about 5-15% monomers, about 5-20% dimers, about 3-10% trimers, about 2-10% tetramers, and about 2-10% pentamer. The total number of low molecular weight phenolic compounds, including monomers, dimers, trimers, tetramers and pentamers, is from about 25 to 50 wt.%, preferably, from about 25 to 40 wt.%, more preferably, from about 30 to 40 wt.%, and most preferably, from about 25 to 35 wt.%. The total content of phenolic compounds is about 80 wt.% or more and preferably about 90 wt.% or more.

In one embodiment of the invention grape extract comprises about 6-15% monomers, about 7-15% monomers, about 8-15% of the monomers, the eye�about 9-15% of the monomers, about 10-15% monomers, about 11-15% monomers, about 12-15% monomers, about 13-15% of the monomers and about 14-15% of monomers. In another embodiment of the invention grape extract comprises about 5-14% monomers, about 5-13% monomers, about 5-12% monomers, about 5-11% monomers, about 5-10% monomers, about 5-9% monomers, about 5-8% monomers, about 5-7% of the monomers and about 5-6% of monomers. In another embodiment of the invention, the number of monomers selected from the group consisting of about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% and 15%.

In one embodiment of the invention grape extract according to the invention includes about 6-20% dimers, about 7-20% dimers, about 8-20% dimers, about 9-20% dimers, about 10-20% dimers, about 11-20% dimers, about 12-20% dimers, about 13-20% dimers, about 14-20% dimers, about 15-20% dimers, about 16-20% dimers, about 17-20% dimers, about 18-20% of dimers and about 19-20% of the dimers. In another embodiment of the invention grape extract comprises about 5-19% dimers, about 5-18% dimers, about 5-17% dimers, about 5-16% dimers, about 5-15% dimers, about 5-14% dimers, about 5-13% dimers, about 5-12% dimers, about 5-11% dimers, about 5-10% dimers, about 5-9% dimers, about 5-8% dimers, about 5-7% of dimers and about 5-6% of the dimers. In another embodiment of the invention, the number of dimers selected from the group consisting of about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 17%, 18%, 19% and 20%.

In one embodiment of the invention grape extract includes �Colo 4-10% of trimers, about 5-10% trimers, about 6-10% of trimers, 7-10% trimers, about 8-10% of trimers and about 9-10% of trimers. In another embodiment of the invention grape extract comprises about 3-9% trimers, about 3-8% trimers, about 3-7% trimers, about 3-6% trimers, about 3-5% of trimers and about 3-4% of trimers. In another embodiment of the invention, the number of trimers according to the invention is selected from the group consisting of about 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%.

In one embodiment of the invention grape extract comprises about 3-10% tetramers, about 4-10% tetramers, about 5-10% tetramers, about 6-10% tetramers, about 7-10% tetramers, about 8-10% tetramers, and about 9-10% tetramers. In another embodiment of the invention grape extract comprises about 2-9% tetramers, about 2-8% tetramers, about 2-7% tetramers, about 2-6% tetramers, about 2-5% tetramers, about 2-4% tetramers, and about 2-3% tetramers. In another embodiment of the invention, the number of tetramers is selected from the group consisting of about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%.

In one embodiment of the invention grape extract comprises about 3-10% pentamer, about 4-10% pentamer, about 5-10% pentamer, about 6-10% pentamer, about 7-10% pentamer, about 8-10% of pentamer and about 9-10% pentamer. In another embodiment of the invention grape extract comprises about 2-9% pentamer, about 2-8% pentamer, about 2-7% pentamer, about 2-6% pentamer, about 2-5% pentamer, about 2-4% pen�me and about 2-3% of pentamer. In another embodiment of the invention, the amount of pentamer selected from the group consisting of about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%.

In one embodiment of the invention, the total content of low molecular weight phenolic compounds, i.e., monomers, dimers, trimers, tetramer and pentamer is about 25% or higher, about 30% or higher, about 35% or higher, about 40% or higher, about 45% or higher, up to about 50 wt.%. In another embodiment of the invention, the total content of phenolic compounds is about 25% or higher, 26% or higher, 27% or higher, 28% or higher, 29% or higher 30% or higher, 31% or more, 32% or above, 33% or higher, 34% or higher 35% or higher, 36% or higher, 37% or higher, 38% or higher, 39% or above 40% or above, 41% or above, 42% or higher 43% or higher, 44% or greater, 45% or higher, 46% or above, 47% or higher, 48% or higher, 49% or higher, up to about 50 wt.%.

Grape extract according to the invention has a profile of phenolic compounds, as determined using normal phase high performance liquid chromatography ("HPLC"), about 5-15% monomers, about 5-20% dimers, about 4-10% trimers, about 2-10% tetramers, and about 2-10% by weight pentamer. Grape extract according to the invention also has a total content of phenolic compounds to about 80 wt.% or more and preferably about 90 wt.% or more as determined by using the Folin-Jikalau. Grape extract according to the invention also� includes about 2 wt.% or less, of terminal units epicatechin-gallate, more preferably, about 1 wt.% or less, as determined using back-phase HPLC after cialisa. Grape extract according to the invention also includes about 12 wt.% or less of the extension units epicatechin-gallate, more preferably, about 8 wt.% and most preferably about 5 wt.% or less, as determined using back-phase HPLC after cialisa.

Grape extract according to the invention is a product obtained by high-temperature water extraction, described in U.S. patent No. 6544581, as shown below. As a rule, high-temperature water extraction, described in U.S. patent No. 6544581, comprising the following steps. At the stage (1) grape seeds, fresh or dried, is heated with hot water during the period of time that is sufficient for the extraction of most of the polyphenols. Can be used temperature of about 140-212°F, preferably about 160-212°F, more preferably about 180-212°F, most preferably about 190-212°F for about 1-6 hours. Heating time may vary depending on the temperature. Generally, lower temperatures require a longer time of extraction. At the stage (2) primary aqueous extract of grape seeds can be separated from seeds used by passing through a metal sieve. Then extract the OHL�claim and handle any commercially available pectolytic enzyme such as Pectinex® Ultra SP-L, produced by Novo Nordisk at a concentration of about 50-200 M. D. (ppm), for destruction of cell walls. Preferably, the aqueous grape seed extract may be enzyme-treated for two hours at a temperature of about 80-120°F. alternatively, the aqueous grape seed extract may be enzyme-treated for 7-14 days or more at a temperature of about 40-50°F. At the stage (3) is obtained, as a result, turbid seed extract may be acidified with an acid, preferably an inorganic acid, more preferably sulfuric acid to a pH of about 1.5 to 2.5, and the reaction takes from about one hour to about two days. Acidified extract can cool for periods of up to several weeks for the deposition of macromolecules, including proteins and other polysaccharides. Then cooled and acidified extract can be filtered using diatomaceous earth to obtain a clarified extract of grape seeds. Can be used with other filters such as perlite.

Stage (2) for U.S. patent No. 6544581 can be modified by treatment with an enzyme aqueous extract of seeds within four to five days at a temperature of about 80-120°F with receiving grape extract according to the invention. Without wishing to be bound by any theory, the authors present and�gained consider what are the long duration in comparison with that used in U.S. patent No. 6544581 at certain temperatures at this stage, as a result, leads to obtaining a new grape. The processing time of the enzyme can vary depending on the applied temperature. Generally, lower temperatures require a longer processing time. Consequently, the aqueous seed extract may be enzyme-treated for two weeks or more at temperatures of about 60-80°F.

