EP1841779A2 - Use of cacao polyphenols for treating a prostate hyperplasia, a specific cacao extract and applications - Google Patents

Abstract

The invention relates to the use of cacao polyphenols for preventing or curing a prostate hyperplasia, to a cacao polyphenol extract which also comprises, in particular lipids and/or xanthines, and to the applications of said extract in food and pharmaceutical products for treating a prostate cancer, cognitive disorders, oxidative stress and cholesterol.

Description

 "Use of Cocoa Polyphenols for the Treatment of Prostatic Hyperplasia, Specific Cocoa Extract and Applications"

The present invention relates to the use of cocoa polyphenols for the treatment of prostate hyperplasia, to a specific cocoa extract and to the applications of said extract in the food and pharmaceutical fields. More specifically, it relates to the use of cocoa polyphenols for the preventive or curative treatment of prostate hyperplasia, to a polyphenol extract of cocoa also comprising, in particular, lipids and / or xanthines, the use of which is particularly effective against prostate cancer, against cognitive disorders, in particular by modulating the level of expression of dopamine, against oxidative stress and cholesterol.

Prostatic hyperplasia is a condition characterized by an increase in the volume of the prostate. This increase in volume may result from a simple benign prostatic hyperplasia or may correspond to a cancer of the prostate.

Benign prostatic hyperplasia (BPH), also known as prostate adenoma or benign prostatic hypertrophy, is characterized by hyperplasia of stromal cells and prostatic epithelium.

BPH affects about 50% of men over 50 years of age. In some cases, it can lead to functional disorders of the lower urinary tract, in particular, voiding disorders (obstruction, irritation). The treatments available today are herbal medicine, the administration of alpha-blockers or 5 alpha reductase inhibitors and surgical treatment. We consider Today, a 40 - year - old man has about 37% chance of getting BPH when he reaches 80 years of age.

The work of the inventors has made it possible to demonstrate that the administration of a composition based on cocoa polyphenols makes it possible to treat prostate hyperplasia in general and more particularly BPH.

According to a first aspect, the invention therefore relates to the use of cocoa polyphenols for the preparation of a medicament for the treatment of prostatic hyperplasia. The term "prostate hyperplasia" refers to benign prostatic hyperplasia and prostate hyperplasia resulting from prostate cancer.

The treatments according to the invention may be intended to relieve the symptoms of hyperplasia, or to be preventive or curative. ^'Examples 1 and 2 demonstrate the activity of a cocoa polyphenol extract in a preventive and curative treatment of hyperplasia chemically induced in rats.

The invention also provides methods of therapeutic treatment comprising administering to a patient a cocoa polyphenol composition in association with pharmaceutically acceptable excipients to prevent / alleviate symptoms / cure prostatic hyperplasia. it is benign or it results from a prostate cancer already diagnosed. Advantageously, the cocoa polyphenols introduced into the composition come from a polyphenol extract of cocoa.

By "polyphenol extract of cocoa" is meant an extract obtained by extraction of cocoa beans or products derived from cocoa beans, comprising cocoa polyphenols.

The resulting drug can be administered alone or in combination with other drugs such as pelvic decongestants, alpha-blockers and / or inhibitors of 5-alpha reductase. The mode of administration can be systemic or topical, preferably orally, parenterally (intravenously or subcutaneously), rectally or topically and be for example in the form of single or coated tablets, capsules, creams, ointments. , suppositories injectible solutions or oral suspensions. The drug can finally be prescribed in addition to surgical treatment.

The dosage is adaptable according to the nature and severity of the condition, the desired therapeutic purpose, the route of administration and the sex and age of the patient.

For example, the drug may be administered at a dose of 15 to 30 mg / kg / day orally, for the purpose of preventive treatment and at a dose of 40 to 60 mg / kg / day. orally, when the treatment is for curative purposes. These doses may advantageously be optionally administered in several doses.

Advantageously, the polyphenol extract of cocoa used in the drug will have the following composition: 20 to 50% by mass in epicatechin equivalent of polyphenols, of which 55% to 80% of flavonoids,

3 to 15% by weight of lipids, of which 1 to 15% of β-sitosterol,

5 to 20% by weight of xanthines.

The extract according to the invention can be used raw or purified.

According to a second aspect of the invention, it also relates to said cocoa polyphenol extract described above. Indeed, this extract is particularly effective in the treatment of other pathologies than hyperplasia. The measurement of the polyphenol content was carried out according to the Folin-Ciocalteu method.

The cocoa extract according to the invention contains a high level of polyphenols, especially polyphenols having a high antioxidant activity such as flavonoids, more especially, flavonoids such as catechin, epicatechin and their derivatives. Advantageously, the extract according to the invention comprises from 10 to 30% of epicatechin. In addition, the extract according to the invention is particularly rich in oligomeric polyphenols composed of 1 to 6 monomers.

Xanthines are mainly caffeine and theobromine.

Examples of extract are given in Example 3, with reference to Figures 12 and 13. Such extracts can be obtained from beans of different origins, preferably beans originating from Cameroon, Tanzania or Côte d'Ivoire. Ivory.

Advantageously, the cocoa extract is obtained from unfermented beans, dried, and then re-wetted. These are then cleaned, peeled to obtain green grain. Extraction in ethanol-water (70:30) is then carried out for about 24 hours with stirring, with a cocoa / solvent ratio of the order of 1/2 to 1/4. The obtained product is then filtered, rinsed, concentrated and evaporated to dryness. It is imperative that no degreasing step be carried out throughout the process.

The extract according to the invention therefore contains both β-sitosterol, xanthines and polyphenols. The presence of β-sitosterol makes it possible to improve the uptake of polyphenols by the body. Moreover, the work of the inventors has made it possible to demonstrate that, contrary to what was suggested in the prior art, the presence of xanthines with the polyphenols did not adversely affect the polyphenolic activity and, on the contrary, provided advantageous effects in some applications, in particular, cognitive applications.

In a third aspect, polyphenol cocoa extract is used to treat prostate cancer. Prostate cancer, an age - related malignancy, is the second most common cause of cancer death among men in the United States and Western Europe. Although the etiology of this cancer is unknown, several risk factors including age, race, environment and diet have been identified. Other risk factors include polymorphic repeats of the androgen receptor gene and high concentrations of circulating androgens. The importance of testosterone in the development of prostate cancer is underscored by the discovery that prostate cancer rarely occurs in castrated males or in males with 5α-reductase deficiency, the enzyme responsible for converting testosterone in its active metabolite, dihydrotestosterone. Several studies have identified various food substances with antioxidant properties and an inhibitory effect on the development and / or progression of prostate cancer. Of these substances, polyphenols are the most abundant natural antioxidants found in fruits, beverages (fruit, wine, tea, coffee, chocolate and beer) and to a lesser extent in vegetables, pulses and cereals. They play a role in various diseases associated with oxidative stress such as cancer, cardiovascular disease and inflammation. Polyphenols are reducing agents, and in combination with other dietary reducing agents, such as vitamin C, vitamin E and carotenoids, they protect body tissues against oxidative stress. The large diversity of their structures makes them different from other antioxidants and affects their biological properties: bioavailability, antioxidant activity, specific interactions with cell receptors and enzymes ... Polyphenols are divided into subgroups such as isoflavones, flavonoids and lignans. It has recently been discovered that several polyphenols extracted from various plants are potent inhibitors of angiogenesis.

Cocoa beans have been identified as a rich source of polyphenols. Cocoa polyphenols modulate immune functions and platelet function, reduce the oxidation of low density lipoproteins (LDL), inhibit the growth of human in vitro cancer cells and experimental tumors in vivo in multi-organ carcinogenesis models. in the rat.

According to a fourth aspect, the invention therefore relates to the use of a cocoa polyphenol extract as described above, for the preparation of a medicament for preventing and / or treating prostate cancer. The invention further relates to the use of a cocoa polyphenol extract as described above for the preparation of a medicament for alleviating the symptoms of prostate cancer.

According to one advantageous aspect, the invention further relates to the use of a cocoa polyphenol extract as described above, for selectively inhibiting the proliferation of human prostatic tumor cells, and this without inhibiting the healthy cells of human prostate.

Thus, the cocoa polyphenol extract of the invention makes it possible, for example, to inhibit the proliferation of prostatic cancer cells 22RvI and DU145, without inhibiting the healthy epithelial cells of human prostate RWEP-I. "22RvI" representing the loco-regional human prostate cancer cell line, "DU145", the human prostate cancer cell line derived from a metastatic site of brain origin and "RWEPl" the healthy human prostate cell line.

The polyphenolic cocoa extract of the invention inhibits the effects of cancer of the prostate chemically. induced in rats, and to the Inventors' knowledge, it is the first time that such results have been obtained.

The present invention also relates to the use as defined above of the cocoa polyphenol extract, in which the extract is administered in a mammal at doses ranging from 15 to 60 mg / kg / day, preferably ranging from 20 to 40 mg / kg / day.

Several thousand polyphenols have been identified in plants, including consumable plants, as well as in beverages. Their role in cancer prevention, osteoporosis or coronary heart disease is currently under study. Their antioxidant and anti-inflammatory effects have been proven by several in vitro and in vivo studies. Flavonoids, the largest class of polyphenols, are found in many fruits, vegetables and beverages.

Numerous epidemiological studies have shown that fruit and vegetable consumption is associated in reverse order with cardiovascular diseases and mortality due to degenerative diseases. The polyphenols contained in the diet have protective and beneficial biological effects in humans under oxidative stress. Oxidative stress in rats exposed to heart attack induces an overproduction of circulating free radicals that adversely affect memory performance and learning.

An imbalance between the formation of reactive oxidizing species and the antioxidant defense is called oxidative stress. Reactive oxidizing species are free radicals including a superoxidant anion, hydrogen peroxide and a hydroxyl radical which are normally produced in small amounts. Under normal conditions, body scavengers such as superoxide dismutase and catalase metabolized these free radicals. With modern life, and under certain physiopathological conditions, Free radicals increase the harmful cellular components. Polyunsaturated fatty acids, phospholipids, free cholesterol, DNA and small molecules are targets of cells reducing the number of free radicals. According to a fifth aspect, the invention therefore relates to the use of a cocoa polyphenol extract as described above, for the preparation of a medicament for preventing and / or treating cognitive disorders. Thus, the invention more particularly relates to the use as described above to improve the learning, storage and orientation capabilities in the space of a mammal.

The invention further relates to the use of a cocoa polyphenol extract as described above, for the preparation of a medicament for preventing the overproduction of free radicals following a heat stroke.

For cognitive applications, the mode of administration can be systemic or topical, preferably orally, parenterally (intravenously or subcutaneously), rectally or topically and be for example in the form of single or coated tablets, capsules , creams, ointments, suppositories, inhalations, injectable solutions or oral suspensions. Cognitive disorders being induced by an overproduction of free radicals, the prevention of the overproduction of free radicals thus makes it possible to prevent the appearance of said cognitive disorders.

Examples of cognitive disorders that may be mentioned include arterial hypotension, cerebral ischemia, neuronal damage, etc.

According to an advantageous embodiment of the invention, the drug for preventing the overproduction of free radicals is administered at a dose of Img / kg / day to 100mg / kg / day, before heat stroke or after heat stroke.

According to a sixth aspect, the invention therefore aims at the use of the cocoa polyphenol extract according to the invention for stimulating the production of dopamine.

Dopamine is a neurotransmitter belonging to catecholamines. In the peripheral nervous system, it acts as a circulatory analeptic (stimulating functions that ensure blood circulation). At the level of the central nervous system, it has a globally stimulating effect. It is particularly involved in addiction phenomena, via the reward system, and in the control of motor functions. Parkinson's disease is, for example, a disease whose cause is the degeneration of a group of neurons producing dopamine.

Increased dopamine levels help maintain poor blood pressure, maintain diuresis by vasoconstricting the renal arteries, and treat the symptoms of Parkinson 's disease. Example 11 demonstrates that oral administration of 24 mg / kg of an extract according to the invention makes it possible to triple the level of dopamine in rats.

