EP2101800A1

EP2101800A1 - Extracts from the skin of fruits of plants from genus vitis, compositions containing the same and a process for its manufacture

Info

Publication number: EP2101800A1
Application number: EP07845476A
Authority: EP
Inventors: Roberto Soares De Moura; Luiz Francisco Pianowski; Artur Beltrame Ribeiro
Original assignee: Ache Laboratorios Farmaceuticos SA
Current assignee: Ache Laboratorios Farmaceuticos SA
Priority date: 2006-12-15
Filing date: 2007-12-14
Publication date: 2009-09-23
Also published as: BRPI0605693A; JP2010512345A; CA2672183A1; BRPI0605693B1; WO2008070948A1; BRPI0605693B8

Abstract

The present invention refers to an enhanced process to obtain a standardized pharmaceutical or veterinary product from the skin of fruits of plants from genus Vitis, which is useful for the treatment of symptoms, dysfunctions and other manifestations of the metabolic syndrome, as well as for prophylaxis or treatment of diseases caused by the syndrome.

Description

Extracts from the skin of fruits of plants from genus Vitis, compositions containing the same and a process for its manufacture.

FIELD OF THE INVENTION The present invention refers to an enhanced process to obtain a standardized pharmaceutical or veterinary product from the skin of fruits of plants from genus Vitis, which is useful for the treatment of symptoms, dysfunctions and other manifestations of the metabolic syndrome, as well as for prophylaxis or treatment of diseases caused by the syndrome.

BACKGROUND OF THE INVENTION Historical records show that the medicinal use of wine, a beverage produced from the fruits of the plants from genus Vitis, has been a practice for more than 2,000 years. Studies from the 1990s showed that red wine has antioxidant effect, acts over the platelet aggregation and has small vasodilatation activity. Despite all the properties of wine consumption, attempts to obtain a pharmaceutical or veterinary product have not been successful over the past few years, both due to low product efficiency and low industrial yielding.

Studies of the University of Rio de Janeiro (UERJ) have concluded that decocts obtained from skin of fruits of two plants of the genus Vitis, Vitis vinifera and Vitis labrusca, when orally given to rodents, presented hypotensive effect. However, the process of manufacture disclosed in the document BR 0106328 presents low yielding, as well as it does not provide the standardization of amount of active principle in the final product. Therefore, it is desired the development of a suitable manufacturing process that yield a standardized pharmaceutical or veterinary product for use as a medicine.

DESCRIPTION OF FIGURES For all figures as listed below, it is understood that A is the product of the present invention.

Figure 1 shows the absolute reduction of blood pressure with the product of the present invention.

Figure 2 shows the percentage reduction of blood pressure with the product of the present invention.

Figures 3 and 4 show the blood sugar profile with the product of the present invention.

Figures 5A and 5B show the increase in insulin secretion. Figures 6A to 9C show the effect of the product of the present invention over glucose metabolism in the model of genetic blood hypertension (SHR) with 400 mg/kg/day.

Figure 10 shows the effect of the product of the present invention over blood pressure in the model of hypertension induced by blocking the synthesis of nitric oxide (L-name) with 400 mg/kg/day. Figure 11 shows the effect of the product of the present invention over relative visceral fat (epididymal) in the model of hypertension induced by blocking the synthesis of nitric oxide (L-name) with 400 mg/kg/day.

Figures 12A to 13C show the effect of the product of the present invention over glucose metabolism in the model of hypertension induced by blocking the synthesis of nitric oxide (L-name) with 400 mg/kg/day.

Figure 14 shows the effect of the product of the present invention over relative visceral fat (epididymal) in the model of neuroendocrine obesity with 400 mg/kg/day. Figure 15 shows the effect of the product of the present invention over relative visceral fat (epididymal) in the model of exogenous obesity (hypercaloric coffee shop diet) with 400 mg/kg/day.

Figures 16A and 16B show blood sugar and insulin curves with 400 mg/kg/day of the product of the present invention in the model of exogenous obesity (hypercaloric coffee shop diet).

Figures 17A to 17C show the areas over the glucose and insulin curves and insulin sensitivity rate with 400 mg/kg/day of the product of the present invention in the model of exogenous obesity (hypercaloric coffee shop diet). DESCRIPTION OF THE INVENTION

In a first aspect, the present invention refers to an enhanced process to obtain a standardized pharmaceutical or veterinary product from skin of fruits of the plants from genus Vitis.

The manufacturing process of the present invention comprises the steps of:

(a) extraction in water of skin of fruits of plants from genus Vitis, keeping constant temperature of about 90 ⁰C to about 120 ⁰C, particularly about 100 ⁰C, occasionally shaking for about 100 to about 140 minutes, particularly about 120 minutes. (b) extraction in an activity column equipped with ionic exchange resin, discharging the water fraction;

(c) recovery of the active principle with a hydroalcoholic solution;

(d) concentration under vacuum and temperature between about 60 ⁰C and about 70 ⁰C; and (e) drying in a spray dryer, with inlet temperature between about

170 ⁰C and about 190 ⁰C, particularly about 180 ⁰C, and outlet temperature between about 80 ⁰C and 90 ⁰C, particularly about 85 ⁰C.