In one embodiment of the invention grape extract can be obtained according to the invention using the following steps. After the extraction stage (1) or after treatment with pektinaza at the stage (2) for U.S. patent No. 6544581 extract may be deposited on the bacteriological agrarian plate. During incubation can be many kinds of yeast, bacteria and/or fungi depending on the source material. A living culture can be isolated as a mixture. Immediately after separation, the mixture can be subjected to subsequent extraction and/or processing stage pectinase enzyme. For example, aqueous seed extract may be subjected to enzymatic treatment with any commercially available pectolytic enzyme and combined with the selected mixture of yeast, bacteria and/or fungi. Combined, the mixture can be�vergota to defend within the time period equal to from about one to ten days, preferably from about two to five days at a temperature of about 70-100°F. the Time of enzyme treatment can vary depending on temperature and the amount of inoculum. The resulting turbid seed extract may be acidified using a suitable acid as described above to a pH of 1.5 to 2.5, and the reaction takes from about one hour to about two days. Acidified extract can be cooled and stored for several days for flocculation of proteins and polysaccharides. Then cooled and acidified extract can be filtered using diatomaceous earth to obtain a clarified extract of grape seeds, which can then be further processed for U.S. patent No. 6544581 with obtaining a purified extract suitable for lowering blood pressure and reducing the concentration of oxidized LDL.

The content of gallium acid in grape extracts obtained according to U.S. patent No. 6544581 comparable to the content of gallium acid in grape extracts obtained according to the invention using a mixture of yeast, bacteria and/or fungi, according to the analysis VEHI. This analysis showed a higher content of gallium acid with about 50-150 M. D. in grape extract for U.S. patent No. 6544581 in comparison with the previous content of�ing gallium acid in grape extract according to the invention using a mixture of up to about 400-1500 M. D. The increase in gallium content of the acid indicates that the terminal and extension units epicatechin-gallate deesterification of procyanidins. Without wishing to be bound by any theory, the authors present invention believe that the mixture of yeast, bacteria or fungi uses grape seed extract as a substrate for growth and activates products tennisnogo of the enzyme, resulting in deesterification procyanidins and the release of gallium acid. Essentially, when using a mixture of yeast, bacteria and/or fungi get grape extract according to the invention containing about 2 wt.% or less, of terminal units epicatechin-gallate, more preferably, about 1 wt.% or less, and about 12% or less by weight of the extension units epicatechin-gallate, preferably about 8 wt.% or less and more preferably about 5 wt.% or less.

In one embodiment of the invention grape extract can be obtained using the following steps. After the extraction stage (1) or after treatment with pektinaza at the stage (2) for U.S. patent No. 6544581 in the extract can be introduced any suitable commercially available tanany enzyme, for example, fungal tanany enzyme, such as trinacional, E. C3.1.1.20 in a concentration of from about 5 to 1000 m. D. depending on the con�entrale used tennisnogo enzyme the mixture can react from about one hour to about two days, preferably, from about one to two days or as long as the contents of the terminal units will be reduced to about 2% or less, preferably 1% or less and the content of the extension units will be reduced to about 8% or less, preferably to about 5% or less. After passing through the reaction for a sufficient period of time extract can be acidified to a pH of from about 1.5 to 2.5 for flocculation of proteins and polysaccharides in cold storage at a temperature of 40-60°F. Then the extract can be filtered for clarification and then can be further processed for U.S. patent No. 6544581 with receiving grape extract with characteristics that reduce blood pressure.

Grape extract according to the invention can be included in formulation of nutritional supplements, including capsules, pills, powders, solutions, gels, suspensions, creams, pastes, gels, suppositories, transdermal patches, etc. These food additives, for example, in the form of powders or solutions can be introduced in nutraceuticals, foods and/or drinks with obtaining functional nutraceutical, food and/or drinks. Nutritional supplements can be obtained in powder form, for example, for mixing with consumable liquids, such as milk, juice, water, or in the form of sweat�allaamah gels or syrups for mixing with other dietary liquids or foods. Food additives according to the invention can be members of other foods or liquids with obtaining portsionirovaniem food additives, such as one-shot packaging in the form of a bar. Generally, foods that can be introduced grape extract according to the invention, include dairy foods such as yogurt, cereals, breads, snacks, fruit juices and soft drinks. If necessary, can be entered flavorings, binders, proteins, complex carbohydrates, vitamins, minerals, etc., Preferably grape seed extract is obtained in the form suitable for oral administration.

The present invention also relates to a food additive containing grape extract according to the invention. Dietary Supplement when administered to mammals, including humans, reduces blood pressure in patients with prehypertension condition or metabolic syndrome. A successful course of treatment, such as patients with prehypertension condition or metabolic syndrome or prevention of these conditions is characterized, essentially, by reducing one or both of the systolic and/or diastolic blood pressure of at least about 2%, preferably by at least about 5% and most preferably at least about 8% and is due�Oh very slight increase in insulin resistance.

Food additives according to the invention are intended for daily use or as needed. Prophylactic or therapeutic dose nutritional supplements for patients with prehypertension condition or metabolic syndrome can vary depending on the severity of the treated condition and the way of introduction. Dose or estimated frequency of administration may also vary depending on the age, body weight and response of the patient. Typically, the total daily dose to the conditions described herein is from about 50 mg to about 1000 mg of grape extract, administered once or divided into doses orally, topically, or transdermally, preferably orally. Daily oral dose is preferably in the range of from about 50 mg to about 500 mg of grape extract (including adjuvant or carrier), more preferably from about 150 mg to about 300 mg. for Example, capsules or tablets can be received in the packaging of 150 mg or 300 mg, while drinks can contain 50 mg of grape extract according to the invention. This mode or reception, it is preferable to maintain for at least one month, more preferably six months or more.

A food additive according to the invention can be obtained by a suitable method (i.e., dry mix�the devices wet or dry granulation, direct compression) in a mixture with pharmaceutically acceptable carriers, excipients, vitamins, minerals and/or other nutrients. Representatives of carriers and excipients include, without limitation starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricating agents, binders, dezintegriruetsja agents and similar in the case of preparations for oral administration such as powders, capsules and tablets).

For introduction to the patient a food additive according to the invention can be used by any suitable route of administration. Suitable routes of administration include, for example, oral, rectal, parenteral, intravenous, local application, transdermal, subcutaneous and intramuscular. Although it may be used any suitable route of administration to the patient of an effective amount of a grape extract according to the invention, preferred is oral administration that includes the use of solid dosage forms such as tablets, capsules or powders. Preferably also, grape seed extract may be included in functional nutraceutical, food product or beverage.

Grape extract according to the invention can also be combined with other active agents, including without limitation diureti�and, beta-blockers, ACE inhibitors, antagonists of angiotensin, calcium channel blockers, alpha blockers, alpha beta blockers, inhibitors of the nervous system, vasodilatory, antioxidants.

Characterization of grape extracts

Recently it was reported that the use of grape seed polyphenols in hypertensive patients does not reduce systolic blood pressure, and, in fact, increases in systolic blood pressure when combined with vitamin C. Cm. Ward et al. "The combination of vitamin C and grape-seed polyphenols increases blood pressure: a randomized, double-blind, placebo-controlled trial," Journal of Hypertension 2005; 23:427-434. Without wishing to be bound by any theory, the authors present invention believe that the profile of phenolic compounds of grape extracts is very important for effective reduction of blood pressure. Grape extract, as measured in the study Ward from Vinlife®the profile of phenolic compounds which has a total content of phenolic compounds at 50.6 wt.%, as determined by using the Folin-Jikalau, 11.2% of terminal units epicatechin-gallate and 11,8% of the extension units epicatechin-Galata, as determined using back-phase HPLC after tiliza, and 7.3% of monomers, 4,4% dimers, 2,0% of trimers, 1.9% of tetramers and 1.1% pentamer with a total content of monomers, dimers, trimers, tetramer and pentamer of 16.7%, as determined using �armelino phase HPLC.