The invention also provides methods of therapeutic treatment comprising administering to a patient a composition based on a polyphenol cocoa extract according to the invention in order to stimulate the production of dopamine. In particular, the administration of this composition may have the object of preventing / relieving the symptoms of Parkinson's disease, of maintaining blood pressure in patients having a deficiency of said arterial pressure or of maintaining diuresis in patients presenting with Parkinson's disease. disorders of diuresis.

The resulting drug can be administered alone or in combination with one or more other drugs. In In particular, in the context of a treatment of Parkinson's disease, the drug may advantageously be administered in combination with L-DOPA, dopaminergic agonists or inhibitors of catechol-O-methyl transferase.

The mode of administration may be systemic or topical, preferably orally, rectally, parenterally (intravenously or subcutaneously) or topically and be in the form of single or coated tablets, capsules, creams, ointments, suppositories , inhalations, injectible solutions or oral suspensions.

The dosage is adaptable according to the nature and severity of the condition, the desired therapeutic purpose, the route of administration and the sex and age of the patient. By way of example, the drug may be administered at a dose of 10 to 30 mg / kg / day orally. These dosages can advantageously be administered in several doses.

According to a last aspect, the invention also relates to the use of the cocoa polyphenol extract described above, for preventing oxidative stress (see Example 12).

The present invention will be more fully described with the aid of the examples which follow, given by way of illustration, with reference to the figures: FIGS. 1 to 5, relating to the preventive treatment of hyperplasia, respectively represent the progress of the experiment ( Fig. 1), weight gain (Fig. 2), food intake (Fig. 3) and drink intake (Fig. 4) and prostate weight (Fig. 5), Figs. of the hyperplasia, represent the progress of the experiment (Fig. β), weight gain (Fig. 7), food intake (Fig. 8) and drink intake (Fig. 9) and the weight of prostate (Fig. 10-11), Figures 12 and 13 are chromatograms of polyphenol extracts of cocoa respectively from Cameroon and Côte d 'Ivoire, Figures 14 to 27, relating to the evaluation of the toxicity of a cocoa polyphenol extract according to the invention. , respectively represent the weight gain of the rats (Figures 14 and 15) and the results of analysis performed on the animals (Figures 16 to 27), Figures 28 and 29, relating to the determination of the LD ₅₀ of a polyphenolic cocoa extract according to the invention, respectively represent the weight curves in male and female rats, Figures 30 to 33, relating to prostate cancer on cell models, represents the morphology of the three RWEP-I cell lines, 22RvI and DU145 (Figure 30), the proliferation of RWEP-I, 22RvI and DU145 cells as a function of dose and time (Figures 31 to 33), Figures 34 to 39, relating to prostate cancer on m co-culture models, show healthy lineage cultures (RWEP-I) (fig. 34a to 34c), tumor line (22RvI) (Fig 35a to 35c), co-culture healthy and tumor lines (Figs 36a to 36c), the results of the proliferation test by Neutral Red release method (Fig. Fig. 37a to 38f), the results of co-culture immunostaining after treatment (Figs. 39a to 39e), Figs. 40 to 42, relating to the chemopreventive effect of the extract in rats, represent the gains body weight of differently treated rats (Fig. 40), food consumption (Fig. 41), water consumption (Fig. 42), Figs. extract on the development of chemi-induced tumors, illustrate the change in body weight of rats (Fig. 43), the consumption of food (Fig. 44), water consumption (Fig. 45), the activity of rats during TESLA tests (Fig. 46-50), the Morris water labyrinth test (Fig. 51-52), Figures 53 to 62, relating to the effects of the extract on the development of chemoinduced tumors, illustrate the change in body weight of rats (Figure 53), the consumption of food (Fig. ), water consumption (Fig. 55), the activity of rats during TESLA tests (Fig. 56-58), the Morris water maze test (Fig. 59-60), the dopamine assay (Fig. Fig. 61 and 62), and Figs. 63-66, relating to the effects of a polyphenolic cocoa extract, as a preventive treatment on the development of chemically induced prostatic tumors, on cognitive performance and on dopamine, present the results obtained on TESLA tests (Figs 63 and 64), Morris (Fig. 65) and the dopamine assay (Fig. 66). Example 1: Evaluation of the effects of a cocoa polyphenol extract as a preventive treatment on the development of prostatic hyperplasia

1. Materials and methods Animals:. 48 Wistar-ϋnilever male rats of 260-27Og under controlled conditions of temperature (22 +/- 2 ° C), humidity (50 +/- 10%), in inverted light cycle of 12h (light from 8h to 20h) ), food and drink ad libi tum. Treatments: Use of polyphenol cocoa extract (CPE) administered daily orally at doses of 24 or 48 mg / kg for 5 weeks.

Progress of the experiment (see figure 1):

Benign prostatic hyperplasia is induced by daily subcutaneous injections for 3 weeks (D14 to D35) of 2 mg / rat testosterone propionate (TP) in solution in corn germ oil.

The monitoring of the parameters is carried out by the daily control of the survival of the animals, the weighing of the animals 3 times a week and the daily monitoring of the food and beverage intake.

The sacrifice of the animals is carried out after 5 weeks after the start of the treatments with the EPC. The prostate is removed and weighed (3 successive weighings).

2. Animal Weight Results (Figure 2)

Treatment of the rats with EPC at 24 or 48 mg / kg made it possible to maintain regular weight gain throughout the experiment. However the dose of 48 mg / kg has limited more weight gain. Food intake (Figure 3)

The treatment of the rats with EPC at 24 or 48 mg / kg made it possible to maintain a food intake comparable to that of the "Control / Vehicle" group throughout the experiment, thus limiting the drop in food intake. induced by the induction of prostate hyperplasia observed for the "TP / Vehicle" group. Drink intake (Figure 4)

Treatment of rats with EPC at either 24 or 48 mg / kg limited the increase in intake of drink due to subcutaneous injections of testosterone observed for the treatment. group "TP / Vehicle", thereby limiting the effects of the induction of prostatic hyperplasia. Prostate weight (Figure 5)

The treatment of rats with EPC is dependent dose: the dose of 24 mg / kg and especially that of 48 mg / kg have limited the increase in weight of the prostate found for the group of rats "TP / Vehicle" which is related to subcutaneous injections of testosterone, thereby greatly limiting the effects of induction of prostate hyperplasia.

3. Conclusion

Pretreatment of the rats with EPC at 24 or 48 mg / kg limited the effects of induction of prostate hyperplasia in a dose - dependent manner. The best efficacy observed under the conditions of the experiment corresponds to the dose of 48 mg / kg.

This dose of 48 mg / kg, however, seems to be relatively strong since it has also limited the weight gain of the animals from the pretreatment phase. Given the effects observed on reducing the weight of prostates, this dose is close to a therapeutic dose.

EXAMPLE 2 Evaluation of the Effects of a Cocoa Polyphenol Extract in Curative Treatment on the Development of Prostatic Hyperplasia 1. Materials and Methods Animals:

48 Wistar-Unilever male rats of 280-30Og under controlled conditions of temperature (22 +/- 2 ⁰ C), humidity (50 +/- 10%), inverted light cycle of 12h (light from 9h to 2Ih) ), food and drink ad libi tum. Treatments:

Use of polyphenol cocoa extract (CPE) administered daily orally at doses of 24 or 48 mg / kg for 2 weeks (D22 to D36). Dose Treatment Group

Vehicle Control + Vehicle -

TP + Vehicle Vehicle -

TP + EPC 24 EPC 24 mg / kg, PO.

TP + EPC 48 EPC 48 mg / kg, bp.

Conduct of the experiment (see figure 6):

Benign prostatic hyperplasia is induced by daily subcutaneous injections for 3 weeks (D15 to D36) of 2 mg / rat testosterone propionate (PT) in solution in corn germ oil.

The monitoring of the parameters is carried out by the daily control of the survival of the animals, the weighing of the animals 3 times a week and the daily monitoring of the food and beverage intake.

The determination of the DHT (dihydrotestosterone) is carried out according to the following protocol:

Collection of 7 to 8 ml of blood from the abdominal aorta on all the animals before the sacrifice on day 36. - Recovery of 2.5 to 4.5 ml of serum and freezing at -20 ^° C.

DHT assays performed by I. R. E. M. in Paris . The sacrifice of the animals is carried out after 2 weeks after the start of the treatments with the EPC, immediately after the blood collection. The prostate is removed and weighed (3 successive weighings). 2. Results

Animal weight (Figure 7)

The average weight of rats in the Control / Vehicle group increased steadily throughout the experiment. That of the "TP / Vehicle" group rats increased steadily until the 20 ^th day of treatment and then stabilized and decreased slightly during the last 15 days of the experiment. The average weight of the rats in the group "TP / CPE 24 mg / kg" also increased steadily j usqu at ^20th j our treatment, then s is stabilized and decreased slightly during the last 15 days bear experimentation, but unless for the group "TP / Vehicle". The mean weight of rats in the "TP / EPC 48 mg / kg" group increased steadily until the 20 ^th day of treatment and then stabilized and increased again during the last 10 days. bear of experimentation. The mean weight of rats in the "TP / EPC 48 mg / kg" group increased more than that of the "TP / EPC 24 mg / kg" group.

Treatment of the rats with EPC at 48 mg / kg made it possible to maintain more regular weight gain throughout the experiment.

Food intake (Figure 8) The average food intake of the 4 groups of rats "Control / Vehicle", "TP / EPC 24 mg / kg", "TP / EPC 48 mg / kg" and "TP / Vehicle" was identical during the first 2 weeks of the experiment, before induction of benign prostatic hyperplasia. The observed decrease is normal since the values correspond to the average of the relations between the food intake and the weight of the animals. The animals eat about the same amount of the diet, but they get bigger, which means a drop in this ratio. The mean dietary intake of the "TP / EPC 24 mg / kg", "TP / EPC 48 mg / kg" and "TP / Vehicle" groups decreased significantly compared to the "Control / Vehicle" group after the start of the study. induction of prostatic hyperplasia and also decreased significantly for all groups after the start of PO treatments, mainly for the TP / Vehicle group and less strongly for the 2 TP / EPC groups. mg / kg "and especially" TP / EPC 48 mg / kg ". During the last week of treatment, the average food intake of the "TP / EPC 48 mg / kg" group increased more than for the 3 other groups. (The statistical treatments remain to be done).

Treatment of the rats with EPC at 48 mg / kg maintained an average food intake closer to that of the "Control / Vehicle" group throughout the experiment, thereby limiting the effects induced by induction of prostate hyperplasia observed at the TP / Vehicle group level.

Drinking intake (Figure 9) The average drinking intake of the 4 groups of rats "Control / Vehicle", "TP / EPC 24 mg / kg", "TP / EPC 48 mg / kg" and "TP / Vehicle" was identical during the first 2 weeks of the experiment, before induction of benign prostatic hyperplasia. After the onset of induction of prostate hyperplasia, the mean drinking intake of the "TP / EPC 24 mg / kg", "TP / EPC 48 mg / kg" and "TP / Vehicle" groups increased by significantly compared to the "Control / Vehicle" group. The average drinking intake of the 2 groups "TP / EPC 24 mg / kg" and especially "TP / Vehicle" continued to increase during the 3 weeks of testosterone SC injections and during the 2 weeks of PO treatment with EPC or the vehicle, whereas it decreased during the last week of treatment PO for the group "TP / EPC 48 mg / kg" to approach that of the group "Control / Vehicle". (The statistical treatments remain to be done).

Treatment of the rats with EPC at 48 mg / kg limited the average increase in mean drinking intake observed for the TP / Vehicle and TP / EPC 24 mg / kg rats in combination with injections. subcutaneous (SC) testosterone, thereby limiting the effects of induction of prostate hyperplasia.

Prostate weight (Figures 10 and 11) The average weight of the prostates of the rats of the group

"TP / Vehicle" (2.26 g / kg body weight) was significantly higher than that of the "TP / EPC 24 mg / kg" groups

(2.06 g / kg body weight), "TP / EPC 48 mg / kg" (1.55 g / kg body weight) and "Control / Vehicle" (1.31 g / kg body weight).