The raw material may comprise, under no limitation, skin of fruits from one or more species of the genus Vitis, particularly Vitis vinifera or Vitis labrusca.

The ionic exchange resin of the process of the present invention is used to concentrate active principles and withdrawal more polar compounds, remaining more nonpolar compounds. A hydroalcoholic solution comprises lower alcohols, particularly methanol, ethanol, propanol, butanol or mixtures thereof.

The step of recovery of the active principle particularly comprises, in sequence:

- (c1) wash the resin with ³A amount of alcohol as used in the process; - (c2) wash the resin with a 1 :1 alcohol-water solution, with the amount corresponding to remaining % alcohol as used in the process and 1/3 of the amount of water as used; and

- (c3) wash the resin with the remaining 2/3 amount of water as used.

Ratio between water and alcohol as used in the process is preferably 5:1.

Specific temperatures at the inlet and outlet of the spray dryer guarantee drying with no need to use additional adjuvants or carriers.

The process of the present invention allows industrial manufacture, since it reduces the processing time, presents suitable yielding and results in a pharmaceutical or veterinary product with standardized amount of polyphenols.

The pharmaceutical or veterinary product of the present invention contains about 0.01% to about 90% by weight, particularly 16 to 20% by weight, of at least one of the polyphenols of formula (I):

Formula (I) wherein Ri, R2, R3, R4, R5 and Re are the same or different and each one is independently selected from YX, wherein Y is a chalcogen, preferably having molecular weight of about 16 and valence between 1 and 3 and X is selected from H, CH₃, COCH₃, halogens, COOH, alkaline metals, sugars, glucosides, glucuronides or (CH₂)_nCH₃, CO(CH)_2nCH₃, wherein n varies between 0 and 16.

The pharmaceutical or veterinary product of the present invention is useful both to be directly given to a patient, and to be used to prepare pharmaceutical or veterinary compositions, under contents varying between about 1 and about 5000 mg/kg/day, particularly between about 200 and about 400 mg/kg/day, divided into one or more times per day.

In another aspect, the present invention refers to pharmaceutical or veterinary compositions containing about 1 to about 5000 mg, particularly about 200 to about 400 mg, of the pharmaceutical or veterinary product of the present invention, as well as pharmaceutically acceptable carriers, or acceptable for veterinary use.

Suitable carriers for the invention are e. g. and under no limitation the ones as mentioned by the book Remington's Pharmaceutical Sciences, from the U. S. publisher Mack Publishing, European Pharmacopaea or Brazilian Pharmacopaea. The pharmaceutical or veterinary product and the pharmaceutical or veterinary compositions of the present invention are useful for the treatment of components of metabolic syndrome, as well as for the prophylaxis and treatment of diseases caused by the syndrome.

The metabolic syndrome comprises a set of metabolic risk factors manifesting in an individual, predisposing him or her to cardiovascular diseases and atherosclerosis. Risk factors or components of the metabolic syndrome as mentioned are abdomen obesity (excess of fat in tissues around the abdomen), dyslipemia (fat dysfunctions in the blood - increase in triglycerides, reduction of HDL cholesterol, increase in LDL cholesterol), increase in blood pressure, resistance to insulin and intolerance to glucose (the organism does not use insulin and sugar from the blood in a suitable manner), increase in fastness blood sugar, hepatic steatosis, pro-thrombotic state and pro-inflammatory state. In another aspect, the present invention refers to a method for treatment of responses involved in the metabolic syndrome, as well as for prophylaxis or treatment of diseases caused by the syndrome, consisting in the administration, to a human or animal patient in need of said treatment, of a daily amount of about 1 to about 5000 mg/kg, particularly about 200 to about 400 mg/kg, of the product of the present invention or a pharmaceutical or veterinary composition containing it.

Merely illustrative examples of specific embodiments of the present invention are presented below, not creating any limitations to its scope aside from those contained in the attached claims. MANUFACTURING PROCESS

EXAMPLE 1A

200 kg of skin of fruits from Vitis vinifera have been added to water at the temperature of 100 ⁰C and, under occasional shaking, left in said condition for 120 minutes. After solid residues were exhausted, the material was passed through an ionic (cationic) exchange resin, discharging the water fraction.

The resin was sequentially washed with 300 liters of ethanol, 200 liters of an ethanol-water solution (1:1) and 200 liters of water.

The product as obtained was concentrated under vacuum at 60 ⁰C and subsequently dried in a spray dryer, not adding adjuvants or carriers, by keeping the inlet temperature at 180 ⁰C and the outlet temperature at 85 ⁰C.

The resulting product is a fine and water soluble powder, with 4% humidity (2 g/105 °C/2 h). Furthermore, by the method of Folin-Ciocalteau, it was verified that the final product as obtained contains 18% polyphenols of formula (I) and pertinent profile by thin layer chromatography (TLC).