Commercially available extracts of grape seed contain various monomers and proanthocyanidins. The profile of phenolic compounds of some commercially available extracts as determined through back-phase HPLC, are shown in Table 1 and defined using straight-phase HPLC, are shown in Table 2. In accordance with these analyses grape extract according to the invention (at the moment produce Polyphenols, Inc. as MegaNatural®-BP) has three factors that distinguish it from other grape extracts:

1. High degree of purity, determined by the total content of phenolic compounds, components higher than about 80 wt.% and more preferably, greater than about 90 wt.%, as determined by using the Folin-Jikalau;

2. High content, for example, about 25-50 wt.% low molecular weight phenolic compounds, and low molecular weight phenolic compounds are monomers, dimers, trimers, tetramers and pentamers; and

3. From very small to zero content, for example, less than about 2%, preferably less than about 1% terminal units epicatechin-gallate and low content, for example, less than about 12%, preferably less than about 5% of the extension units epicatechin-gallate.

Without wishing to be bound by any theory, the authors present invention believe that the profile pheno�of intelligent connection of grape extracts is very important for effective treatment or prevention prehypertensive status or metabolic syndrome. In particular, it is believed that the lack of an epicatechin-gallate terminal units, and small content of epicatechin-gallate in extension units in grape extract according to the invention, along with the presence of high content of low molecular weight compounds, is responsible for the increased vasodilation, which is responsible for the drop in blood pressure in a clinical study of a person with metabolic syndrome and prehypertension condition described below.

Back-phase HPLC to determine the percentage of monomers, oligomers and polymers

To determine the proportions of monomers, oligomers and polymers used back-phase HPLC analysis of grape extract, based on the 280 nm peak area.

The HPLC conditions:

Mobile phaseA: 2% glacial acetic acid
B: 80% acetonitrile, 0.4% of acetic acid
GradientTime (min)% % Curve
0.001000-
3,00 10006
6,009646
15,0090106
30,0085156
50,0077236
60,0075256
66,0070306
80,0050506
83,0020806
85,0010006
105,0010006
11010006
Column250 mm × 4.6 mm, Prodigy 5µ ODS (3) 100 Å (Phenomenex, Torrance, CA)
Flow rate1.0 ml/min
Wavelength detector280 nm
Temperature30°C
Volume injection25 µl

A sample of the drug. 0.1 g of grape extract was carefully weighed into a 100 ml volumetric flask. The sample was dissolved in a small amount of methanol (≤ 5 ml) were dispersively using ultrasound, if required. Volume 18 complement-timegames water. The sample was centrifuged (14000 rpm, 10 min) or filtered through 0.45 µm glass filter prior to injection. Determined the percentage by weight of monomers, oligomers and polymers based on standard peak area and concentration.

The method of determination of terminal and extension units of proanthocyanidins based on HPLC analysis after cialisa

Cialis is a way to determine the average molecular size (degree polyester�polarization) and the basic structure of proanthocyanidines grape extract. The received information may indicate the biological quality of grape extract for adsorption of nutrients grape extract.

Tiality reagent:5% phenyl methanethiol (benzyl mercaptan) in methanol containing 0.2 N HCl.

Condition:A 0.1% methanol solution of grape extract was mixed with an equivalent amount tilingo reagent, thoroughly stirred and heated to a temperature of 90°C for 2 min To stop the reaction of the injected water. Then the reaction product was centrifuged at 14000 rpm for 2 min. the Supernatant was directly analyzed by HPLC.

The HPLC conditions:

Mobile phase:A: 10% acetic acid/0.1 to TPV/5% acetonitrile/to 84.9% water (vol./about./about./about.)
In: acetonitrile
Gradient:0-30 minutes 0-50%
30-35 minutes 50-100%
Column150 cm × 2.0 mm, inner diameter, Synergi 4µ hydro-RP 80 Å (Phenomenex, Torrance, CA)
Flow rate0.3 ml/min
Wavelength detectorHP 1100 FLD with emission/excitation at 276 nm and emission/ emission pri nm and HP DAD 280 nm
Temperature30°C
Volume injection1-3 µl

For analysis of grape extracts were dissolved in methanol, was mixed with an equal volume of tilingo reagent and heated to a temperature of 90°C for 2 min, the Released units were identified using mass spectrometry, quantification was performed using HPLC conditions described above. The average degree of polymerization was measured by calculate the molar ratio of all flavan-3-measuring units (thioether adducts plus terminal units) to the catechin, epicatechin and epicatechin-gallate, which represents a terminal unit. Percentage of the epicatechin-gallate terminal units was determined based on the molar ratio of epicatechin-gallate, summing moles of all the terminal units, which include catechin, epicatechin and epicatechin-gallate. Percentage of the epicatechin-gallate extension units have been determined based on the molar ratio of thioether adducts of the epicatechin-gallate, summing moles of all extension units of thioether adducts that include thioether adducts of catechin, epicatechin and epicatechin-gallate. The total content of phenolic compounds was determined in terms of n� equivalent gallium acid (GAE) per gram using the method of Folin-Jikalau. A more detailed description of the procedure of analysis by the method of Folin-Jikalau given in Waterhouse, A. L., Determination of Total Phenolics, in Current Protocols in Food Analytical Chemistry, 11.1.1-11.1.8, Wrolstad, R. E., Wiley, 2001, or Singleton, V. L; Orthofer, R.; Lamuela-Raventos, R. M. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu Reagent," Methods in Enzymology 1999, 299, 152-178, each of which is introduced here by reference in full.

73,3
Table 1
Comparative characteristics of MegaNatural®-BP and other grape extracts in the market, some using back-phase HPLC
Country of originProduct nameTotal phenolThe epicatechin-gallateThe epicatechin-gallateDefined back-phase HPLC using peak area
g GAE/100g (as is)Terminal-nye (%)Lengthen-ing (%)Monomers (%)Oligomers (%)Polymers (%)
USAMegaNatural®-BP93,86,812,8And 62.624,6
USAMegaNatural®-BP91,00,05,49,269,6Of 21.2
USAMegaNatural®-BP95,20,06,913,864,7A 21.5
USAMegaNatural®-BP98,60,08,211,368,0A 20.7
USAMegaNatural®-BPTo 91.10,05,45,471,523,2
USAMegaNatural®-BP95,50,05,16,520,3
USAMegaNatural®-BP92,70,03,08,569,821,7
USAMegaNatural®-BP93,50,04,85,469,924,7
USAMegaNatural Gold®93,010,511,77,874,717,6
USAMegaNatural Gold®91,94,314,612,376,711,0
USAMegaNatural Gold®92,411,011,910,277,712,1
USAMegaNatural Gold®89,15,212,99,973,7A 16.4
USAMegaNatural Gold®90,13,614,511,173,4The 15.6
USAMegaNatural Gold®90,32,88,813,265,521,3
USAMegaNatural Gold®To 89.68,711,810,065,224,8
AustraliaVinlife®50,611,211,86,360,633,1
EuropeMasquelier OPC® 98,08,57,512,168,419,5
EuropeNaturex®78,58,36,26,364,229,5
EuropeIndena®93,010,58,810,164,425,5
ChinaLycome®88,510,67,55,3For 63.131,7
ChinaRecovery®The 95.83,95,49,158,432,5
ChinaGrape PE®92,69,86,8 3,651,944,4
ChinaMA®70,18,710,54,655,340,1
USAME®68,912,16,31,952,645,4
USASan Joaquin®74,917,76,72,256,1Of 41.8
USAActivin®Of 84.314,811,13,055,941,1
JapanKIKKOMAN®44,55,213,18,152,938,9

Normality HPLC analysis of proanthocyanidins

HPLC analysis of proanthocyanidins:Chromatographic analysis was performed on a HP 1100, HPLC equipped with autoposition/dispenser, double pump, column heater, diode array detector, fluorescence detector, and HP ChemStation for data collection and manipulation. Normal phase separation of oligomers of proanthocyanidins was performed on a column (Phenomenex Luna Silica (2).