The average weight of the prostates of the rats of the 2 groups "TP / EPC 24 mg / kg" and "TP / EPC 48 mg / kg" is also significantly higher than that of the rats of the "control / vehicle" group, but it is lower than that of the rats of the "TP / Vehicle" group, especially for the "TP / EPC 48 mg / kg" group.

It is also observed that the average weight of the prostates of the "TP / EPC 48 mg / kg" rats is significantly lower than that of the "TP / EPC 24 mg / kg" group (P <0.0001).

Treatment of the rats with EPC at a dose of 48 mg / kg strongly limited the increase in mean weight gain of the prostate observed in rats in the "TP / Vehicle" group which is related to SC injections. testosterone, thereby greatly limiting the effects of induction of prostate hyperplasia. 3. Conclusion

The treatment of rats with hyperplasia of the prostate already present with the EPC at the dose of 48 mg / kg made it possible to limit the effects of this pathology.

Example 3: Example of polyphenolic cocoa extracts Analysis polyphenol extract of cocoa

Parameters Units Results Methods

Starch% <0, 5 Enzyme Kit

Caffeine mg / 100g 759 HPLC / UV

Cellulose% 0, 2 WENDE

Ashes% 4, 9 Order of 16/10/75

Soluble and insoluble fibers AOAC 985-29

Soluble fiber% 0, 7

Insoluble fibers% 3, 3

Humidity% 3, 7 Order of 16/10/75

Mg mg / lO Og 242 Ar .8, 9, 77 (MO0.147)

Total fat% 13, 9 Order of 16/10/75 Phosphorus mg / 100g 197 Order of the 08/09/77

Total Polyphenols% 36, 9 Folin-Ciocalteu Method

Potassium mg / 100g 1984 Ar .8.9.77 (MO0147)

Proteins (Nx6, 25)% 31, 1 DUMAS

Sodium mg / 100g 21 Ar .8.9.77 (MO0147)

Pectic substances

(in galacturonic acid)%> 1, 5 NF V 05-128

HPLC Sugar / Refractometry

Fructose% 1, 4

Glucose% 1, 2

Sucrose% 10, 4

Maltose% 0, 6

Lactose% <0, 5

Theobromine (on chocolate)% 12, 25 HPLC / UV

Vitamin A (Retinol) μg / 100g <20 NF V18-401 (1987)

Vitamin Bl

(Thiamine) mg / lO Og <0, 10 Order of 12.01.99

Vitamin B12

(Cyanocobolamine) μg / 100 g 0.6 AOAC 952-20 (1990)

Vitamin B2

(Riboflavin) mg / lOOg 0, 4 Order of 12.01.99

Vitamin B5 (Panthothenic acid) mg / 100g 3, 2 AOAC 945-74 (1990)

Vitamin B6 ME 0099

(Pyridoxine) mg / 100 g <0.1 (HPLC / Fluorimetry)

Vitamin C mg / 100g <1 HPLC / UV

Vitamin D3 μg / lO Og <0.4 HPLC / UV

Vitamin E (dl alpha tocopherol) mg / lOOg 1, 9 NF V18-402 (87)

Vitamin H (Biotin) μg / 100g <1 HPLC

Vitamin Kl μg / 100g 6, 58 HPLC

Parameters Units Results Methods

Vitamin K3 mg / 100

(Menadione) g <0, 40 EEC 5th Directive

Vitamin PP mg / 100

(Niacin) g 3, 6 HPLC

Total vitamin B9 μg / 100

(Folic acid) g 79 Pr EN14131

Hemicellulose% <0, 2 Internal

Lignin% 1, 0 Internal

- METALS mg / 100

Calcium g 4 Ar .8.9.77 (MO0147)

- MYCOTOXINS

Ochratoxin A μg / kg <1.0 MC 155 HPLC / FLUO Composition of a polyphenolic extract from Cameroon

Humidity (% by mass) 3.62%

Supplementation of the diet with an extract according to the invention Medium polyphenolic composition of cocoa extract

Flavonoids 68%

Other polyphenols 30%

Recommended daily doses in: ^op C 125 mg / day / person Or 1.79 mg / day / kg

Flavonoids 150mg / day / person Or 2, 15 mg / day / kg

OPC: Proanthocyanidin oligomer (to be verified)

Rats averaging 350g, we must give each rat:

OPC 0, 63 mg / d

Flavonoids 0, 75 mg / d

Taking into account the polyphenolic composition of cocoa extract:

- 2, 10 mg of pure polyphenol to reach the OPC dose, recommended,

1, 10 mg of pure polyphenol to reach the recommended epicatechin dose.

A quantity of 3 mg / day / rat of pure polyphenols can be chosen in order to respect the recommended daily dose for each type of polyphenols. 2 Specific extracts:

Polyphenolic extract Ivory Coast rich in β-sitosterol: 37.45% polyphenols.

Polyphenol extract (Cameroon): 35.5% polyphenols. In the first case, give 8 mg / day / rat of the Côte d 'Ivoire extract. In the second case it is necessary to give 8, 5 mg / day / rat of the Cameroon extract.

A chromatrogram of each of these two extracts is given in Figures 12 and 13.

Analysis of polyphenols (oligomers)

Example 4: Evaluation of the toxicity of a cocoa polyphenol extract (CPE) 1. Materials and methods

Animals :

30 adult male rats and 30 female Wistar rats, 120-130 g, temperature-controlled conditions (22 + 2 ⁰ C), humidity (50 + 10%), light cycle reversed 12 h (light 8 h to 20 h ), food and drink ad libitum. Treatments:

Polyphenol Cocoa Extract (CPE) administered for 28 days orally at doses of 50 (FaD), 200 (DI) or 800 (FoD and Fod Sat) mg / kg.

Treatment Male Rats Female Rats

Vehicle 6 rats 6 rats

EPC 50 mg / kg 6 rats 6 rats

EPC 200 mg / kg 6 rats 6 rats

EPC 800 mg / kg 12 rats 12 rats

Animal tracking:

Daily clinical examination of all animals before treatment,

Daily checks of animal survival after treatment, weighing animals 3 times a week for 4 to 6 weeks.

Sacrifice, samples and autopsy of animals:

Sacrifice of half of the animals after 28 days of daily oral administration of EPC at doses of 50, 200 or 8000 mk / kg and the other half 15 days after the last administration of EPC at these same doses,

Blood samples for hematological and biochemical analyzes, Complete autopsy of all the animals and samples of 4 to 20 organs per rat for histopathological analyzes. 2.Results Mortality of animals. No mortality observed in male and female rats after repeated oral administrations of EPC at 50, 200 and 800 mg / kg.

Animal weight (Figures 14 and 15)

. No weight loss observed in male and female rats after repeated oral administrations of EPC at 50, 200 and 800 mg / kg.

Behavior of animals

. No strange behavior observed in male and female rats after repeated oral administrations of EPC at 50, 200 and 800 mg / kg.

Autopsy of animals (Figures 16 to 27)

. No evidence of macroscopic toxicity observed in male and female rats after repeated oral administration of EPC at 50, 200 and 800 mg / kg. 3. Conclusion

=> After 28 days of oral administration of EPC: - no toxicity for doses of 50 and 200 mg / kg in both sexes but on the contrary immunostimulation (increase of the weight of the spleen and the thymus), - effect toxic for the 800 mg / kg dose in both sexes: increased ASAT and decreased ALT and liver weight

=> 15 days after the last oral administration of EPC: no toxicity for doses of 50 and 200 mg / kg in both sexes, plus toxicity for the dose of 500 mg / kg in both sexes = reversibility of toxic effects observed for this dose after 28 days of oral administration of EPC. Example 5: Determination of the LD ₅₀ of a Cocoa Polyphenol Extract (CPE) in Rats 1. Materials and Methods

Animals: 18 adult male rats and 18 female Wistar rats, 150-175 g,

Controlled conditions of temperature (22 ± 2 ° C), humidity (50 + 10%), inverted light cycle of 12 h (light from 8 am to 8 pm), food and drink ad libitum. Treatments: Polyphenol Cocoa Extract (EPC) administered orally at the dose of 2g / kg = dose limit according to OECD standards.

Treatment Male Rats Female Rats

6 rats 6 rats

Vehicle 6 rats 6 rats EPC 2g / kg 6 rats 6 rats

Experimental procedure: Animals placed at least one day before the administration of EPC,

Animals weighed before the administration of EPC,

Oral administration of EPC at a dose of 2 g / kg,

Animals kept for 3 hours after oral administration of EPC. Animal tracking:

Daily clinical examination of all animals before treatment,

Daily checks of animal survival after treatment, weighing animals 3 times a week for 2 weeks.

Sacrifice and autopsy of animals:

Animal sacrifice 14 days after single oral administration of EPC at a dose of 2 g / kg,

Complete autopsy of all animals. 2. Results

Mortality of animals

. No mortality observed in male rats and females after single oral administration of EPC at a dose of 2 g / kg.

Animal weight

. No weight loss observed in male and female rats after single oral administration of EPC at a dose of 2 g / kg. Autopsy of the animals

. No evidence of macroscopic toxicity observed in male and female rats after single oral administration of EPC at a dose of 2 g / kg.

3. Conclusion -> The EP ₅₀ LD50 can not therefore be determined according to OECD standards.

EPC in single oral administration is therefore not toxic.

4. Materials and methods Animals:

. 6 male rats (290-300 g) and 6 female rats (200-210 g)

Wistar of the control group,

Controlled temperature conditions (22 ± 2 ° C), humidity

(50 + 10%), inverted light cycle 12 hours (light 8 am to 8 pm), food and drink ad libitum.

Treatments:

Polyphenol Cocoa Extract (CPE) administered orally at a dose of 4 g / kg = 2 times the OECD recommended limit dose.

Treatments Male Rats Female Rats

EPC 4 g / kg 6 rats 6 rats

Experimental procedure: . Animals placed one day before the administration of EPC,

. Animals weighed before the administration of EPC,

. Oral administration of EPC at a dose of 4 g / kg,

. Animals kept for 3 hours after oral administration of EPC.

Animal tracking:

. Daily clinical examination of all animals before treatment,

. Daily checks of animal survival after treatment,

. Weighing animals 3 times a week for 2 weeks.

Sacrifice and autopsy of animals:

. Animal sacrifice 14 days after single oral administration of EPC at a dose of 4 g / kg,. Complete autopsy of all animals.

5. Results Animal mortality:

No mortality observed in male rats, but 67% (4/6) mortality in female rats after single oral administration of EPC at a dose of 4 g / kg. Animal weights (Figures 28 and 29)

. Slight weight loss observed in male rats and surviving female rats (~ 10 g on average in both cases) after single oral administration of EPC at a dose of 4 g / kg. Behavior of animals:

. No strange behavior observed in male rats and surviving female rats after single oral administration of EPC at a dose of 4g / kg. Autopsy of the animals:. No evidence of macroscopic toxicity observed in surviving male and female rats after single oral administration of EPC at a dose of 4 g / kg.

6. Conclusion 7

The LD50 of the EPC has not been reached in male rats and is therefore greater than 4 g / kg. EPC in single oral administration is therefore not toxic in male rats at a dose of at least 4 g / kg. The LD ₅₀ of the EPC has been reached and even exceeded in female rats and is therefore between 2 and 4 g / kg.

EXAMPLE 6 Prostate Cancer on Cellular Models

In vitro effects of cocoa and β-sitosterol polyphenols on the growth of cancerous and normal human prostate cells.

The antiproliferative properties of cocoa extract polyphenols have been studied on human colon cancer cells, but not on cancerous or normal human prostate cells.

The effects of different extracts of cocoa polyphenols from different countries, as defined above, were therefore examined, alone or in combination with β-sitosterol, on the growth of human prostate cancer cells (DU145 and 22RvI cells) and normal (RWEP-I cells).

The results obtained in this in vitro study show that some polyphenols from specific countries reduce cell proliferation in a dose - and time - dependent manner, and that other polyphenols inhibit cell growth in a dose - dependent manner. dependent.