EXAMPLE 1 B

In a tub with an extractor provided with mechanical shaking, 200 kg of skin of fruits from Vitis vinifera dried in warming chambers with controlled temperature at 60 ⁰C were added and crushed in an electrical mill.

Subsequently, 280 liters of water at the temperature of 100 ⁰C were added under frequent shaking, for the period of 72 to 144 hours (two to four days). After that process, the product was filtered under vacuum through

100 μm filters. The obtained yielding was approximately 240 liters of product solution of the present invention.

After solid residues were exhausted, the obtained solution was passed through an ionic (cationic) exchange resin, discharging the water fraction.

The product as obtained was concentrated under vacuum at 60⁰C and subsequently dried in a spray dryer, without adjuvants or carriers, by keeping the inlet temperature at 180 ⁰C and the outlet temperature at 85 ⁰C.

The resulting product is a fine and water soluble powder, with 4% humidity (2 g/105 °C/2 h). Furthermore, by the method of Folin-Ciocalteau, we verified that the final product as obtained contains 30% polyphenols of formula (I) and pertinent profile by high pressure liquid chromatography (HPLC). ACUTE TOXICITY f DLsn) OF THE PRODUCT

Specific pathogen free mice (SPF) were used to carry out this test. Animals were orally treated with the pharmaceutical or veterinary product of Example 1 under dosages varying between 0 and 5000 mg/kg. Control groups received carrier (0.9% NaCI, 10 mi/kg, v. o.).

Through the experiment, the possible influence of acute administration of the pharmaceutical or veterinary product over water and food consumption and gain of weight was researched, as well as the effects over some vital organs.

Data of Table 1 shows that the pharmaceutical or veterinary product as acutely given orally was well tolerated and did not cause significant toxic effects which could lead to death. DL₅0 was not obtained until the dosage of 5000 mg/kg.

Body weight of animals and their water and food consumption were not significantly changed, and also no macroscopic change was noticed over color and size of some vital organs.

TABLE 1

CHRONIC TOXICOLOGIC ASSAY

Chronic treatment of mice with the product of the present invention (100 to 1000 mg/kg), daily given for 90 consecutive days orally, caused reduction in plasma levels of triglycerides and total cholesterol in comparison with control animals, as shown by the table below:

TABLE 2

HYPOTENSIVE EFFECT OF THE PRODUCT

For this experiment, hypertensive adult male rats from the line Spontaneously Hipertensive Rats (SHR) were used. Animals received a cannula over their left femoral artery with a PE-10 catheter connected to another PE-50 catheter. The PE-10 end advanced through the femoral artery until the abdomen aorta and the PE-50 end was used for connection to Statham pressure transductors coupled to a Gould polygraph for continued record of the blood pressure.

The pharmaceutical or veterinary product obtained as per Example 1 was diluted in distilled water and the volume of solution as orally given varied between 0.8 and 1.3 ml.

As it can be seen from the graphs as presented by Figures 1 and 2, the pharmaceutical or veterinary product, given orally under the dosage of 300 mg/kg, caused reduction in blood pressure of SHR rats, which efficiency was three times higher than placebo.

Such acute hypotensive effect is similar to the one obtained with amlodipine (0.5 mg/kg) or enalapril (1 mg/kg), traditionally used products for the treatment of hypertension. EFFECTS IN DIABETES INDUCED BY ALOXANE Two groups of twelve animals, adult male mice weighing 25 and 30 g, were used for this study (control group and experimental group). Initially, animals were analyzed for blood sugar after twelve hours of fastness. Once blood sugar values were obtained, animals received food and water at will.

On day zero, after obtaining basal blood sugar, animals were treated with aloxane, diluted in saline, under the initial dosage of 300 mg/kg and later two dosages of 150 mg/kg applied on successive days.

In the first protocol, the control group was solely treated with aloxane along 25 days. The experimental group was treated with aloxane, but on day zero, after measuring sugar blood, animals received 100 mg/kg/day of the product of Example 1 with drinking water and the dosage was later increased to 200 mg/kg/day. Blood sugar was monitored for 25 days.

In the second protocol, animals were treated with aloxane as disclosed above. One of the groups received only aloxane for ten days and, from that date, was treated with the product of Example 1 under the dosage of

200 mg/kg/day in drinking water. The other group was treated with the product of Example 1 under the dosage of 200 mg/kg/day in drinking water until day 10, when the treatment was halted. Sugar blood was measured until day 13. Figure 3 shows the evolution of blood sugar from two groups of mice, control, only treated with aloxane, and experimental, treated with aloxane and the product of Example 1. As we can see, the group treated only with aloxane became diabetic, with blood sugar reaching values above 400 mg/dl.

However, the group treated with aloxane and the product of Example 1 showed significantly lower blood sugar levels than the group treated only with aloxane.