Mobile phase:A: dichloro methane, methanol, water and acetic acid (83:13:2:2 (about./about.))
In: methanol, water and acetic acid (96:2:2 (about./about.))
Gradient:0-30 minutes linear 0-17,6%
30-45 minutes linear to 17.6 30.7 percent In
45-50 minutes linear a 30.7 87.8% In
50-60 minutes linear 87,8%
ColumnPhenomenex Luna Silica (3,0×150 mm; 3.0 mm)
Flow rate0.5 ml/min
Wavelength detectorHP 1100 FLD with emission/excitation at 276 nm and emission/emission at 316 nm
Temperature25°C
Volume injection3 µl

Column trim the bottom side o�shivali every time between sprays equivalent to 5 ml of initial mobile phase. Standards of catechin were obtained and examined to establish the response of the calibration curve, which was calculated concentration of proanthocyanidins in the samples. The relative sensitivity factors of dimers, trimers, tetramer and pentamer to monomer was determined using fluorescence, as described in R. L. Prior and L. Gu, Occurrence and biological significance of proanthocyanidins in the American diet," Phytochemistry 2005, 66(18) 2264-2280, using standards, isolated and purified from cocoa beans. These sensitivity factors used for calculation of the content of dimers, trimers, tetramer and pentamer relative to the monomers.

Table 2

Comparative characteristics of MegaNatural®-BP and other grape extracts in the market that are defined by straight-phase HPLC

Country of originProduct nameDefined straight-phase HPLC using the equivalent of catechin and epicatechin ( wt.%)
MonomersDimersTrimersTetramersPentane-ryThe monomers to pentane-RAMOther
USAMegaNatural®-BP6,38,74,04,42,826,273,8
USAMegaNatural®-BP9,113,65,66,23,538,161,9
USAMegaNatural®-BP9,214,85,25,42,737,362,7
USAMegaNatural®-BP10,014,25,45,22,837,662,4
USAMegaNatural®-BP7,511,95,25,53,4 The 33.466,6
USAMegaNatural®-BP8,212,15,45,73,534,965,1
USAMegaNatural®-BP9,011,35,44,72,532,967,1
USAMegaNatural®-BP5,29,84,14,82,626,473,6
AustraliaVinlife®7,34,42,01,91,116,783,3
EuropeMasquelier OPC®15,514,26,2 4,82,943,656,4
EuropeNaturex®8,36,43,63,02,023,276,8
EuropeIndena®16,612,46,24,83,343,356,7
ChinaLycome®7,66,23,42,81,922,078,0
ChinaRecovery®17,58,14,42,82,034,865,2
ChinaGrape PE® 3,93,21,91,51,111,688,4
ChinaMA®4,13,81,91,70,812,387,7
USAME®1,81,80,90,80,55,894,2
USASan Joaquin®5,35,82,61,81,316,883,2
USAActivin®5,54,82,11,31,315,1To 84.9
Japan KIKKOMAN®0,91,10,70,70,43,796,3

The results presented in Tables 1 and 2, were obtained using different methods, which explains the various limits, for example, the percentage of monomers. For example, to determine the percentage content of monomers, oligomers and polymers used back-phase HPLC, based on the peak area of these three groups of compounds. The monomers included Galic acid. For the determination of carbon by weight in grape extract of monomers, dimers, trimers, tetramer and pentamer when conducting a straight-phase HPLC as the standard used catechins and epicatechin. The relative sensitivity factors of dimers, trimers, tetramer and pentamer to monomer defined by R. L. Prior and L. Gu, used to calculate the content of dimers, trimers, tetramer and pentamer relative to the monomers.

2. Effects of grape extract on blood pressure in individuals with metabolic syndrome.

Effects of grape extract according to the invention on blood pressure was investigated in twenty-four patients diagnosed with metabolic �Ingram. The study involved an equal number of men and women from 20 to 50 years. Metabolic syndrome was diagnosed using criteria defined by the National Educational Program on cholesterol (The National Cholesterol Education Adult Treatment Panel III). Each entity has identified at least three of the following grounds: 1) blood sugar > 110 mg/DL, 2) HDL (<40 mg/DL in men and < 45 mg/DL in women), 3) blood pressure > 130/85 and 4) abdominal obesity (>102 cm for men and > 88 cm for women). Patients did not participate in the study if they smoked at the time of the study or quit Smoking (< 3 years); took anti-inflammatory or pressure lowering drugs; or had used over-the-counter antioxidant compounds.

Patients randomly divided into three groups of eight people, and they received one of the following capsules, depending on the group into which they entered:

Group 1 received a capsule containing a placebo.

Group 2 received a capsule containing 150 mg of grape extract.

Group 3 received a capsule containing 300 mg of grape extract.

For the next twenty-eight days patients received capsules with the same dose. At the end of this period were the results of blood pressure measurement. Outpatient� blood pressure was recorded 12 hours after the start of the study and again - through four weeks. The procedure was non-invasive with the use of a cuff for measuring blood pressure placed on the hand. A cuff which is provided with a locking tape was connected to an automatic device for supercharging approved by Management on sanitary inspection behind quality of foodstuff and medicines of the USA.

Table 3 summarizes the data on blood pressure of three groups of patients with metabolic syndrome. Those who received daily doses of 300 mg and 150 mg of grape extract according to the invention, there was a significant decrease, both systolic and diastolic blood pressure. The group treated with placebo, no significant changes were observed.

Table 3
The results of the use of grape extract according to the invention in patients with metabolic syndrome
Daily dose of 300 mgDaily dose 150 mgPlacebo
SystolicDiastolicSystolicDiastolicSystolic Diastolic
Start129±479±3137±484±3,3124±474±4
4 weeks117±371±3125±478±1,9123±471±4
R*0,0070,0060,0030,009minorminor
R*-the possibility that the initial and final assessment are the same. Typically, R of 0.05 or less (5%) is considered significant.

Fig. 1 and 2 show the relationship between baseline blood pressure and reduction in both pressure - systolic and diastolic. Blood pressure was measured in mmHg. article Since the diagnosis of metabolic syndrome is based on the presence of three factors from the list of risk factors (one of which is blood pressure), the study patients were divided randomly in blood pressure. By sheer�TSS, the average pressure in the three groups differed significantly (but varied in a narrow range).