The origin of polyphenols is an important factor for the effectiveness of these compounds. In addition, the tested cocoa extracts are more effective on androgen-sensitive cells (22RvI cells) than on non-androgen-sensitive cells (DU145 cells). It therefore appears that interactions exist between polyphenols and intracellular steroid receptors. The results obtained show that polyphenols and β-sitosterol have an antiproliferative effect on the growth of prostatic cancer cells.

The effect of polyphenols and β - sitosterol was tested on the growth of RWEP - I and 22RvI cells in a dose - dependent manner, and on the growth of DU145 cells as a function of dose and time.

Each product (1-5) was applied for 1-72 hours, at different concentrations. The number of cells was compared to a control, that is cells cultured in the absence of the test compound.

Table 1 below illustrates the final concentration of cocoa extracts and β-sitosterol for each product tested on prostate cells DU145, 22RvI and RWEP-I.

TABLE I.

Product 1 Product 2 Product 3 Product 4 Product 5

[β-sitosterol] "Extract" [β-sitosterol] Extract [β-sitosterol] Extract Extract [β-sitosteiol]

2 10 ² 0.2 1.6 10- * 0.2 1 10 ^"1 0.2 0.2 I ₁ IO ^{" 1}

1 10 "0.1 8 10 ° 0.1 0.5 10 ^{" 1} 0.1 0.1 0.5 10 ^"1

5 10 ⁴ 0.05 4 10 ^" 0.05 2.5 10 ⁴ 0.05 0.05 2.5 10 ⁴

2.5 Î O ^"4 I0- 0.025 2 ^{1 0.025 1.2 10 4 0.025 0.025 1.2} 10 4

1.2 10 ⁴ 0.0125 1 10 "0.0125 0.6 10 ⁴ 0.0125 0.0125 0.6 10 ⁴

6.2 10 "0.00625 5 10 ⁴¹ 0.00625 3 10 ^" '0.00625 0.00625 3 10 ^" '

3.1 10 ° 0.003125 2.5 10 * 0.003125 1.5 10 "0.003125 0.003125 1.5 10 '

1.5 10 "0.001562 1, 25 IQ- ⁶ 0.001562 7.5 IQ- * 0.001562 0.001562 7.5 Î O * a values in%

A) MATERIALS AND METHODS

Cell lines and culture conditions

DU145 and 22RvI human prostate carcinoma cells were provided by the American Type Culture Collection (ATCC, Rockville, Maryland). DU145 cells originate from a cerebral metastatic site and are not sensitive to androgens. 22RvI cells are derived from prostatic carcinoma and are androgen sensitive. The cells were maintained in the form of a monolayer culture according to the usual techniques, in flasks 75 cm ³ (Corning), at 37 ^° C., in an atmosphere containing 5% CO ₂ and 95% humidity, in RPMI-1640 medium free of phenol red and 10% fetal calf serum. inactivated by heating and 1% L-glutamine. RPMI-1640 medium, fetal calf serum and L-glutamine were provided by Gibco BRL (Cergy Pontoise, France). Cell cultures were subcultured every 5 days to ensure exponential growth. Normal human RWEP-I prostate cells were provided by the American Type Culture Collection (ATCC, Rockville, Maryland). The cells were maintained in the form of a monolayer culture according to the usual techniques, in 75 cm ³ flasks, at 37 ^° C., in an atmosphere containing 5% CO ₂ and 95% humidity, in a Serum free, keratinocyte medium supplemented with 2.5 μg of recombinant human EGF and 25 mg of calf pituitary extract. The medium was provided by Gibco BRL (Cergy Pontoise, France).

Preparation of media supplemented with the tested products Five products were tested for this study and were kept in the dark at +4 ^° C. Β-Sitosterol (product 1), obtained from Sigma Chemicals, was first dissolved in absolute ethanol to a final concentration of 24.1 mM. Product 2, supplied by Barry Callebaut, France, is a defatted cocoa extract containing 35.5% polyphenols and 0.081% β-sitosterol. Product 3 was reconstituted from products 1 and 2 and contains 35.5% polyphenols and 0.72% β-sitosterol. Products 4 and 5 were manufactured by Barry Callebaut, France. The composition of the product 4 is unknown and does not contain β-sitosterol. Product 5 contains 35.27% polyphenols and 0.72% β-sitosterol. Products 2, 3, 4 and 5 were first dissolved in complete medium. For products 2, 4 and 5, the total polyphenols in cocoa extracts come from different countries. Just before use, the final product concentrations (Table 1) used are prepared for the different experiments from stock solutions, by diluting the stock solution in complete medium. 64, 2 μM of β-sitosterol is the maximum dose used in this solubility range and in the physiological range (4 to 70 μM). Absolute ethanol was at a constant concentration of 0.26% in the solvent control, in the negative control, and in all eight concentrations tested. The ratios of β-sitosterol and polyphenols for products 2, 3 and 5 are as follows: Product 2: 2: 1000 Product 3: 2: 100 Product 5: 2: 100 Growth and cell viability assays

The cells were seeded on 96-well plates at an initial density of 15,000 cells / well for DU145, at an initial density of 30,000 cells / well for 22RvI, and at an initial density of 25,000 cells / well for RWEP. I, with 200 μL of medium per well. The cell density was optimized according to the time needed to double the cell population, in order to obtain a significant measurement of the Optical Density (OD) and to be able to compare the different cell lines. After 24 hours, in order to ensure uniform fixation of the cells from the start of the experiments, the products were added to the cell culture at different concentrations and at different times. After incubation with the products, cell growth and viability was measured using combined Neutral Red (RN) and Suiforhodamine B (SRB) (In Cytotox, Vandoeuvre, France). The uptake of the vital RN dye was used by the lysosomes / endosoreas and the vacuoles of living cells as a quantitative indication of the number and viability of the cells after treatment with a chemical test. The anionic SRB dye was used to quantify viable cells by measuring the protein content of the cells.

Cytotoxicity was assessed at 1, 3, 6, 24, 48 and 72 hours after incubating the 22RvI, RWEP-I and DU145 cells with the products. The cells were washed with 200 μl of washing solution in order to eliminate the culture medium supplemented with the products. 200 μl of Neutral Red medium was added and incubated with the cells at 37 ^° C. for 3 hours. The Neutral Red medium was then removed and the cells were washed twice with phosphate buffered saline (PBS). The cells were fixed for 1 minute with 200 μl of fixing solution. 200 μL of a desorption solution was added to extract the Neutral Red from the cells. After 15 minutes at room temperature, the OD, which reflects the number of viable cells, was determined at 540 nm with a 690 nm reference filter, using a microplate reader (Genios, Tecan SA, France). Then the cells were washed four times with PBS. 50 μL of 0.4% SRB solution was added to the culture and 1 hour later the monolayers were washed with 200 μL of rinse solution to remove the unbound dye. 200 μL of solubilization solution was then incubated with the cells to extract the SRB from the cells. After overnight incubation at room temperature, the plates were read at 540 nm with a reference filter at 690 nm. Each experiment was performed three times to obtain 18 values per test condition.

To document changes in cell morphology and growth observed during treatment, cells with maximum concentrations of each product were examined (x40 magnification) by microscopy. phase contrast (Leica DM IL microscope) and photographed using a digital photographic system

(Trinitron ® Color Video Monitor). The RWEP-I, 22RvI and DU145 cells were examined with media supplemented with products after 48 hours of incubation.

B) RESULTS

Morphological alterations

The effect of each product on the morphology of the three cell lines is shown in Figure 30. The cells were treated with the maximum concentration of product for up to 48 hours.

Rounding of the 22RvI cells and the absence of attached cells suggested the induction of apoptosis by the addition of the products 2, 4 and 5. A priori, the product 3 seems to be less efficient on the 22RvI cells. Indeed, a smaller number of rounded cells was observed as well as attached cells with loss of morphology. No significant differences in density and morphology between β-sitosterol-treated 22RvI cells and untreated cells were observed. However, rounded cells on the monolayer were observed, suggesting a very weak inhibition of cell proliferation.

DU145 cells were found to be less sensitive to product 2, 3, 4 and 5 treatments than 22RvI cells. Indeed, no rounded cell has been observed, but many attached cells show a loss of morphology. However, the number of attached cells is lower with the products 4 and 5 than with the products 2 and 3. The DU145 cells seem to be more sensitive to the products 4 and 5. There was no significant difference in terms of density and morphology between β-sitosterol-treated DU145 cells and untreated cells. For normal RWEP-I prostate cells, no significant morphological difference was observed between treated and untreated cells for each product. However, all products were sufficient to slightly reduce the number of cells by day 2. At maximum concentrations, the products affect the growth of normal prostate cells by slowing cell proliferation.

Inhibition of prostatic cell growth as a function of dose and time

The effect on the proliferation of RWEP-I, 22RvI and DU145 cells was studied, depending on the dose and time, at a percentage between 1.56.10 ^{~ 5} % and 2.10 ^{~ 2} % for product 1. and between 0, 2 and 1.562 × ³ % for the other products, after 1, 3, 6, 24, 48 and 72 hours of treatment (Table 1, Figures 31, 32 and 33).

Cytotoxic effect of the products on the growth of RWEP-I cells.

The IC ₅₀ was not reached in the presence of β-sitosterol between 1 and 72 hours of treatment. Β-Sitosterol thus has no cytotoxic effect on RWEP-I cells. In addition, after 48 hours of treatment and for the SRB assay, a dose-dependent inhibitory effect of β-sitosterol on the normal prostate cell line was observed. At this stage, β-sitosterol treatment caused a significant reduction in protein synthesis in RWEP-I cells (Figure 31). For products 2, 3, 4 and 5, the IC ₅₀ after 24 hours of treatment was not reached. However, a diphasic curve was observed after 48 and 72 hours of treatment. In general, all these products inhibited cell proliferation at concentrations between 0.003125% and 0.025% of extract (Figure 31). No significant difference in inhibition was observed between the products and Clj. ₀₀ have never been reached (Table 3). Anticancer effect of the products on the growth of 22RvI cells.

At l, 3 and β hours of treatment, β-sitosterol did not inhibit lysosonal activity or protein synthesis in 22RvI cells. In addition, the IC ₅₀ was reached at 24 and 72 hours. 22RvI cells are the only cancer cell line for which CIs ₀ values were reached with β-sitosterol. At 48 hours of treatment, a dose-dependent inhibitory effect was induced by β-sitosterol on the protein synthesis activity (Figure 31).

In addition, the 22RvI cell line was the only one to display IC _50s during short-term treatment with products 2, 3, 4 and 5 (Table 2). Thus, this line of cancer cells seems to be the most sensitive to cocoa extracts. The IC ₅₀ at 24, 48 and 72 hours of treatment with all products are shown in Table 3. The effect as a function of dose and time is shown in Figure 33 to allow comparison of the anticancer activity of the product. 2 to that of product 3, and that of product 4 to that of product 5. All these products had an effect that depends on the dose and time.

TABLE 2

Hours Test Product 2 Product 3 Product 4 Product 5

CI ₅₁₁ "CI ₃₁ , CI ₅₀ CI ₃₀

RN 0.049 0.060 0.029 0.031

SRB 0.052 0.061 0.076 0.069

RN 0.035 0.036 0.023 0.030

SRB 0.036 0.051 0.041 0.043

RN 0.034 0.034 0.025 0.026

SRB 0.030 0.024 0.032 0, 039

¹ Values of CIw expressed in% of extract TABLE 3.