Figure 4 shows that, after blood sugar increased with the treatment with aloxane for ten days (control group), treatment of animals with the product of Example 1 orally under the dosage of 200 mg/kg/day produced significant reduction in glucose levels. On the other hand, when the treatment with the product of Example 1 was halted, blood sugar levels increased. EFFECT TO THE SECRETION OF INSULIN BY GLUCOSE IN ISLETS OF LANGERHANS IN

VITRO To isolate islets, five adult male Wistar rats were sacrificed by tiopental administration (60 mg/kg of weight) intraperitoneal^ and laparatomized to locate the biliar duct. Said duct was occluded in its terminal portion (near duodenus). Later, it was dissected near the hepatic pediculum, where a polyvinyl catheter (Bio vida) was introduced towards the pancreas. Through that catheter, 10-20 ml of Krebs solution was injected with the purpose to inflate pancreas, thus allowing better visualization and removal. Lymph nodes and fats present were taken off.

The removed pancreas was chopped (reduced to small fragments) and washed various times with Krebs solution to separate pancreas tissue from the remaining adipose tissue, which was eliminated after centrifugation. Krebs solutions was bubbled with carbogenic gaseous mixture (to keep pH 7.4) and in ice after the end of its use.

Islets were isolated by the collagenase technique (Lacy & Kostianovsky, 1967). Pancreas fragments were put in a graduated test tube, adding for each one 1 ml of pancreas, 4 mg of collagenase (Collagenase P Boehringer Mannheim Biochemicals) diluted in 500 μl of Krebs solution. Test tubes with pancreas and collagenase were put under the carbogenic mixture and bath at 37 ⁰C for eight minutes. After aeration, the tube was well closed to be slowly shaken for four minutes and vigorously for one minute, still in bath at 37 ⁰C. After shaking, pancreas was washed with Krebs solution and 1% albumin (bovine serum albumin - SIGMA®) in a test tube and centrifuged at 500 rpm/min for 30 seconds, disposing supernatant. Part of the precipitated material was separated and, with the help of a magnifying glass and silicone pipette, islets were observed and collected. Approximately 120 islets were obtained from each animal.

After collected, the islets were packed in a perfusion chamber with flow kept at 1 ml/min in a peristaltic pump (Bomba Buchler Polystaltic®) at the temperature of 37 ⁰C. To perfuse the islets, Krebs solution was used with different glucose concentrations (2.8 and 16.7 mM for the control group; 2.8 and 16.7 + product of Example 1 (50 μg/ml) for the experimental group). Initially, Krebs solution was used with 2.8 mM of glucose (keeping 20 minute rest) and soon afterwards nine perfusates were collected with a five-minute interval (time: -40, -35, -30, -25, -20, -15, -10, -5, 0) to obtain basal values for insulin secretion. By exchanging effluent by unidirectional valve, glucose concentration for Krebs solution was changed only with 16.7 mM glucose for the control group or with 16.7 mM glucose + 50 μg/ml of the product of Example 1 for the experimental group, and the perfusate was collected each two minutes during thirty minutes. Perfusates were frozen at -70 ⁰C for subsequent insulin dosage.

Insulin (as secreted by islets in vitro) contained in the perfusates was analysed in duplicates by the radioimmunoassay method, by using kit Insulin 125 I RIA (MP Biomedicals, LLC - Orangeburg NY 10962).

Figures 5A and 5B show that the increase in insulin secretion as induced by increasing glucose concentration in the perfusing liquid of Langerhans islets in vitro was not neither reduced nor increased by treating animals with the product of Example 1 within the period of up to 25 minutes of perfusion. However, insulin secretion as observed thirty minutes after the start of perfusion with high glucose and the product of Example 1 (55.5 ± 0.89 ng/ml) was significantly higher than the secretion as observed when the islets were perfused with high glucose and without the product of Example 1 (49.7 ± 1.02 ng/ml) (Figure 5A). The analysis of the area under the curve of insulin values, as secreted along thirty minutes, also showed that the product of Example 1 increased insulin secretion (Figure 5B). STUDIES OF METABOLIC EFFECTS AND OVER ATHEROSCLEROSIS AS OBTAINED BY

CHRONIC ADMINISTRATION Experimental Models A) Blood Hypertension Models

1 - Genetics: Adult male SRH rats were used (12 to 14 weeks old). The animals were divided into three groups: a) Group 1 (control): treated for twelve consecutive weeks with carrier, n = 10 for blood pressure measurement and n = 7 for metabolism. b) Group 2: treated with 400 mg/kg/day of product of Example 1, for twelve consecutive weeks, added to drinking water (n = 9). c) Group 3: treated for twelve consecutive weeks with 400 mg/kg/day of product of Example 1 in four daily takings (n = 6).