This study demonstrates that a grape extract according to the invention in a daily dose from 150 mg to 300 mg lowers both blood pressure - systolic and diastolic in patients with metabolic syndrome. Blood pressure reduction was statistically significant for both doses used grape extract. Indeed, changes in blood pressure observed in the use of grape extract, comparable to the changes observed in most clinical trials with the use of pharmaceutical agents.

3. The effects of grape extract on oxidized LDL in patients with metabolic syndrome.

The effects of grape extract according to the invention the oxidized LDL was investigated in twenty-four patients diagnosed with metabolic syndrome, as described above. The concentration of oxidized LDL was measured after the start of the study and again after four weeks of application. To measure the concentration of oxidized LDL in each patient took a blood sample and analyze it.

Changes in the concentration of LDL three groups are summarized in Fig.3. Fig.3 shows a slight change in oxidized LDL for placebo - gradual change in oxidized LDL for patients who received 150 �g of grape extract according to the invention, and a statistically significant variation (p<0.05) and oxidized LDL for patients receiving 300 mg of grape extract according to the invention. Fig.4 shows the relationship between the initial concentration of oxidized LDL and changed the concentration of oxidized LDL in patients receiving 300 mg of grape extract according to the invention. The regression coefficient R2=0,52. Fig.4 shows a larger change in the concentration of oxidized LDL in patients who had early higher levels of oxidized LDL.

This study demonstrates that a grape extract according to the invention in a daily dose from 150 mg to 300 mg reduces the concentration of oxidized LDL in the plasma of patients with metabolic syndrome. In addition, there is a statistically significant decrease in the concentration of oxidized LDL in patients receiving 300 mg of grape extract according to the invention.

4. The effects of grape extract on patients with prehypertension condition

The effects of grape extract according to the invention was investigated in twenty-four patients diagnosed with prehypertensive state. The study involved an equal number of men and women 30 to 60 years. Prehypertensive condition was diagnosed using criteria defined by the Seventh report of the joint national Committee on pre�prevention, the identification, assessment, and treatment of high blood pressure Seventh Report of the Joint National Committe on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure). Systolic blood pressure of each subject ranged from 120 to 139 mm Hg. CL. and/or diastolic blood pressure of each subject ranged from 81 to 89 mm Hg. article Patients did not participate in the study if they smoked at the time of the study or quit Smoking (< 3 years); took anti-inflammatory or pressure lowering drugs; or had used over-the-counter antioxidant compounds.

Patients randomly divided into two groups of twelve people, and they received one of the following capsules, depending on the group into which they entered:

Group 1 received a capsule containing a placebo.

Group 2 received a capsule containing 300 mg of MegaNatural®-BP.

Over the next eight weeks, the patients received capsules with the same dose. At the end of this period were the results of blood pressure measurement. Ambulatory blood pressure was recorded 12 hours after the start of the study and again after four weeks. The procedure was non-invasive with the use of a cuff for measuring blood pressure placed on the hand. A cuff which is provided with a locking tape was connected to an automatic device for Nadu�and, approved by Management on sanitary inspection behind quality of foodstuff and medicines of the USA.

Table 4 summarizes the data on blood pressure of two groups of patients with prehypertension condition. The initial pressure of the two groups differed slightly. Those who received daily doses of 300 mg of the grape extract of the present invention, there was a significant decrease, both systolic and diastolic pressure, however, in those who received placebo, no significant changes were observed. For example, the average change in systolic blood pressure in the treated group MegaNatural®-BP, was 7.2±2.5 mm Hg. the article, while systolic blood pressure in the group receiving placebo, increased by 0.03±1.5 mm Hg. senior Data summarized below. Values are given in mmHg. article (mean ± standard error of the mean).

Daily dose of 300 mgPlacebo
SystolicDiastolicSystolicDiastolic
Start133±2 80±2134±279±2
8 weeks126±273±2134±280±2
R*Of 0.0210,042minorminor
R*-the possibility, that the initial and final assessment are the same. Typically, R of 0.05 or less (5%) is considered significant.

This study demonstrates that a grape extract according to the invention at a daily dose of 300 mg lowers both blood pressure - systolic and diastolic in patients with prehypertension condition. The decrease in blood pressure was statistically significant. Indeed, changes in blood pressure observed in the use of grape extract, comparable to the changes observed in most clinical trials with the use of pharmaceutical agents.

Examples

The present invention is additionally described by the following examples of methods for producing grape extract and food supplements. The examples only illustrate the present invention but not ogranichivayas goals the nature and volume.

Example 1

A method of manufacturing grape

Dry grape seeds were extracted with water at 200°F within 2 hours and the extract was separated from the seeds on a metal sieve. The extract was cooled to a temperature of 90-100°F and introduced the pektinaza at a concentration of 200 ppm the Extract was divided into two portions. In the first portion introduced commercially available tanany mushroom enzyme (candidacidal, E. C3.1.1.20) at a concentration of 1000 M. D. In the second portion introduced tannase at a concentration of 50 M. D. residual concentration of gallium acid in the original extract was 117 M. D. from 18.9% terminal units and 11.1% of the extension units. When processing 1000 M. D. tennisnogo enzyme for about 2 hours, the concentration of gallium acid increased to 904 M. D. with 0% terminal units and about 5.5% of the extension units. When machining with 50 M. D. tennisnogo enzyme for about thirty-four hours, the concentration of gallium acid increased to 810 M. D. with less than 1% of the terminal units and less than 6% of the extension units. After about two days both extract was acidified with to pH from 1.5 to 2.5 for flocculation of proteins and polysaccharides during cold storage at a temperature of 40-60°F. the Extract was filtered and further processed for U.S. patent No. 6544581 with receiving grape extract characterizing�I ability to lower blood pressure and reduce the concentration of oxidized LDL.

Example 2

Capsules

Grape seed extract MegaNatural®-BP (150 mg or 300 mg) were mixed by dry blending with magnesium stearate (3 mg or 6 mg, respectively) and filled into hard gelatin capsules (derived from gelatin and water). At a dose of 150 mg grape seed extract contains a minimum of 90% phenol or 135 mg of phenols on 150 mg of grape extract. At a dose of 300 mg of the grape extract containing a minimum of 90% phenol or 270 mg of phenols on 300 mg of grape extract. The daily dose was one capsule per day.

Example 3

Powder

Grape seed extract MegaNatural®-BP was obtained by dry mixing with the filler, as shown in Table 4, for use as a beverage, the beverage ingredients were mixed by dry blending. To obtain the final beverage for 9.47 g of dry mixture was combined with 500 ml of cold water and thoroughly mixed. A 500 ml portion contains 16 calories. The final beverage contains 100 mg of grape extract, MegaNatural®-BP and 120 mg of vitamin C per serving 1 l with indicator ORAC 2200 TE. ORAC was measured in mmol Trolox (water-soluble non-commercial derivatives of Tocopherols) equivalents (TE) per gram "the Degree of adsorption of oxygen radicals". This is the standard for measuring antioxidant activity in foods and supplements. A single serving of fresh or cooked fruit�o or vegetable additives are, on average, 600 to 800 ORAC units. It is believed that the increase in the consumption of food or supplements from 2000 to 5000 ORAC units per day can have beneficial effects on the body.