Produce3 ^d Product4 ' ¹

Β-silosterol line Product2 ^J Prodιπt5 ^u

1 cell dose Inhibilion

Cl ₁₁ ,,, Clioo ma \ Ck, CIs, CI ₅₀ CI ₅₁ , Clin,]

RWEPl RN I5 ° o "no no no no no no no no

SRB 9% no no no no no no no no

24 h 49% 0.0080 0.05

22RV1 RN 0.025 0.0169 0.0098> 0.01 0.0173> o, ι

SRB 49 ° o 0.0085 0.025 0.0170 0.05 0.0098> 0.2 0.0152> 0, l

RN 11% 0.0535> 0.2 0.0835> 0.2

DU 145 0.105> 0.2 0.0940> 0.2

SRB 42% 0.0478> 0.2 0.0874> 0.2 0.0952> 0.2 0.0810> 0.2

0.021 0.021 0.022

RN 29% no no no 0.073 0.078 no 0.024 0.087 0.103

RWEPI

0.017 0.017

SRB 48% no no no 0.086 0.089 no 0.021 0.021 0.081 0.138

RN 16% 0.0080 0.025 0.0076 0.0125

22RV 1 0.0074 0.0125 0.0086 0.0125

SRB 47% 0.0078 0.025 0.0070 0.0125 0.0088 0.0125 0.0083 0.0125 n / a RN 21% 0.0507 ^; 0.2 0.0551> 0.01 0.084> 0.2 0.0350> 0.2

SRB 44% 0.0509> 0.2 0.0504> 0.2 0.062> 0.2 0.0353> 0.2

0.039 0.034 0.030

RN 11% 0.036 0.153 no no no no 0.081 0.096 0.104

RWEPI

0.03 0.024 0.021 0.021

SRB 26% No No No No 0.1 0.065 0.075 0.071

7211 RN CI ,,, = I7.84 ^ι 0.0080 0.025 0.0061 0.025 0.0077

22RV I 0.025 0.0110 0.025

SRB CU = 14.01 0.0078 0.025 0.0055 0.025 0.0062 0.025 0.0090 0.025

RN 0.0498> 0.2 0.0612> 0.2

DU145 42% 0.0594> 0.2 0.0310> 0.2

SRB 40% 0.0450> 0.2 0.0496> 0.2 0.0454> 0.2 0.0297> 0.2

¹ was incubated RWEP-I cells, and 22RvI DUI4 ₅ for I, 3, 6, 24, 48 and 72 hours in the presence of various concentrations of β-sitosterol and cocoa extracts (Table I) Average of three experiments carried out in triplicate ^b Maximum inhibition expressed in%, ^c CI values "expressed in μM, ^d IC values ₅ O expressed in% of extract, not obtained

Anticancer effect of the products on the growth of DU145 cells.

In contrast to 22RvI cells, β-sitosterol did not induce IC ₅₀ in DU145 cells during short-term and long-term treatments. In addition, this phytosterol had a dose-dependent inhibitory effect on the protein synthesis activity at 48 hours of treatment (Figure 31). In addition, no IC ₅₀ was observed with the products 2, 3, 4 and 5 during the short-term treatment. DU145 cells appear to be more resistant to cocoa extracts than 22RvI cells. The IC ₅₀ values of all products at 24, 48 and 72 hours of treatment are shown in Table 3. The dose and time dependent effects are shown in Figure 32 to allow comparison of the anticancer activity of Product 2 to that of product 3, and that of product 4 to that of product 5. All these products presented a dose and time dependent effect, except product 2, which exhibited constant activity at all three times of treatment.

C) DISCUSSION AND CONCLUSION Certain dietary factors may influence the development of cancer by altering cellular signal transmission pathways. In recent years, attention has been focused more and more on fruit and vegetable polyphenols because of their powerful antioxidant power. In addition, β-sitosterol may play a protective role in the development of cancer. This notion is supported by in vivo and in vitro studies.

The purpose of the present in vitro study was to analyze the effects of cocoa polyphenols present in the diet on the growth of RWEP - I, 22RvI and DU145 cells, as a function of time and dose.

The morphological and proliferative changes of the three human prostate cell lines are shown in Figure 30. The results obtained reveal a direct inhibitory effect of the products 2, 3, 4 and 5 on the proliferation of cell lines 22RvI and DU145. Androgen - sensitive 22RvI cells are more sensitive to the action of cocoa extracts than non - androgen - sensitive DU145 cells. Indeed, the 22RvI cells do not adhere and form aggregates in suspension, whereas the DU145 cells are well attached, although the number of cells observed is low. These results suggest that steroid receptors are involved in the action of polyphenols. The presence of polyphenol - steroid receptor (AR) interactions may explain a potent inhibition of 22RvI cells. In this regard, it has been reported that certain polyphenols would be steroid receptor agonists. Cocoa polyphenols can inhibit DU145 cells by altering cellular signaling pathways.

The present example also demonstrates that the three cell lines react differently to each product. Indeed, for the RWEP-I cells, no cytotoxic effect is observed before 48 hours of treatment with all the products. The results obtained demonstrate that the normal RWEP-I cell line is the one that best resists each dose-time effect of the products. However, at 48 and 72 hours, the results obtained form a diphasic curve for RWEP-I cells incubated with the cocoa extracts. At minimal product concentrations, there is no inhibition of cell proliferation. The higher the concentration of the extracts, the stronger the inhibition, up to 70%. Then, the rate of inhibition decreases, even if the concentration of the extracts increases.

For the 22RvI prostatic carcinoma cell line, the results obtained from dose-dependent experiments indicate that these cells are more sensitive to products 3 and 4 and products 2 and 5 have been found to be the most effective products for treatment. inhibition of the growth of DU145 cells. The difference in sensitivity to the cocoa extracts of the two tumor cell lines can be explained by the different origins of the tumors. 22RvI cells are hormone sensitive and are derived from prostatic carcinoma. Conversely, DU145 cells are hormone resistant and are metastatic cells. It has also been found that the membranes of metastatic cells tend to be more fluid than non-metastatic tumors. Since their properties are different, it can be assumed that the molecular mechanisms of inhibition induced by polyphenols are not the same in both tumor cell lines. To explain the different inhibition profiles of the extracts, we can hypothesize that the origin of the polyphenols is very important - for the effectiveness of the cocoa extracts. Indeed, each tested extract contains polyphenols from different countries. In addition, the effectiveness of anti-proliferative and anti-tumor properties of polyphenols is likely related to their degree of ^{polymerization.} It has been reported that the antioxidant potency of flavonols and procyanidins in cocoa and chocolate may depend on the length of the oligomeric chain.

Conclusion:

For each cacao extract and at the maximum concentration, the growth of the 22RvI and DU145 cells is completely inhibited while that of the RWEP-I cells is not inhibited. In addition, 22RvI cells are more sensitive to cocoa extracts than DU145 cells. These results demonstrate that cocoa extract polyphenols exhibit specific anticancer activity against metastatic cells and prostatic carcinomas. In addition, these results are the first to describe a potent anti-proliferative effect of cocoa polyphenols on human prostate cancer cells. All cocoa extracts, at maximum concentrations, induce complete inhibition of tumor cells without any effect on RWEP-I cells. Cocoa extracts are more effective on non-metastatic cells than on metastatic tumors. It is likely that the inhibition of steroid receptor - mediated cell growth is not a general mechanism, but rather an element of the action of polyphenols.

EXAMPLE 7 Prostate Cancer on Co-Culture Models

In this example, the biological effects of polyphenol cocoa extract (EPC) and β-sitosterol are studied. on a co-culture of healthy and cancerous human prostate cells.

The effects of the EPC and the reference phytosterol are visualized by an immunolabeling technique proposed by BlOaternatives by the expression of the galectin 8 glycoprotein at the co - culture of human cancerous and healthy prostate cell lines. respectively the 22RvI and RWEP-I cells.

1) MATERIALS AND METHODS a) Cells and culture media tested

Healthy Human Epithelial Cells of Human Prostate (RWEP-I, ATCC No. CRL-11609)

- Human Prostate Carcinoma (22RvI, ATCC No. CRL-2505) Culture:

37 ⁰ C, 5% CO ₂ , 95% humidity

Mediums tested: Medium Ml = Recommended medium for healthy cells (RWEP-

D: Keratinocyte-Serum Free Medium (KSFM) (Invitrogen, 17005-042)

Epidermal Growth Factor, 5 ng / ml (Invitrogen, 10450-013)

Cattle extract, 0.05 mg / ml (Inv. Trogen, 13028-014)

Penicillin 50 IU / ml-Streptomycin 50 μg / ml 1% (v / v,

Invitrogen 15070-063) Medium M2 = Preferred medium for tumor cells

(22RvI):

Medium RPMI 1640 (Invitrogen, 31870-025)

Fetal calf serum decomplemented, 10% (v / v Invi trogen 10270-

098) L-Glutamine, 1% (v / v Invi trogen 25030-024)

Penicillin 50 IU / ml-Streptomycin 50 μg / ml 1% (v / v,

Invi trogen 15070-063)

Medium M3 = Mixed medium:

Complete Keratinocyte-Serum Free medium, 50% (v / v) Medium RPMI 1640 complete, 50% (v / v) b) Products under test and reference

c) Primary antibody used Galectines belong to the lectin family. These are glycoproteins devoid of enzymatic activity, of variable molecular weight, which can specifically bind oligosaccharide ligands.

The first group consists of proteins formed of two tandem lectin domains located at C- and N-terminal, linked by a peptide sequence of variable size called peptide linker or peptide linker. Galectins 4, 6, 8 and 9 belong to this category.

Galectin 8, usually absent in healthy prostate, is found in prostate carcinomas at a significantly higher rate than in adenomas. As a result, an anti-galectin 8 human monoclonal antibody (R & D Systems, Ref: MAB1305) was selected to specifically fluoresce the human prostatic tumor cell line. d) Selection of co-culture medium → Immunostaining

The selection of the co-culture medium was carried out in two steps :

labeling of the two independently seeded cell lines to determine the concentration of the anti-galectin 8 primary antibody allowing specific labeling of tumor cells (22RvI) without background noise on healthy cells (RWEP-I),

labeling of the two cell lines in co-culture to determine whether the labeling of the tumor cells (22RvI) is identical to that obtained in culture alone, even in the presence of healthy cells (RWEP-I).

Prostate healthy and tumor cells were inoculated, independently or in co-culture, into 96-well microplates at a rate of 30,000 cells per well in each culture medium (M1, M2 or M3). After 48 hours of culture in an oven at 37 ° C. and 5% CO ₂ saturated with moisture, the cellular mats were fixed in alcohol / acetic acid and then labeled with the human anti-galectin 8 monoclonal antibody for 1 hour. The primary antibody was then revealed by a goat anti-mouse immunoglobulin goat conjugate, Alexia Fluor 488 (Interchim, Ref: A-11029). The primary and secondary antibody concentrations were strictly identical for independently grown and co - cultured cultures. After extensive PBS washes, the labeled cell mats were observed using a Nikon DXM1200F camera driven by LUCIA 6.0 software. All images were made under the same conditions and with identical camera settings.

→ By a cell proliferation test: Neutral Red release method

The method of release of the Neutral Red made it possible to determine:

the culture medium making it possible to obtain IC _50s close to those obtained previously (IN CYTOTOX Laboratory, Vandoeuvre-Lès-Nancy) for the two independently grown prostatic cell lines, two concentrations of β-sitosterol and cocoa polyphenol extract applied to healthy prostate and tumor cells for the final step of co-culture immunostaining .

The healthy and tumorous prostatic cells were seeded, independently, in 96-well microplates, at the rate of 30,000 cells per well in each culture medium (M1, M2 or M3) and incubated for 24 hours in an incubator at 37 ^° C. C and 5% CO ₂ saturated with moisture.

After 24 hours of growth, the healthy and tumor cell lines were incubated in the presence of β-sitosterol (reference product) at 8 concentrations (see paragraph b) above) or at the EPC at 8 concentrations (see paragraph b). above). Each concentration tested was performed in 6 wells. The incubation of healthy cells and tumors with the products is 24 hours. At the end of the incubation time with the products, the cell mats were subjected to the Neutral Red release test. Neutral red is a vital dye with the ability to pass passively into lysosomes at physiological pH. During a deterioration of the membrane, the neutral red comes out of the cytoplasm. The amount of dye incorporated into the cells is therefore proportional to the number of cells still having an intact membrane. The amount of dye incorporated in the cells is measured by a spectrophotometric reading at 540 nm and is directly proportional to the number of living cells. The results were expressed as percentage inhibition with respect to the untreated control. e) Treatment with test products and immunostaining of co-culture

The healthy cells and tumors were seeded in co-culture, at the rate of 30 000 cells per well for each cell line, in the selected medium to obtain a homogeneous and specific labeling of tumor cells.