2 - Induced (L-Name Model): Adult male Wistar rats were used (12 to 14 weeks old). Blood hypertension was induced in all animals, by administration of the nitric oxide synthesis inhibitor L-NAME, at the dosage of 50 mg/kg/day, added to drinking water, for eight consecutive weeks. The animals were divided into two groups: a) Group 1 (control): treated for eight consecutive weeks with vessel (n = 5). b) Group 2: treated for eight consecutive weeks with 400 mg/kg/day of product of Example 1 added to drinking water (n = 5). B) Obesity Models

1 - Neuroendocrine model - Monosodium Glutamate - MSG

This obesity model consists in the induction of chemical hypothalamic injury (satiety centers) by the subcutaneous administration of monosodium glutamate in the neonatal period. Such model is followed by hyperchortisolism, presents increase in visceral fat content, but low increase in body weight. 00350

14

To induce said model, we used male rats from the Wistar line, which received within the first eleven days of life daily subcutaneous injections of monosodium glutamate under the dosage of 4 g/kg.

After inducing hypothalamic injury, animals were fed with standard rat food until reaching adult stage (twelve weeks old), when they were divided into two groups: a) Control: treated for twelve consecutive weeks with vessel (n = 8). b) Group 1 : treated for twelve consecutive weeks with 400 mg/kg/day of the product of Example 1 added to drinking water (n = 9). 2- Exogenous Models - Coffee Shop Diet

This model consists in giving the animals a hypercaloric diet. Therefore, male rats from the Wistar line, after weaning, started to receive coffe shop diet (hypercaloric and highly palatable) at will until the adult stage (twelve weeks). The animals were divided into two groups: a) Control: animals kept in coffee shop diet and treated with carrier for twelve consecutive weeks (n = 9). b) Group 1: animals kept in coffee shop diet and treated with 400 mg/kg/day of the product of Example 1 at drinking water for twelve consecutive weeks (n = 9).

All the animals in experimental and control groups had, during the whole period of treatment (usually twelve weeks, except for L-NAME groups which were followed by eight weeks), determining two times per week the blood pressure of the tail by sphygmoelectromanometrics and the body weight.

Product of Example 1 was given in all experiments under the dosage of 400 mg/kg/day diluted in drinking water, except for a subgroup of SHR animals to which the product was given orally in four daily takings.

At the end of the treatment period, the animals received a cannula at their left femur artery with a PE-10 catheter connected to another PE-50 catheter to collect blood, aiming to determine levels of sugar blood and insulin levels during the Oral Glucose Tolerance Assay (TOTG).

Twenty-four hours after surgical procedures to implant the catheter, the first blood collection was made to determine basal glucose and insulin and lipid levels in plasma (total cholesterol, HDL-cholesterol, triglycerides). Glucose overcharge (170 g/kg) was orally given and new blood collections were made to determine blood sugar and insulin levels after 15, 30,

60, 90 and 120 minutes from the overcharge.

Blood sugar levels were determined in a digital glucose meter and insulin by radioimmunoassay methods. At the end of the oral glucose tolerance test, animals were sacrificed and the fat around epididymis was dissected and weighed.

Subsequently, the weight of said fat, representing visceral fat, was corrected for given 100 g of body weight, being presented as the relative content of epididymal fat. Results

A) Blood Hypertension Models

1 - Group 1 : as in this group, the treatment with the product of Example 1 at the dosage of 400 mg/kg/day was made in two different ways, i. e. with drinking water and orally in four takings, metabolic results will be separately presented for each subgroup according to the mode of administration of the product as also as a sum of said subgroups. Other results (blood pressure, body weight and epididymal fat) will be presented just as a sum of the subgroups.

Figures 6A to 7C present blood sugar and insulin curves, the areas of said curves and insulin sensitivity rates as obtained for the control and treated groups.

We can observe that control SHR rats, from blood sugar levels of normal fastness, present a diabetic blood sugar curve to TOTG, reaching values which are much higher than 200 mg/dl. On the other hand, treatment with the product of Example 1 , no matter which was the mode of administration, provided significant reduction of blood sugar during fastness and an important improvement of the blood sugar profile, which values during TOTG were located within a glucose intolerance level. Concerning the insulin curve, we can observe that the control group presented normal levels of insulin during fastness, but they did not increase during glucose overcharge, thus suggesting deficiency in insulin release by the pancreas, which even justifies the diabetic blood sugar curve. On the other hand, animals treated with the product of Example 1, no matter which was the mode of administration of said product, started to present higher insulin levels as a response to glucose overcharge, thus suggesting that the treatment with the product of Example 1 restored at least in part the pancreas response to glucose stimulation, which was translated by significant improvement in the blood sugar curve, which passed from diabetic to intolerant level. These effects are also clearly shown when areas under the glucose and insulin curves are evaluated. Therefore, we can observe that the product of Example 1 , no matter which is the mode of administration, caused reductions in the area of glucose curve around 42% that observed in the control group, but was followed by an over 50% increase in the area of the insulin curve. As a consequence, we noticed about 24% increases in the insulin sensitivity rate with the two modes of chronic administration of the product of Example 1.

Figures 8A to 9C present the effects of treatment with the product of Example 1 over the metabolism of glucose of SHR glucose, considering the sum of both modes of administration of said product in comparison to the control group receiving just the carrier.