Table 4
Ingredients% dry mix (g)
Maltodextrin37,48
Citric acid29,99
Mud agent (Purity Gum 2000)*The 5.25
Aspartame3,85
Sodium citrate, FCC GradeOf 3.75
UltraGuar**Of 3.75
N&A orange flavor (SN 313897)***7,5
Nat FF flavor passion fruit (SN 313898)***4,27
FD&C yellow #6 (20:1 in maltodextrin)2,24
FD&C yellow #5 (20:1 in maltodextrin)0,75
Ascorbic acid 0,64
Grape seed extract MegaNatural®-BP (Polyphenolics, Inc.)0,53
Total100
*Available from National Starch & Chemical Corporation, Bridgewater, NJ
**Available from P. L. Thomas & Co., Inc. Morristown, NJ
***Available from International Flavors & Fragrances, Dayton, NJ

Example 4

Drink

Grape seed extract MegaNatural®-BP has received in the form of a drink with a filler, as shown in Table 5. The beverage contains 50 mg of grape extract, MegaNatural®-BP and 60 mg of vitamin C (100% RDI) per serving 8 FL oz. Serving 8 FL oz contains 0 calories and 0.15 g of total carbohydrates. Serving in 16 FL oz will have ORAC THOSE 2200:

Table 5
Ingredientswt.%
Water99,4373
Citric acid0,2640
Magenta dye MegaNatural (Canandaigua Conc.)0,0528
Flavoring system Sethness-Greenleaf0,0867
0,0448
Potassium sorbate0,0448
Ascorbic acid0.0338 per kWh
Grape seed extract MegaNatural®-BP (Polyphenolics, Inc.)0,0211
Aspartame0,0147
Total100,0000%

Example 5

Drink

Grape seed extract MegaNatural®-BP has received in the form of a drink with a filler, as shown in Table 6. The beverage contains 50 mg of grape extract, MegaNatural®-BP and 60 mg of vitamin C (100% RDI) per serving 8 FL oz. Serving 8 FL oz contains 15 calories and 4G of total carbohydrates. Serving in 16 FL oz will have ORAC THOSE 2200:

Table 6
Ingredientsweight. %
Water95,8778
The orange juice concentrate 651,3973
The cranberry juice concentrate 500,8691
P�corny dye MegaNatural (Canandaigua Conc.) 0,5032
Flavoring system Sethness-Greenleaf1,1074
Sodium benzoate0,0444
Potassium sorbate0,0444
Ascorbic acid0,0357
Grape seed extract MegaNatural®-BP (Polyphenolics, Inc.)0,0210
Neotame (The NutraSweet Co.)0,0997
Total100,0000%

Example 6

Vitamin/mineral Supplement

Grape seed extract MegaNatural®-BP (150 mg) were mixed by dry blending with a filler that is listed in Table 7, and extruded in the form of a pill with obtaining a multi-vitamin/mineral supplements. The daily dose was one tablet daily, preferably with food.

Table 7
Ingredients% daily value
Vitamin a 3500 IU (29% as beta carotene)70
Vitamin C 60 �g 100
Vitamin D 400 IU100
Vitamin E 45 IU150
Vitamin K 10 mcg13
Thiamin 1.5 mg100
Riboflavin 1.7 mg100
Niacin 20 mg100
Vitamin B6 3 mg150
Folic acid 400 mcg100
Vitamin B12 25 mg417
Biotin 30 mcg10
Pantothenic acid 10 mg100
Calcium 299 mg20
Phosphorus 48 mg5
Iodine 150 mcg100
Magnesium 100 mg25
Zinc 15 mg100
Selenium 20 mgCopper 2 mg100
Manganese 2 mg100
Chromium 150 mcg125
Molybdenum 75 mcg100
Chlorine 72 mg2
Potassium 80 mg2
Grape seed extract MegaNatural®-BP 150 mg*
Boron 150 mcg*
Nickel 5 mcg*
Silicon 2 mg*
Vanadium 10 mcg*
Lutein 250 mcg*
Lycopene 300 mcg*
*Daily value (% SN) not established.

Example 7

Vitamin/mineral Supplement

Grape seed extract MegaNatural®-BP (150 mg) were mixed in a V blender to obtain a smooth mixture with the following ingredients and fillers listed in Table 8. Press the mixture�Wali in the form of tablets with a weight of 755 mg ±2% with getting a multivitamin/mineral Supplement. Tablets are spray-applied transparent coating of a water soluble gum, such as hydroxypropyl cellulose, and dried. The daily dose was one tablet a day. The size of the batch according to the recipe shown in table 8, 500,000 tablets.

Table 8
Ingredients (units)As indicated on the labelSurplus (%)*Quantity/ Tablet (mg)Amount/Batch (kg)
Vitamin a palmitate at 500 IU/g (IU)5000 IU3013,0006,500
Vitamin D3at850 To IU/d (IU)400 IU300,6120,306
Vitamin E succinate (D-α) at 1210 IU/g (IU)15 IU513,0176,508
Vitamin C (mg)30 mg230,600 15,300
Thiamine HCl at 89,2%1.5 mg21,7150,858
(mg)
Riboflavin (mg)1.7 mg21,7340,867
Niacinamide (mg)10 mg210,2005,100
Pyridoxine HCl 82,3% (mg)2 mg52,5521,276
Folic acid, powder of 1.0% (µg)400 mcg2550,00025,000
Vitamin b-12, powder of 1.0% (µg)6 mcg200,7200,360
Pantothenic acid (Pan Cal) (mg)10 mg 510,5005,250
Biotin, powder of 1.0% (µg)30 µg203,6001,800
Calcium (dicalcium phosphate) 29,46% (mg)100 mg0344,119172,060
Phosphorus (dicalcium phosphate) 22,77% (mg)75 mg00,0000,000
Magnesium (MgO) 60,32% (mg)20 mg033,15616,578
Zinc (ZnO) 80,34% (mg))5 mg06,2243,112
Iodine (KI) 76,45% (µg)150 mcg00,1960,098
Copper (gluconate) 14,00% (mg)2 mg014,2867,143
Manganese (gluconate) 12,34% (mg)2 mg016,2078,104
Grape seed extract MegaNatural®-BP150 mg150,00025,000
Microcrystalline cellulose33,75016,875
Cross-carmelose sodium20,25010,125
Stearic acid
Magnesium stearate
13,5006,750
Magnesium stearate5,0632,531
Total775,000337,500
*The percentage content of an ingredient sverhuspeshnogo on the label �ispolzovano for to obtain the amount specified on the label.

1. Polyphenolic grape extract containing about 5-15% monomers, about 5-20% dimers, about 3-10% trimers, about 2-10% tetramers, and about 2-10% by weight pentamer.

2. The extract according to claim 1, wherein the total content of monomers, dimers, trimers, tetramer and pentamer is about 25-50 weight. %.

3. The extract according to claim 1, having a total content of phenolic compounds to about 80 weight. percent or more.

4. The extract according to claim 3 containing about 2 weight. % or less of terminal units epicatechin-gallate.

5. The extract according to claim 4 containing about 12 weight. % or less of the extension units epicatechin-gallate.

6. The extract according to claim 3, having a total content of phenolic compounds to about 90 weight. percent or more.

7. The extract according to claim 6 containing about 1 weight. % or less of terminal units epicatechin-gallate.

8. The extract according to claim 7, containing about 5 weight. % or less of the extension units epicatechin-gallate.

9. The extract according to claim 1, wherein the total content of monomers, dimers, trimers, tetramer and pentamer is about 25-50 weight. %; the total content of phenolic compounds is about 80 weight. percent or more; wherein the content of terminal units epicatechin-gallate is about 2 weight. % or less, and the content of the extension units epicatechin-gallate is about 12 weight. % or less.