After 24 hours in an oven at 37 ^° C. and 5% CO ₂ saturated with moisture, the coculture was incubated in the presence of β-sitosterol at two concentrations and EPC also at two concentrations.

At the end of the incubation time with the products, the cell mats were fixed in alcohol / acetic acid and then labeled with human anti-galectin monoclonal antibody 8 for 1 hour. The primary antibody was then revealed by mouse goat anti-mouse immunoglobulin conjugate, Alexia Fluor 488 (Interchim, Ref: A-11029). After extensive PBS washes, the labeled cell mats were observed using a Nikon DXM1200F camera driven by LUCIA 6.0 software. All images were captured under the same conditions and with identical camera settings. For this experiment, in order to improve the intensity of this immunostaining and for a better quality of visual rendering, the concentration of the primary anti - galectin 8 antibody was increased from 1/50 to 1/20. In addition, this primary antibody was incubated overnight at + 4 ° C. for better fixation of the antibody and therefore for a better labeling intensity.

2) RESULTS a) Selection of the co-culture medium → Immunolabeling

The results of immunostaining on independently grown healthy and tumoral prostate cell lines are shown in Figures 34a, 34b, 34c and 35a, 35b, 35c.

Figures 34a, 34b and 34c show the healthy line (RWEP-I) grown respectively in the medium M1 (Figure 34a), ^

M2 (Fig. 34b) and M3 (Fig. 34c).

Figures 35a, 35b and 35c show the tumor line (22RvI) grown respectively in medium M1 (Figure 35a), M2 (Figure 35b) and M3 (Figure 35c). The results of immunostaining on healthy human and tumor prostate cell cultures cultured in co-culture are respectively shown in Figures 36a, 36b and 36c.

Figures 36a, 36b and 36c respectively show the co-culture of healthy and tumor lines in the medium M1 (Figure 36a), M2 (Figure 36b) and M3 (Figure 36c).

For prostatic cells grown independently (Figs 34a-c and 35a-c), the healthy cell line

(RWEP - I), grown in the three M1, M2 or M3 media, was not labeled with the primary anti - galectin 8 antibody and the Alexia Fluor 488 secondary antibody. In addition, the tumor cell line ( 22RvI) was specifically recognized by the anti-galectin 8 antibody, which labeling was more homogeneous and more intense under the culture conditions of the M3 medium.

The results obtained for the immunostaining of the cells in co-culture (Fig. 36a-c) confirm those obtained for the cells grown independently. Indeed, the labeling of the tumor cells (22RvI) is more homogeneous in the culture conditions of the M3 medium. In the Ml and M2 media, heterogeneous staining was observed at the level of the co-culture ranging from low to intense.

Thus, the M3 medium allowed precise and specific observation of tumor cells (22RvI) without labeling healthy cells (RWEP-I).

→ By a cell proliferation test: Neutral Red release method

The results of the proliferation test by Neutral Red release method are shown in FIGS. 37a, 37b, 37c, 37d, 37e, 37f and 38a, 38b, 38c, 38d, 38e, 38f. This assay was performed on independently grown healthy and tumor cell lines to determine the percentage of proliferation inhibition induced on each cell line by the products.

Figures 37a to 37f show the effects of β-sitosterol on healthy and cancer-tolerant human cell lines grown independently in Ml, M2 or M3 media. Figures 37a and 37b show the effects of β-sitosterol on human healthy prostate (Fig. 37a) and cancerous cell lines (Fig. 37b) cultured in Ml medium.

Figures 37c and 37d show the effects of β-sitosterol on human healthy prostate (Fig 37c) and cancerous cell lines (Fig. 37d) cultured in M2 medium.

Figures 37e and 37f show the effects of β-sitosterol on human healthy prostate cell lines (Fig. 37e) and cancerous cell lines (Fig. 37f) grown in M3 medium.

According to the results obtained in FIGS. 37a to 37f, β-sitosterol induces a weak inhibition of the proliferation of healthy (RWEP-I) and tumor (22RvI) cell lines grown in the three culture media Ml, M2 or M3. The red dots shown in FIGS. 37a to 37f correspond to the concentrations selected for the co-culture immunolabeling treated with β-sitosterol, at

-4-4 namely 2, 5.10% and 1, 2.10%. Figures 38a to 38f show the effects of EPC on healthy human and prostate cancer cell lines grown independently in Ml, M2 or M3 media.

Figures 38a and 38b show the effects of the EPC on healthy human prostate (Fig 38a) and cancerous cell lines (Fig 38b) cultured in Ml medium.

Figures 38c and 38d show the effects of EPC on human prostate healthy (Figure 38c) and cancerous cell lines (Figure 38d) cultured in M2 medium.

Figures 38e and 38f show the effects of EPC on human prostate healthy (Fig 38e) and cancerous cell lines (Fig 38f) cultured in M3 medium.

From the results obtained in Figures 38a to 38f, a dose - dependent effect of EPC was observed. In addition, the complete inhibition of the proliferation of tumor cells by this EPC was observed for the culture conditions in M2 and M3 medium. This inhibitory effect was induced at the lowest EPC concentrations of 0.025% and 0.0125%. At these same concentrations, the EPC induced a low inhibition of healthy cell proliferation (RWEP-I) in M2 medium and no inhibition in M3 medium. The M3 medium was therefore selected to test the effects of the EPC in immunostaining. Thus, according to the results of this Neutral Red release method proliferation assay, the M3 medium was selected for immunolabeling on co-culture. Indeed, this mixed medium makes it possible to obtain the same inhibitory effects as those observed in the previous study: EPC induced complete inhibition of tumor cell proliferation and did not show any effect on the cell line healthy;

β-sitosterol induced a moderate inhibition of cell proliferation on both healthy and tumor cell lines. b) Immunolabelling of the co-culture after treatment The results of the co-culture immunostaining after treatment of the reference product (β-sitosterol) and the test product (EPC) are presented in FIGS. 39a to 39e. FIGS. 39a to 39e show the co-culture of β-sitosterol or EPC-treated tumor and healthy prostate cell lines in M3 medium.

Figure 39a shows the untreated co-culture (= proliferation control) (X 20).

Figure 39b shows the co-culture treated sitosterol β- (2, 5.10 ^{~ 3%)} (X 20).

Figure 39c shows the co-culture treated with EPC (0.025%) (X 20). Figure 39d shows the co-culture treated with β-sitosterol (1.2 ± ³ %) (X 20).

Figure 39e shows co-culture treated with EPC (0.0125%) (X 20).

During labeling, the Hoechst dye was also used to highlight the nuclei of healthy prostate and tumor cells. The tumor cells were labeled with anti-galectin antibody 8 (in green) and the

Hoechst; healthy cells were marked by Hoechst

(in blue) .

-4 At the two concentrations of β-sitosterol tested (2, 5.10%

-4 and 1, 2.10%), the labeling surface revealed by immunostaining and Hoechst is equivalent to that of the untreated proliferation control. This lack of significant difference in labeling is due to a moderate inhibitory effect of β-sitosterol on tumor cell lines and healthy.

At both tested concentrations of EPC (0.025% and 0.0125%), tumor cell aggregation was observed, greatly reducing the labeling area. This cellular aggregation could explain a higher labeling intensity mainly related to the accumulation of the primary antibody at the antigenic site galectin 8 tumor cells. This aggregation phenomenon is dose - dependent since the density of aggregation and the The marking area is less important at 0, 025% EPC than at 0, 0125%. The proliferation of the tumor cell line is effectively inhibited (see paragraph above on the method of release of Neutral Red) but always presents the antigen in its integrity since this cell line has been labeled. 3) CONCLUSION

The two concentrations of EPCs tested (0.025% and 0.0125%) induce an inhibition of cell proliferation of about 90% without inhibiting healthy cells and also allow a decrease in tumor cell adhesion and total area of the tumor cell mat. This high percentage of inhibition of proliferation and this ability to limit the adhesion of tumor cells are not observed for the reference product, β-sitosterol. The difference in biological effect between β - sitosterol and EPC is clearly visualized in fluorescence by immunostaining. Thus, by observing these images, the EPC, in addition to its ability to inhibit the proliferation of tumor cells, reduces the adhesion of these cells.

Example 8: Chemopreventive effect of the polyphenol cocoa extract (CPE) according to the invention in rats

The carcinogenesis model of the prostate using the direct-acting chemical carcinogen, N-methylnitrosourea (MNU), followed by chronic androgen stimulation with testosterone in the Wistar-Unilever rat, has been used in several chemoprevention studies. Recent It is an anatomically and physiologically relevant model for the preclinical evaluation of substances that are thought to inhibit or enhance prostate carcinogenesis in humans.

In this example, the chemopreventive effects of cocaine polyphenol extract (CPE), administered orally in the Wistar-Unilever rat at doses of 24 and 24 hours, were evaluated. ^

48 mg / kg against chemically induced prostate tumors.

Treatment of rats with EPC at a dose of 24 mg / kg inhibited the development of prostate tumors with histological features similar to those of clinical human prostate cancer. 2. MATERIALS AND METHODS Chemical substances The carcinogen MNU (N-methylnitrosourea) and an androgenic hormone, testosterone propionate (PT) (CAS No. 57-85-2), were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France).

The solvent free EPC was provided by Barry Callebaut France (Louviers, France). It was isolated from Ivory Coast cocoa beans by extraction, filtration and drying. The total polyphenol composition of this extract is 88.5% procyanidins and 11.5% catechin, epicatechin and epicatechin gallate. Animals Seventy-five male Wistar-Unilever rats (HsdCpb: WU), weighing 250-275 g at the start of the experiment, were obtained from Harlan Nederland (Horst, The Netherlands) and divided into 5 groups of 15 rats each. They were housed in polypropylene cages (3 rats per cage) equipped to provide food and water. The animals were kept in an air-conditioned room under conditions of controlled temperature (22 ± 2 ° C), relative humidity (50 ± 10%), with an inverted 12-hour day / night cycle (extinction of light at 8 in the morning) . They had access to a standard M20 diet (Dietex, France) and had ad libi tum tap water available. The animal shop has the authorizations issued by the French Ministries of Agriculture and Research and the experiments on animals have been carried out in accordance with European recommendations for animal testing (Council Directive 86/609, 1986) and the UK animal welfare guidelines for experimental neoplasia.

Induction of prostate tumors

The induction of prostate tumors was carried out in two phases. In the initiation phase, the rats received a single intravenous injection into the tail vein of carcinogen MNU at 35 mg / kg body weight prepared with sterile saline immediately prior to injection. The controls received only one intravenous injection of sterile saline. For the development phase, one week after the administration of MNU, the rats received subcutaneous implantation of a silastic tube (Sani-Tech ¹⁰ 50, Saint-Gobain Performance Plastics, Taunton, MA, USA) 2 cm long filled with 40 mg PT and sealed with medical glue (3M Vetbond ^™ , St. Paul, MN, USA). The silastic tube was implanted by making a small incision in the subcutaneous space within the abdominal ventral wall area of the rats under acepromazine anesthesia (2 mg / kg Calmivet, Vetoquinol, Lure, France) with ketamine (50 mg / kg of Ketamine 1000, Virbac, Carros, France). The silastic tube was replaced every two months during the first six months of the experiment. On the controls, hermetically closed empty silastic tubes were implanted. Treatments After an acclimation period of seven days from their arrival, the 75 male Wistar - Unilever rats were randomized into 5 groups of 15 rats each according to their body weight.