As we can observe, the sum of the results of the groups of SHR animals with different modes of product administration of Example 1 show that the increase in the insulin curve area is now significant over the control group. We can therefore see that the treatment of SHR rats with the product of Example 1 reduced the glucose curve area in 42.8%, increased the insulin curve area in 52.8% and increased the insulin sensitivity rate in 24.1 %. Figure 10 presents blood pressure values for the tail in the basal period and during eight weeks of treatment with the product of Example 1.

We can observe that, in this model, the product of the present invention partially prevented blood hypertension as induced by blocking the synthesis of nitric oxide with L-NAME. We therefore observe significantly lower pressure values between the third and the sixth weeks of treatment, and tail pressure values in the seventh and eighth weeks of the treated group; although numerically lower than the control group, do not present statistically significant differences between the groups. Therefore, we detect significant chronic hypotensive effect in the product of Example 1. Figure 11 presents relative average values for visceral fat

(epididymal) in L-NAME and control groups and treated with the product of Example 1.

As we can notice, the content of visceral fat in this model of blood hypertension was increasing and the chronic administration of the product of the present invention promoted significant reduction (-15.7%) of the relative content of fat in epididymis.

Figures 12 and 13 present the standards of glucose metabolism in this experimental model of induced blood hypertension and the effects of treatment with the product of Example 1. As we can observe in Figures 12A and 12B both groups of animals from the L-NAME model presented fastness blood levels within normal, and the chronically treated group with the product of the present invention was slightly lower than the control group. Glucose overcharge induced significant and progressive increases in blood sugar in the L-NAME group which surpassed the 200 mg/dl level and is therefore classified as diabetic. Treatment with the product of Example 1 significantly reduced the blood sugar curve. Concerning insulin curve as presented on the right panel of that same graph, we can observe that the insulin levels in fastness were within normal levels, but slightly higher in the group treated with the product of Example 1. During TOTG, we noticed in both groups significant and progressive increases, similar to insulin in response to glucose overcharge.

In Figures 13A to 13C, average results of areas under the glucose and insulin curve during TOTG and the insulin sensitivity rate are calculated.

We can therefore see that the chronic treatment of L-NAME animals with the product of Example 1 reduced the insulin curve area in 24.4%, not significantly modifying the insulin curve area. As a consequence, we noticed considerable increase in the insulin sensitivity rate, which more than doubled over the control L-NAME group (+ 101.8%). Therefore, for one single insulin release, the glucose rate in the blood was reduced during TOTG, and these results mean a real improvement in the peripheral collection of glucose (increase in insulin sensitivity).

In blood hypertension models presenting change in glucose metabolism (resistance against insulin and intolerance to glucose), the chronic administration of the product of the present invention seems to exert effects both for the peripheral collection of glucose, increasing the sensitivity to insulin (L-NAME model), and in insulin release in models presenting unsuitable release of said hormone by the pancreas in case of glucose overcharge (SHR model). B) Obesity Models

1- Neuroendocrine Model

Figure 14 presents the relative content of visceral fat (epididymis) in studied groups. We can notice that the relative content of epididymal fat of animals with visceral obesity was very high (about three times the normal content), characterizing visceral obesity. Treatment for twelve weeks with the product of Example 1 significantly reduced the deposit of visceral fat in that model, with reduction of about 7% in the relative content of fat as present in animal epididymis and neuroendocrine obesity induced by monosodium glutamate. 2 - Exogenous Models - Coffee Shop Diet

Figure 15 presents the relative content of visceral fat of both groups of obese animals by coffee shop diet. It can be noticed that animals with obesity induced by coffee shop diet present, besides general obesity, an important increase in the relative content of visceral fat as represented by the fat as found around the epididymis. Chronic administration (twelve weeks) of the product of Example 1 significantly reduced (-17.7%) the relative content of epididymal fat in animals fed with coffee shop diet.

Figures 16A to 17C present standards for glucose metabolism of both groups of animals with obesity induced by the hyper caloric coffee shop diet.

We observe that animals with obesity as induced by coffee shop diet, despite presenting fastness blood sugar levels still within normal range, show poorly diabetic response when submitted to glucose overcharge with blood sugar levels well above 200 mg/dl. When these animals were chronically treated with the product of Example 1 , we observe reduction in blood sugar levels in fastness and considerable improvement in the blood sugar profile during TOTG, which presented a slightly intolerant curve, but near normality. Parallel to said blood sugar profile, we observe that the treated animals presented usual fastness insulin levels, although slightly higher than the control group, but very lower pancreas response to glucose overcharge, showing insulin levels during overcharge which were significantly lower than observed in the control coffee shop group. Therefore, the product of Example 1 facilitated the collection of glucose by peripheral tissues of said animals, providing reduction in blood sugar values when lower levels of plasma insulin were available.

Thus, when we calculate the areas under the glucose and insulin curves and also the sensitivity rate to insulin, we observe that chronically treated animals with the product of Example 1 showed significant reductions both of the area of glucose curve (-30.8%) and the insulin curve (-28.4%) in comparison to the animals of the control coffee shop group. Calculation of sensitivity rate of peripheral tissues to the action of insulin allowed us to observe the important increase in sensitivity of peripheral tissues to the action of insulin. Therefore, the sensitivity rate to insulin in the group of obese animals treated with the product of Example 1 was almost double (93.4% more) that obtained in the control coffee shop group.