10. The extract according to claim 1, containing 400-1500 ppm Gallic acid.

11. The composition for oral administration comprising polyphenolic extract according to any one of claims. 1-10.

12. The composition according to claim 11, wherein the polyphenolic content of the extract is about 50-1000 mg.

13. The composition according to claim 11 or 12, containing the specified extract with at least one acceptable excipient.

14. A food product comprising polyphenolic extract according to any one of claims. 1-10.

15. A food product according to claim 14, wherein the polyphenolic content of the extract is about 50-1000 mg.

16. A food product according to claim 14 or 15, containing the specified extract with at least one acceptable excipient.

17. Beverage, comprising polyphenolic extract according to any one of claims. 1-10.

18. Beverage according to claim 17, wherein the polyphenolic content of the extract is about 50-1000 mg.

19. Beverage according to claim 17 or 18, containing the specified extract with at least one acceptable excipient.

20. Dietary Supplement containing polyphenolic extract according to any one of claims. 1-10.

21. Food Supplement according to claim 20, in which the polyphenolic content of the extract is about 50-1000 mg.

22. Food Supplement according to claim 20 or 21, containing the specified extract with at least one acceptable excipient.

23. Nutraceutical product containing polyphenolic extract according to any and� PP. 1-10.

24. The nutraceutical product according to claim 23, in which the polyphenolic content of the extract is about 50-1000 mg.

25. The nutraceutical product according to claim 23 or 24, containing the specified extract with at least one acceptable excipient.

26. Revitalizing composition comprising a polyphenol extract from about 5-15% monomers, about 5-20% dimers, about 3-10% trimers, about 2-10% tetramers, and about 2-10% by weight of pentamer and at least one acceptable excipient.

27. Therapeutic agent for reducing blood pressure in patients with prehypertension condition or metabolic syndrome, or improvement of conditions, including metabolic syndrome, containing polyphenolic grape extract containing about 5-15% monomers, about 5-20% dimers, about 3-10% trimers, about 2-10% tetramers, and about 2-10% by weight pentamer.



 

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4 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of organic chemistry, namely to novel derivatives of pyrazole pyridine of formula , as well as to its tautomers, geometrical isomers, enantiomers, diastereomers, racemates and pharmaceutically acceptable salts, where G1 represents H; G2 represents -CHR1R2; R1 and R2 independently on each other are selected from H; C1C6-alkoxy-C1C6-alkyl; C1-C6-alkyl; optionally substituted phenyl; optionally substituted phenyl-C1-C6-alkyl; optionally substituted morpholine-C1-C6-alkyl; or -CHR1R2 together form a ring, selected from an optionally substituted C3-C8-cycloalkyl and substituted piperidine; G3 is selected from an optionally substituted C1C6-alkoxy -C1-C6-alkyl; C1-C6-alkyl; substituted phenyl; substituted phenyl-C1C6-alkyl; G4 is selected from a substituted acyl-C1C6-alkyl, where acyl represents a group -CO-R and R stands for H or morpholine; optionally substituted C1-C6-alkyl; optionally substituted phenyl or indene; substituted phenyl-C1-C6-alkyl; optionally substituted pyridine- or furanyl-C1C6-alkyl; morpholine- or piperidine-C1-C6-alkyl; G5 represents H; where the term "substituted" stands for the groups, substituted with 1 to 5 substituents, selected from the group, which includes a "C1-C6-alkyl," "morpholine", "C1-C6-alkylphenyl", "di-C1-C6-alkylamino", "acylamino", which stands for the group NRCOR", where R represents H and R" represents a C1-C6-alkyl, "phenyl", "fluorine-substituted phenyl", "C1-C6-alkoxy", "C1-C6-alkoxycarbonyl", "halogen". The invention also relates to a pharmaceutical composition based on the formula (I) compound and particular compounds.

EFFECT: obtained are the novel derivatives of pyrasole pyridine, useful for the treatment and/or prevention of disorders or states, associated with NADPH-oxidase.

12 cl, 3 tbl, 21 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to a citrate of a compound described by formula (II) below, and a pharmaceutical composition containing said citrate.

EFFECT: experimental results of the present inventions prove that said citrate can inhibit activity of phosphodiesterase type 5 and can be used for treating erectile dysfunction, for inhibiting thrombocyte aggregation and treating thrombosis, for reducing pulmonary hypertension and treating cardiovascular diseases, asthma and diabetic gastroparesis.

2 cl

FIELD: medicine.

SUBSTANCE: group of inventions relates to field of therapy and/or prevention of diseases in mammals, in particular humans. Group of inventions includes medication for treatment and/or prevention of cardiovascular disease, and/or inflammatory disease, and/or liver disease, and/or neurological disease, and/or steatosis by increasing content of polyunsaturated fatty acids in mammal's blood, representing dairy product of ruminants with reduced cholesterol content, where cholesterol content constitutes from 10 mg/100 g of fat to 150 mg/100 g of fat, as well as application of dairy product of ruminants with reduced cholesterol content, in which cholesterol content constitutes from 10 mg/100 g of fat to 150 mg/100 g of fat, for treatment and/or prevention of cardiovascular disease, and/or inflammatory disease, and/or liver disease, and/or neurological disease, and/or steatosis by increasing content of polyunsaturated fatty acids in mammal's blood.

EFFECT: obtaining medication for treatment and/or prevention of cardiovascular disease, and/or inflammatory disease, and/or liver disease, and/or neurological disease, and/or steatosis.

18 cl, 5 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new compounds of formula I, wherein R1 and R2 are identical or different and specified in an alkyl or alkenyl hydrocarbon chain; the R3 group values split by lipase are specified in the patient claim. R4 and R5 are independently hydrogen or C1-C7alkyl; R6 represents hydrogen or C1-C7alkyl; and R7 and R8 are independently hydrogen or C1-C7alkyl. The invention also refers to using compounds of formulas ,

which are introduced into the mammalian biological system and increase the cell concentrations of specific sn-2 substituted ethanolamine-plasmalogens.

EFFECT: compounds are applicable in treating or preventing the age-related disorders associated with high membrane cholesterol, high amyloids and low plasmalogens, such as neurodegeneration, cognitive disorder, dementia, cancer, osteoporosis, bipolar disorder and vascular diseases.

11 cl, 18 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular to cardiology, and represents a method of the drug treatment of patients in late rehabilitation period after aortocoronary bypass surgery, consisting in the combined administration to the patient of medications thrombo ASS 100 mg, atorvastatin 20 mg and additionally amlodipine in a daily dose from 5 to 10 mg and losartan in a daily dose from 25 to 100 mg.

EFFECT: invention ensures the long-term preservation of results of surgical myocardium revascularisation.

3 ex

FIELD: medicine.

SUBSTANCE: minor amputation of the foot with the further necrectomy is performed. After the application of an antimicrobial bandage and drainage, the wound is hermetised from the environment by the creation of a negative pressure above the wound in a combination with drug treatment. The reatment is performed in two steps. At the first step the wound with the antimicrobial bandage and drainage is first hermetised from above with an adhesive film, with the creation and support of the negative pressure not lower than 80 mm Hg. Urokinase 500000 U is additionally introduced daily intravenously by drop infusion per 100 ml of physiological solution, Vessel-Due-F in a dose of 600 LU per 100 ml of physiological solution and VAP 20 - alprostadil in a dose of 40 mcg per 100 ml of physiological solution. In addition Antistax in capsules is introduced to the patient. At the second stage active 24-hour vacuum aspiration with the change of the negative pressure from 10 to 80 mm Hg within a day is carried out. Additionally introduced is Vessel-Due-F in a dose of 1 capsule with 250 LU 2 times per day between meals and Antistax. At the first and second stages Antistax is introduced in a dose of 2 capsules in the morning 30-40 minutes before meal, daily. Duration of each stage constitutes not less than 7 days.