A control group was induced chemically and was not treated ("no treatment" group). Another control group received an intravenous injection of sterile saline, subcutaneous implants of empty silastic tubes and was treated with a vehicle (spring water) ("No induction + vehicle" group). The other three groups of rats were induced to induce prostate tumors according to the protocol described above and were treated with EPC at 24 or 48 mg / kg body weight ("Chemo" groups). -induced + CPE 24 "and" Chimio-induced + CPE 48 ", respectively) or with a vehicle (" Chimio-induced + vehicle "group). Treatment with EPC and vehicle started 2 weeks before the administration of MNU. The EPC was dissolved in the vehicle each day before being administered to the animals. EPC and vehicle were administered orally based on body weight of rats, weekly from Monday to Friday, for the duration of the experiment. Animal Surveillance and Autopsy

The viability, behavior and body weight of the rats were recorded three times per week throughout the duration of the experiment. Food and water consumption was recorded weekly from Monday to Friday for each cage in order to estimate the amount of food and water ingested by rats in the cages. During the experiment, animals were euthanized under anesthesia if they showed signs of suffering (cachexia, weakness, difficulty moving or feeding), toxicity related to the compounds (gibbosity, convulsions), a loss 25% body weight for three consecutive days. An autopsy was performed in each case.

Nine months after induction of prostate tumors, six rats from each group were euthanized under anesthesia for histopathological evaluation. The nine Remaining rats in each group were maintained in a survival study and treated as described above.

Histopathological evaluation

At autopsy, all marked lesions were excised. The accessory sex glands and urinary bladder were carefully removed en bloc from each rat and fixed in a 4% formalin solution (Roti ^® -Histofix

4%, Carl Roth GmbH & Co, Germany). After fixation, the accessory sex glands were embedded in paraffin and thin sections (5.0 μm thick) were prepared from all the blocks and stained with hematoxylin and eosin for evaluation. histopathological. The sections were evaluated blindly and independently twice by the same investigator to classify the lesions. Lesions of the prostate and seminal vesicles have been cataloged as inflammation, hyperplasia (focal or diffuse), adenocarcinoma and lymphoma.

Statistical analysis

Analysis of variance (ANOVA) was used to compare the mean body weight change (MBWC) with the food and water consumption of the five groups of rats. When a significant event was observed, analysis of variance (ANOVA) was followed by a post hoc Scheffé test to compare the chemoinduced groups to the control groups. The results were expressed as standard ± SEM error calculation. For all comparisons, the differences were considered significant at the P <0.05 level. All statistical analyzes were performed using the StatView ^® 5 statistical software (SAS, Institute, Inc., Cary, NC, USA). United ) .

2. RESULTS

Average body weight change in rats All animals remained healthy throughout the experimental period.

As shown in Figure 40, the body weight gains of the five groups of rats were not statistically different during the first two weeks of treatment, prior to induction of prostate tumors (between D5 and D19). . After the induction of prostate tumors (between J19 and J278), the body weight gain of the groups "Without treatment" (+ 249, 9 ± 27, 0 g) and "Without induction + vehicle" (+ 250, 6 ± 28.7 g) were considerably higher than those in the "chemoinduced + vehicle" (+116, 1 ± 23, 1 g), "chemoinduced + CPE 24" groups (+ 140, 3 ± 17, 6 g) and "Chemo-induced + CPE 48" (+ 147, 1 ± 50, 3 g) (P <0, 0001 in all cases). This difference in body weight gain is due to combined treatments of MNU and PT. No statistical difference in body weight gains was observed between the "no treatment" and "no induction + vehicle" groups or between the "chemo-induced + vehicle", "chemo-induced + CPE 24" and "no-induction" groups. Chemo-induced + CPE 48 "during the same period. However, the body weight gains of the "chemoinduced + CPE 24" and "chemoinduced + CPE 48" groups were greater than those of the "chemo-induced + vehicle" group. Feed and water consumption of rats As shown in Figure 41, the consumption of foods in the "chemoinduced + vehicle" group (50.0 ± 0.5 g / kg / day) was significantly higher. than that of the groups "Without treatment" (46, 8 ± 0.9 g / kg / day) and "Without induction + vehicle" (46, 1 ± 1.2 g / kg / day) (P = 0, 019 and P = 0.003, respectively). The mean intake of foods in the "Chemoinduced + CPE 24" group (49, 5 ± 1, 3 g / kg / day) was significantly higher than that of the "No induction + vehicle" group (P = 0, 011). The average consumption of foods of the group "Chimio-induced + CPE 48 "(51, 4 ± 1.0 g / kg / day) was significantly higher than that of the groups" Without treatment "and" Without induction + vehicle "(P = 0, 0007 and P = 0, 0001, respectively ). No significant difference was observed in the average food consumption between the "no treatment" and "no induction + vehicle" groups and between that of the "chemoinduced + vehicle", "chemoinduced + CPE 24" groups. and "Chimio-induced + CPE 48".

As shown in Figure 42, the mean water intake of the "chemo-induced + vehicle" group (117, 2 ± 12, 9 g / kg / day) was significantly higher than that of the "chemo-induced" groups. CPE 24 "(83, 4 ± 16, 2 g / kg / day)," Without treatment "(66, 8 ± 3, 3 g / kg / day) and" Without induction + vehicle "(64, 4 ± 6 9 g / kg / day) (P = 0.028, P = 0.007 and P = 0.004, respectively). No significant difference was observed in water consumption between groups

"Chimio-induced + vehicle" and "Chimio-induced + CPE 48"

(93.3 ± 13.3 g / kg / day). No statistical difference in mean water consumption was observed between the "Chemoinduced + CPE 24", "Chimioinduced + CPE 48", "No treatment" and "No induction + vehicle" groups.

Incidence of prostatic lesions

Table 4: Incidence of prostatic lesions after nine months.

Treatment Number Inflammation Hyperplasia Hyperplasia Adenocarcinoma Diffuse focal rat lymphoma examined ______ g ^ J _{3 2 2} induced + vehicle

Chimio- 6 2 1 2 0 0 induced +

EPC 24

Chimio- 6 4 1 3 1 1 induced +

EPC 48

Not induced 6 1 1 1 0 0

Without 6 0 1 1 0 0 treatment

Table 4 above summarizes data on the incidence of prostatic lesions observed nine months after initiation of treatment with the vehicle or EEC at 24 or 48 mg / kg and induction of prostate tumors with MNU and PT. The highest incidence of prostatic neoplasia was observed in the "chemoinduced + vehicle" group where two of the six rats had adenocarcinomas and two other rats had prostatic lymphomas. Four inflammations of the prostate and three diffuse hyperplasias were also observed in this group. The lesions induced by combination treatments of MNU and PT appear to occur in the dorsolateral and / or anterior prostate. A reduction in the incidence of prostatic neoplasia was observed in the "chemoinduced + CPE 48" group where adenocarcinoma and lymphoma were observed in the dorsolateral and / or anterior prostate in two rats. The same number of prostate inflammations and diffuse hyperplasias (four and three, respectively) were observed in this group, as for the "chemoinduced + vehicle" group. No adenocarcinoma and no lymphoma were observed in the "chemo-induced + CPE 24" group, as for the two groups "Without treatment" and "Without induction + vehicle". Only two inflammations of the prostate and two diffuse hyperplasias were observed in the group "Chemo-induced + CPE 24" whereas a single inflammation of the prostate was observed in the group "Without induction + vehicle" and a diffuse hyperplasia a observed in both the "no treatment" and "no induction + vehicle" groups. Focal hyperplasia has been observed in the five groups of rats. No tumors were observed in the seminal vesicles or tissues other than the accessory sex glands in all groups of rats, nine months after the induction of prostate tumors.

3. CONCLUSION

The EPC of the invention, administered orally to rats prior to induction of carcinogenesis of the prostate and for the following nine months, significantly reduces the incidence of prostate lesions, particularly at the low dose of 24 mg / kg.

This is the first time, to the Inventors' knowledge, that it has been shown that an EPC has inhibitory effects on chemically induced prostate carcinogenesis in rats.

Treatments with the EPC, at doses of 24 and 48 mg / kg increased the body weight gain in rats in comparison to the treatments with a vehicle but with a body weight gain significantly lower ^• by comparison with control groups.

EPC treatments also reduced the feed and water consumption of rats after induction of prostate tumors compared with vehicle treatments. However, a dose - related effect of EPC treatment in the number of prostate lesions was not observed nine months after induction of prostate tumors. More tumors were observed in the dorsolateral and / or anterior prostate of the EPC treated rats at 48 mg / kg per dose than in the 24 mg / kg treated group.

The effects of the diffusion of PT through silastic tubes and its activity in the body of rats must be limited by the action of low doses of the EPC. I / EPC administered at 24 mg / kg can inhibit a 5α-reductase, thus limiting the conversion of testosterone to dihydrotestosterone, as reported by Willis and Wians in 2003, and can thus slow the development of prostate tumors that are androgen-dependent during the first months. A higher dose of EPC, ie 48 mg / kg, may involve a regulatory mechanism such as a negative feedback that may slow the EPC inhibitory activity on a 5α-reductase over time, and with the combination of carcinogenic MNU, may increase the development of prostate tumors instead of inhibiting them. These observations may explain the fact that the feed and water consumption of rats in the EPC 48 treatment group are higher than those in the EPC 24 treatment group and closer to those in the group. treated with a vehicle for the first eight months.

In this example, the efficacy of EPC appears to be inversely related to dose although it can be assumed that its bioavailability is dose dependent. An earlier study indicated that the bioavailability of epicatechin was dose dependent in the range of doses tested (1-10 mg / kg body weight). 30 to 50% orally administered epicatechin was absorbed through the gastrointestinal tract and distributed in conjugated blood and had antioxidant activity in rat blood.

The same observation has been made, in an even earlier study, in humans after the high consumption of different amounts of a procyanidin - rich chocolate. In 2003, it was shown by Record et al. _r that the consumption of chocolate containing either 200 mg of flavonoids or related procyanidins reduced the production of free radicals in faecal water, which suggested the effectiveness of polyphenols derived from chocolate on the health of the intestine.

The strong antiproliferative effect of cocoa flavonoids and procyanidins on human colon cancer cells has also been described. The efficacy of the antiproliferative and antitumor properties of flavonoids and procyanidins is likely related to their degree of polymerization. It has been reported that the antioxidant properties of these compounds in cocoa and chocolate may be affected by the length of the oligomeric chain.

In other studies, polyphenols in cocoa liquor have been shown to inhibit 12-0-tetradecanoylphorbol-13-acetate-induced tumor development, suggesting antiproliferative and anti-inflammatory mechanisms. Subjecting male rats to a diet containing polyphenols in cocoa liquor has been associated with significant inhibition of lung carcinogenesis and a tendency towards inhibition of thyroid carcinogenesis. Based on the results of this and other studies, it appears that the daily consumption of small amounts of flavonoids and procyanidins from cocoa or chocolate, in conjunction with a typical intake of flavonoids from other dietary sources, is likely to contribute to protection against carcinogenesis in various organs including the prostate.

In conclusion, EPC treatments at 24 mg / kg per dose protect rats against chemically induced prostate tumors when administered prior to the initiation phase of induction.

EXAMPLE 9 Effects of polyphenol cocoa extracts (EPC) on the σognitive performance of rats after heat stroke. The objective of this example is to evaluate the preventive antioxidant effects of two cocoa phenolic extracts (EPCl and EPC2) on the increase of circulating free radicals after heat stroke in male Wistar rats and their protective effects on cognitive performance.

Several authors have observed that prolonged heat stroke in rats induces cerebral ischemia, including lesions in the cerebellum, cortex, striatum, and thalamus, and oxidative stress with overproduction of free radicals circulating in the form of cerebral ischemia. nitric oxidant.

Large amounts of reactive oxidative species cause damage to the brain and rats have difficulty learning and memorizing. The light extinction test is used to evaluate the ability of rats to discriminate between an active lever and an inactive lever in an operant conditioning situation. The water labyrinth test is used to determine short - term and long - term spatial storage in rats. Rats with large amounts of free radicals in circulation are unable to pass this test successfully in a short time and memorize it correctly.