Thus, in both studied obesity models, which main features are large resistance to insulin and increase in the deposition of visceral fat, the chronic administration of the product of the present invention brought important benefits with significant improvement in the sensitivity of peripheral tissues to insulin, resulting in higher tissue collection of glucose and reduction of the deposition of visceral fat which, as we know, is considered an important factor in cardiovascular risk.

Table 3 summarizes the effects of the product of the present invention over glucose metabolism in these four experimental models presenting resistance to insulin, in comparison to the corresponding control groups.

TABLE 3

CHRONIC EFFECT OF THE PRODUCT OF THE PRESENT INVENTION METABOLIC STANDARDS: PERCENTUAL VARIATION OVER CONTROL GROUPS

We can therefore observe:

1 - Fastness blood sugar: 11.5% to 19.4% lower than the control groups;

2 - Area over the glucose curve in TOTG: unchanged in one model (MSG) and 24.4% to 42.9% lower than control in the other three models;

3 - Area over the insulin curve in TOTG: increased (SHR) or unchanged (L- NAME) in hypertension models and 25.4% to 28.4% lower in obesity models than corresponding controls;

4 - Insulin sensitivity rates: 24.1% to 101.8% higher than detected for corresponding controls;

5 - Visceral Fat: unchanged (SHR) and 7.0% to 17.7% lower than the control groups;

6 - Weight increment: similar (L-NAME) and 3.2% to 3.6% lower in other groups than corresponding controls.

Claims

1. PROCESS TO MANUFACTURE A STANDARDIZED PHARMACEUTICAL OR VETERINARY PRODUCT FROM THE SKIN OF FRUITS OF PLANTS FROM GENUS VITIS characterized by comprising the steps of:

(a) water extraction of skin of fruits of plants of the genus Vitis, keeping constant temperature between about 90 ⁰C and about 120 ⁰C, under occasional shaking, for about 100 to about 140 minutes;

(b) extraction in an activity column equipped with ionic exchange resin, subsequently discharging the water fraction;

(c) recovery of the active principle with a hydroalcoholic solution;

(d) concentration under vacuum and temperature between about 60 ⁰C and about 70 ⁰C; and

(e) drying in a spray dryer, with inlet temperature between about 170 ⁰C and about 190 ⁰C and outlet temperature between about 80 ⁰C and about 90 ⁰C.

2. PROCESS according to claim 1 characterized by the fact that plants are Vitis vinifera and/or Vitis labrusca.

3. PROCESS according to claim 1 , characterized by the fact that the temperature of step (a) is kept around 100 ⁰C and the time is about 120 minutes.

4. PROCESS according to claim 1 , characterized by the fact that the ionic exchange resin is a cationic resin.

5. PROCESS according to claim 1 , characterized by the fact that the hydro alcoholic solution comprises one or more lower alcohols.

6. PROCESS according to claim 5, characterized by the fact that lower alcohols are selected from methanol, ethanol, propanol, butanol or mixtures thereof.

7. PROCESS according to claim 1 , characterized by the fact that the step of recovery of active (c) sequentially comprises washing the resin with (c1) ³A of the amount of alcohol as used in the process, (c2) washing the resin with a 1:1 hydro alcoholic solution comprising the remaining % of the amount of alcohol as used in the process and 1/3 of the amount of used water and (c3) washing the resin with the remaining 2/3 of the amount of water.

8. PROCESS according to claim 1, characterized by the fact that the inlet temperature in the spray dryer is about 180 ⁰C and the outlet temperature is about 85 ⁰C.

9. PROCESS of any of claims 1 or 8, characterized by the fact that the ratio from water to alcohol as used in the process is 5: 1.

10. STANDARDIZED PHARMACEUTI CAL OR VETERINARY PRODUCT obtained according to one of claims 1 to 9, characterized by the fact that it comprises from about 0.01 to about 90% of polyphenols useful for the treatment of metabolic syndrome components.

11. STANDARDIZED PHARMACEUTICAL OR VETERINARY

PRODUCT obtained according to claim 10, characterized by the fact that it comprises from about 16 to about 20% of polyphenols useful for the treatment of metabolic syndrome components.

12. PHARMACEUTICAL OR VETERINARY PRODUCT obtained according to one of claims 10 and 11, characterized by the fact that it comprises at least one of the polyphenols of formula (I):

Formula (I)

Wherein Ri, R₂, R₃, R₄, R₅ and R₆ are the same or different and each one is independently selected from YX, and Y is a chalcogen, preferably having 00350

24

molecular weight of about 16 and valence between 1 and 3 and X is selected from H, CH₃, COCH₃, halogens, COOH, alkaline metals, sugars, glucosides, glucuronides or (CHa)_nCH₃, CO(CH)_2nCHs, and n varies between 0 and 16.