EFFECT: increase of the treatment efficiency due to the complete and timely purification of the wound from pathological exudates, elimination of the progression of the purulent-necrotic process, increase of the regenerative activity of tissues, activation of local immunity, recovery of microcirculation and oxygenation of the affected tissues.

2 cl, 2 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to nanoparticles for encapsulating biologically active compounds. The nanoparticle contains a casein matrix, a basic amino acid and a metal selected from a group including a divalent metal, a trivalent metal and combinations thereof. Disclosed is a method of producing nanoparticles, which includes preparing an aqueous solution of a casein source and a basic amino acid and adding to the prepared solution an aqueous solution of a metal selected from a group including a divalent metal, a trivalent metal and combinations thereof to obtain a suspension containing formed nanoparticles. Another version of the method of producing nanoparticles includes mixing an aqueous solution of a casein source and a basic amino acid with a solution of a biologically active compound and adding to the obtained mixture an aqueous solution of a metal selected from the group to obtain a suspension containing formed nanoparticles. Nanoparticles obtained using said methods are used in combination with a carrier to prepare food products, as well as in pharmaceutics and cosmetics.

EFFECT: invention enables to obtain nanoparticles with high stability and low polydispersity.

40 cl, 17 dwg, 13 tbl, 11 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to a two-phase mouth wash liquid and a method for use thereof. The disclosed two-phase mouth wash liquid contains a hydrophilic phase, a hydrophobic phase, a hydrotrope and a preservative, wherein the preservative contains (i) sodium benzoate and (ii) potassium sorbate and/or methylisothiazolinone (MIT), and the hydrotrope component contains glycerine and/or propylene glycol. The mouth wash liquid contains (a) 0.05-0.11 wt % sodium benzoate and (b) 0.05-0.2 wt % potassium sorbate and/or 0.0005-0.01 wt % MIT. The hydrophilic phase of the mouth wash liquid can additionally contain cetylpyridinium chloride in amount of 0.01-0.1 wt %, wherein the disclosed method of improving oral health includes using an effective amount of said liquid in the mouth of a subject to reduce the level of cariogenic bacteria.

EFFECT: use of said combination of preservatives coupled with said hydrotrope provides good resistance to microbial contamination without any adverse effect on taste properties or appearance of the mouth was liquid.

12 cl, 1 ex

FIELD: medicine.

SUBSTANCE: combined phyto- and physiotherapy is conducted. The phytotherapy involves administering Prolit Super 2 capsules two times a day (at 8 and 14 o'clock) for one month; the physiotherapy provides rectal electrical stimulation by means of AndroGyn for 8 minutes a day, 15 procedures in the therapeutic course.

EFFECT: invention enables improving the sexual function and prostatic microcirculation, and can be used in ambulance situation.

1 dwg, 1 ex

FIELD: veterinary science.

SUBSTANCE: method includes the complex application of systemic antimycotic agents, surface antifungal agents, additionally the preparation Forvet is injected in a dose of 0.2 ml per 1 kg of mass subcutaneously, for 7 days.

EFFECT: increased efficiency of treatment, no side effects and reduced remission time.

2 tbl

FIELD: chemistry.

SUBSTANCE: method includes treating crushed Echinacea purpurea (L.) Moench roots and rhizome with steam, extraction with ethyl alcohol, then steeping, stirring, steeping, draining a portion of the extract which is equal to the amount of the loaded Echinacea purpurea (L.) Moench roots and rhizome, after draining a portion of the extract, adding ethyl alcohol to the treated material, draining the whole extract; extracting crushed Echinacea purpurea (L.) Moench herbs with ethyl alcohol, steeping, then stirring, steeping, draining a portion of the extract which is equal to the amount of the loaded Echinacea purpurea (L.) Moench herbs, after draining a portion of the extract, adding ethyl alcohol to the treated herbs, draining the whole extract; all obtained extracts are mixed, cooled and filtered under certain conditions. An Echinacea purpurea (L.) Moench tincture. Use of the method to obtain an Echinacea purpurea (L.) Moench tincture.

EFFECT: method preserves the biological activity of components of the tincture and medicinal properties.

3 cl

FIELD: medicine.

SUBSTANCE: burn-treating composition for local application based on Vaseline lanolin, or carbopol, or hydrogel; as an active substance, the composition contains 5% dry extract of the herbal raw material big-flowered self and self-heal containing 60% rosmarinic acid.

EFFECT: above composition is effective for treating burns; it is non-toxic.

7 tbl, 10 ex, 12 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to synthesis of N-mono fluoroalkyl tropanes with application of fluoroalkyliodides, as well as to application of such method for obtaining non-radioactive tropane intermediate compound of formula and its further conversion into 123I-labelled radiopharmaceutical substance DaTSCAN (123I-ioflupane). Method consists in alkylation in presence of base of formula compound with alkylating agent of formula F-(CH2)mX, where m equals 2, 3 or 4, X represents I. .

EFFECT: obtaining non-radioactive tropane intermediate compound of formula and its further conversion into 123I-labelled radiopharmaceutical substance DaTSCAN (123I-ioflupane).

10 cl, 1 ex

FIELD: chemistry.

SUBSTANCE: composition of preparation for teeth cleaning contains effective quantity of arginine in free form or in form of salt; abrasive substance, containing (i) natural sodium carbonate (NSC) with the average size of particles 3-7 mcm and moisture absorption 12-25 g/100 g and (ii) precipitated calcium carbonate (PCC) with the average particle size 1-5 mcm and water absorption higher than 25 g/100 g. Ratio of natural calcium carbonate to precipitated calcium carbonate constitutes from 1:2 to 1:3. Composition has pellicle cleaning ratio (PCR), at least, 70 and value of abrasivity of isotope-labelled dentin (RDA) lower than 140. Method of oral cavity care includes application of effective quantity of said composition in oral cavity of individual requiring it.

EFFECT: effective teeth cleaning and strengthening without injuring abrasive action, which makes it possible to apply it, in particular, for individuals with increased teeth sensitivity.

13 cl, 2 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: composition contains a) surface-active substances, including a salt of C10-16 alcohol ethoxylate sulphate, a betaine surface-active substance and alkylpolyglicoside, and b) a fatty C12-18 acid, constituting at least 15% of the total composition weight.

EFFECT: composition, possessing an increased viscosity and capable of producing a stable foam, is created.

10 cl, 1 tbl, 4 ex, 2 dwg

FIELD: food industry.

SUBSTANCE: task, being solved by way of the said invention, consists in development of a compound of functional drinking water containing biologically active substances and natural extracts of several medicinal herbs healthily affecting the organism in general. To obtain the compound one uses the following components in an amount of 1 dm3 of water: 0.3-0.5 g of amber acid, 0.02-0.04 g of citric acid, 0.001 - 0.003 g of silver nitrate in conversion to silver ions, 0.1-0.2 g of dry water-soluble extract of holy thistle seeds, 0.05-0.1 g of dry water-soluble extract of balm leaves and artesian water - balance.

EFFECT: obtainment of drinking water with a soft balanced taste and immune-stimulating, general tonic, calming and antioxidant action.

3 ex

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