13. STANDARDIZED PHARMACEUTICAL OR VETERINARY PRODUCT according to claim 12, characterized by the fact that it comprises from about 0.01 to about 90% of at least one of the polyphenols of formula (I).

14. STANDARDIZED PHARMACEUTICAL OR VETERINARY PRODUCT according to claim 13, characterized by the fact that it comprises from about 16 to about 20% of at least one of the polyphenols of formula (I).

15. STANDARDIZED PHARMACEUTICAL OR VETERINARY

PRODUCT characterized by the fact that it comprises at least one of the polyphenols of formula (I):

Formula (I) wherein Ri, R₂, R₃, R₄, R5 and R₆ are the same or different and each one is independently selected from YX, and Y is a chalcogen, preferably having molecular weight of about 16 and valence between 1 and 3 and X is selected from H, CH₃, COCH₃, halogens, COOH, alkaline metals, sugars, glucosides, glucuronides or (CH₂)_nCH₃, CO(CH)_2nCH₃, and n varies between 0 and 16.

16. STANDARDIZED PHARMACEUTICAL OR VETERINARY

PRODUCT according to claim 15, characterized by the fact that it comprises from about 0.01 to about 90% of at least one of the polyphenols of formula (I).

17. STANDARDIZED PHARMACEUTICAL OR VETERINARY PRODUCT according to claim 16, characterized by the fact that it comprises from 16 to 20% of at least one of the polyphenols of formula (I).

18. USE OF STANDARDIZED PHARMACEUTICAL OR VETERINARY PRODUCT obtained according to one of claims 10 to 17, characterized by the fact that it is intended for the preparation of a medicine useful for the treatment of components of metabolic syndrome, as well as the prophylaxis or treatment of diseases caused by the syndrome.

19. USE according to claim 18, characterized by the fact that said components of the metabolic syndrome include one or more from abdomen obesity, dyslipemia, hypertension, resistance to insulin and intolerance to glucose, blood sugar levels in fastness, hepatic steatosis, pro-thrombotic state and pro-inflammatory state.

20. USE according to claim 19, characterized by the fact that diseases caused by metabolic syndrome include one or more from cardiovascular diseases, diabetes and atherosclerosis.

21. PHARMACEUTICAL OR VETERINARY COMPOSITION, characterized by the fact that it comprises from about 1 to about 5000 mg of a standardized pharmaceutical or veterinary product comprising at least one of the polyphenols of formula (I):

Formula (I) wherein R₁, R₂, R₃, R₄, R₅ and R₆ are the same or different and each one is independently selected from YX, and Y is a chalcogen, preferably having molecular weight of about 16 and valence between 1 and 3 and X is selected from H, CH₃, COCH_3, halogens, COOH, alkaline metals, sugars, glucosides, glucuronides or (CH₂)nCH₃, CO(CH)_2nCH₃, and n varies between 0 and 16.

22. PHARMACEUTICAL OR VETERINARY COMPOSITION according to claim 21 , characterized by the fact that it comprises from about 200 mg to about 400 mg of at least one of the polyphenols of formula (I) and pharmaceutically acceptable carriers or acceptable for veterinary use.

23. USE OF PHARMACEUTICAL OR VETERINARY PRODUCT according to one of claims 21 and 22, characterized by the fact that it is intended for the preparation of a medicine useful for the treatment of components of metabolic syndrome or the prophylaxis and/or treatment of diseases caused by the syndrome.

24. USE according to claim 23, characterized by the fact that components of the metabolic syndrome include one or more from abdomen obesity, dyslipemia, hypertension, resistance to insulin and intolerance to glucose, blood sugar levels in fastness, hepatic steatosis, pro-thrombotic state and pro-inflammatory state.

25. USE according to claim 24, characterized by the fact that diseases caused by metabolic syndrome include one or more from cardiovascular diseases, diabetes and atherosclerosis.

26. METHOD OF TREATMENT OF COMPONENTS OF THE METABOLIC SYNDROME OR PROPHYLAXIS OR TREATMENT OF

DISEASES CAUSED BY THE SYNDROME, characterized by the fact that it comprises the administration to a patient in need of a daily amount of from about 1 to about 5000 mg of the pharmaceutical or veterinary product according to claims 10 to 17 or pharmaceutical or veterinary compositions containing it.

27. METHOD according to claim 23, characterized by the fact that it comprises the administration of from about 200 to about 400 mg of the standardized pharmaceutical or veterinary product or pharmaceutical or veterinary compositions containing it.

28. METHOD according to one of claims 26 or 27, characterized by the fact that the components of the metabolic syndrome include one or more from abdomen obesity, dyslipemia, hypertension, resistance to insulin and intolerance to glucose, blood sugar levels in fastness, hepatic steatosis, pro-thrombotic state and pro-inflammatory state.

29. METHOD according to one of claims 26 or 27, characterized by the fact that diseases caused by metabolic syndrome include one or more from cardiovascular diseases, diabetes and atherosclerosis.