To evaluate the preventive effects of cocoa polyphenols on the production of free radicals after heat stroke, rats are treated daily orally with two EPCs (EPC 1 and EPC 2) for 14 days before exposure to a stroke. heat (40 ^° C., 120 min). Two learning tests are used to evaluate the preventive effects of products on cognitive impairment: the light extinction test and the Morris water labyrinth test. Free radicals are determined in serum after heat stroke and rats are tested in cognitive tests to evaluate their performance in comparison with two control groups (treated with a vehicle, with or without 6 (J without heat stroke). Vitamin E (α-tocopherol) administered daily orally at 200 mg / kg body weight / day is used as the reference antioxidant. Free radical content in the blood in rats treated with EPCl, EPC 2 or vitamin E and then exposed to heat stroke was found to be significantly lower than in control rats. Rats treated with EPCl or 1 ΕPC2 had a low total leverage count in the light extinction test. In addition, rats treated with EPCl distinguished between the active lever and the inactive lever. In the Morris water labyrinth, latency to reach the platform of rats treated with EPC1 and CP2 decreased during testing.

Thus, the daily oral and preventive administration of EPC1, CP2 or vitamin E for 14 days protects rats against overproduction of free radicals after heat stroke and preserves them from cognitive impairments.

1) Materials and methods Animals

Eight male rats of Wistar strain / Han IGS (Charles River Laboratories, 69-St-Germain on Arbresle - France), weighing 300 to 320 g are used. At reception, the rats are housed, four per cage, in 48 x 27 x 20 cm polycarbonate cages (ϋ.AR, 91 - Epinay-Sur-Orge, France) in a controlled environment (humidity 50 ± 5%, temperature 22 ± 1 ° C, light from 20:00 to 08:00, a reverse light / dark cycle: 12h / 12h). Rats can freely access a standard diet (M20 pellets, Dietex, 95 - Saint Gratien, France) and tap water up to the time of the experiment. After an acclimatization period of 7 days from their arrival, the rats are weighed and randomly divided into five groups according to treatment and exposure to heat stroke (n = 16). heat (spring water), vehicle / heat stroke (spring water), vitamin E / heat stroke (200 mg / kg body weight / day vitamin E in olive oil, Xinchang Pharmaceuticals Factory, France ); EPCl / heat stroke (22.9 mg / kg body weight / day EPCl in spring water, Barry Callebaut, France), EPC2 / heat stroke (24.3 mg / kg body weight / day) EPC2 in spring water, Barry Callebaut, France). Thus, vitamin E is dissolved in olive oil unlike EPCl and EPc2 which are dissolved in water, and this for the sake of increasing energy input. All rats given EPCl, PC2 or vitamin E are exposed to heat stroke.

Rats are weighed each day for fourteen days of treatment to determine the change in body weight. The tests were double-blind and the recorded behaviors are evaluated by the experimenters without knowing the products administered. The rats used in this study are treated according to the rules established by the ASAB Ethical Committe (1993) and the Canadian Council on Animal Care (1993). All of the standard operating procedures used by ETAP complied with the European Communities Council Directive. Products EPCl and EPC2 are solvent free products and contain 37, 45% and 35, 40% total polyphenols, respectively.

EPC1, PC2, vitamin E or the vehicle are orally administered to rats for 14 days (days 1 to 14). After each daily administration of the product, rats can freely access standard food pellets.

Exposure to heat stroke

The 16 ^th j our after a j eun a night awake rats of different groups, with the exception of those in the group with vehicle, exposed to heat stroke by placing them in a controlled room temperature 40 ⁰ C with a relative humidity of 50% for 120 minutes. The rats were able to express their normal thermoregulatory behavior. Immediately after the heat stroke, the rats having a rectal temperature between 41.5 ° C and 42 ⁰ C are included in the study. The rats were able to recover their cage at 22 ^° C.

Free radicals in the blood plasma

The 16 ^th j our 500 .mu.l samples blood are collected from the vein of tail 12 rats from each group in tubes containing heparin to check the increase of free radicals after exposure to heat stroke. Based on the chemiluminescence of pholasine, a photoprotein of the marine mollusk Pholas dactylus, the production of leukocyte free radicals was measured by means of a kit (Knight Scientific, Cell Activation Test Kit for Whole Blood with Adjuvant K). ) by activation of the NADPH oxidase complex in diluted blood (In CytoTox laboratory,

Nancy, France). Cognitive performance

Light extinction test (avoidance test of an aversive light stimulus)

Three days after exposure to heat stroke, at day 18 (D18), the rats are tested for the preventive antioxidant effects of the two EPCs on learning performance, using the light extinction test. The experimental setup consists of a brightly lit cage (50 x 40 x 37 cm). The light level in the test cage is 1200 lux. Two levers are included in the device: W

an active lever that allows access to a dark period of 30 seconds when pressed and an inactive lever. During darkness, squeezing the active lever does not prolong the period of darkness. During the test session, rats are individually placed in the cage for 20 minutes to learn how to control the light environment. The number of active lever and inactive lever presses is recorded as well as the ability of the rats to distinguish between the two levers.

Morris Water Labyrinth Test (Water Labyrinth Test)

The Morris Water Labyrinth (1984) tests the ability to orient in space, learning and memorizing processes. The 20 ^th and 21 ^th j bear (J20 and J21), the rats were tested in the maze device for water to evaluate the performance of spatial learning and long-term memory. The experimental procedure involves training rats on the 20 ^th day to find a hidden platform (10 mm below the surface of the water) at a fixed location in the water tank (150 cm). diameter) in a series of five trials from the same starting position.

During the first test, the platform is placed against the wall of the tank. In the second test the platform is placed 5 cm from the tank wall to allow the rats to easily find the platform. During the following three tests, the platform is placed 10 cm from the tank wall. Each test is followed by a rest period of 30 seconds on the platform.

On the 21st day, the platform is placed away from the tank wall at the same location as during the 20 ^th day session and the long-term memory of the rats is tested in a single test. o4

The variable recorded in the water tank is the lag time required to get out of the water and reach the platform used as a refuge in the basin.

Statistics Analysis of variance (ANOVA) is used to compare the five groups. When a significant result is observed, the ANOVA is followed by the Dunnett t test to compare the treated groups with the control groups. For repeated measures, a paired t-test (two-sided) is used in each group. The results are expressed as an average ± standard deviation (SEM). All statistical analyzes are performed using the StatView®5 statistical package (SAS, Inc., USA). For all comparisons, the differences are considered significant at the P <0.05 level.

2) Results

Weight variation

As shown in Table 5 below, no significant difference was observed during the 14 day treatment period in the body weight of the rats in the groups.

Table 5

Body Weight Variation (g) (Mean ± SEM)

Day 1 3 4 5 6 7 8 9 10 11 12 13 14

-π m Vehicle 328.9 336.8 339.8 342.8 346.9 349.2 352.3 356.3 359.7 363.3 366.9 371.0 373.9

Ą ± ± ± ± ± ± ± ± ± ± ± ą ± 13.6 13.6 12.8 13.5 13.8 13.6 13.8 13.8 13.3 12.6 12.9 13.1 11.8 m Vehicle / HS 322.2 329.3 331.9 334.6 339.3 342.4 345.3 348.9 352.8 355.5 359.0 363.2 368.1

± ± ± ± ± ± ± ± ± ± ± ± ± ±

TJ 13.8 13.4 13.0 12.5 12.5 13.6 14.2 14.0 14.0 13.9 14.8 15.4 15.2 m - CPE 1 / HS 325.8 333.4 336.1 340.0 344.0 346.8 350.6 354.1 358.2 361.4 363.6 368.4 371.8

≤ ^

± ± ± ± ± ± ± ± ± ± ± ± ±

Ys 8.8 1 1.1 10.6 11.1 11.6 11.0 11.5 12.6 12.6 11.9 12.6 12.7 12.6

O CPE 2 / HS 325.1 332.5 335.6 340.0 344.2 346.9 349.0 352.8 355.5 358.6 361.9 366.3 368.8 ± ± ± ± ± ± ± ± ± ± ± ± ± m 12.1 12.3 1 1.6 10.9 9.7 11.0 11.1 11.1 10.0 9.9 11.0 10.9 11.2

Z H Vitamin E / HS 320.9 328.9 330.6 333.9 336.6 339.4 342.1 344.3 348.7 351.6 354.6 357.6 361.0

± ± ± ± ± ± ± ± ± ± ± ± ± 15.4 15.8 15.7 16.5 15.9 17.3 17.2 15.6 17.8 18.0 18.8 19.4 18.4

O ANOVA m F (4,75) 0.64 0.62 0.84 0.93 1.1 1 0.91 0.93 1.26 1.06 1.22 1.10 1.34 1.27 ro σ> Character NS NS NS NS NS NS NS NS NS NS NS significant

As a result, the products did not induce any toxicological effects after the 14 - day subchronic administration.

Heat stroke and free radical content in the plasma

The j ^th lβ our, analysis of variance showed a significant heterogeneity in the levels of free radicals in the five groups.

As shown in Table 6 below, the "vehicle / heat stroke" rats show a significant amount of free radicals circulating relative to those of the other groups.

Table 6 Content of free radicals in the plasma the ^day 16 (m V)

(Average ± SEM)

Groups (*) Vehicle / HS> Vehicle CPE 1 / HS CPE 2 / HS Vitamin

E / HS

(n = 13) (π = 14) (n = 14) (n = 14)

(n = 13)

Radical content 1257.46 ± 163 .79 310.07 ± 300.79 ± 281.68 ± 44.84 31 1.15 ± 20.56 free 19.55 1 1.31

ANOVA: F ₍₄₁₅ ) = 421.66; p <0.001

Dunett's t test

(vs. Vehicle / HS) - t = 21.51 t = 21.83 t = 21.47 t - 20.67

Character p <0.001 p <0.001 p <0.001 p <0.001 significant

(*) Some samples were hemolysed and could not be analyzed.

After the heat stroke, the free radicals circulating in the vehicle / heat stroke rats increased significantly in comparison with those of the vehicle rats, indicating the ability of heat stroke to induce significant oxidative stress. The free radical content in EPCl / heat stroke, EPC2 / heat stroke and Vitamin E / heat stroke rats are significantly lower than those in vehicle / heat stroke rats, which shows a protection of the body against production of free radicals after subchronic administration of EPCl, 1ΕPC2 or vitamin E. The difference in free radical content between the Vehicle / Heatstroke and Vitamin E / heat stroke groups or between the EPCl / heat stroke or EPC2 / heatstroke groups should be due to the antioxidant effects of vitamin E, vitamin E EPCl or 1 ΕPC2 themselves.

Cognitive Performance Light Extinction Test Total Number of Leverage Presses: As shown in Table 7 below, no significant difference was observed in the total number of leverage presses for the rats of the five groups.

Table 7

Total number of lever presses (Mean ± SEM)

Groups Vehicle Vehicle / HS CPE 1 / HS CPE 2 / HS Vitamin E / HS (n = 16 / group)

Total Pressings 32.88 ± 6.34 42.75 ± 5.84 26.81 ± 4.52 24.69 ± 4.71 42.38 ± 5.76 Leverage

ANOVA: F, 4. ₇₅ ) = 2.39; NS

Unpaired t-test (vs. Vehicle) t = 1.15 t = 0.78 t = 1.04 t = 1.11

Significant Character NS NS NS NS

Unpaired t-test (vs. Vehicle / HS) t = 2.16 t = 2.41 t = 0.05 Significance p <0.05 p <0.05 NS

Discrimination of the levers:

As shown in Table 8, group rats

Vehicle, Vitamin E / heat stroke and EPCl / heat stroke pressed the active lever more than the inactive lever.

EPC2 group rats / heat stroke only attempted to distinguish between active and inactive leverage. The vehicle / heat stroke rats made no distinction between the two levers.

Table 8

Distinction between levers

Number of Active Leverage (ALP) vs. Number of inactive lever presses

(ILP) (mean ± SEM)

Groups Vehicle Vehicle / HS Vitamin / HS CPE 1 / HS CPE 2 / HS

(n = 16 / group) #

ALP 20.38 ± 3.96 23.19 ± 3.35 27.28 ± 3.54 15.63 ± 2.82 14. 19 ± 2.76

ILP 12.50 ± 3.13 19.56 ± 2.93 17.60 ± 2.47 11.19 ± 1.87 10 .: 50 ± 2.41

Paired t test = 2.40 t = 1.55 t = 3.81 t = 2.85 t = 1.71

(ALP vs. ILP) p <0.05 NS p <0.005 P <0.05 P = 0.10

# one rat was excluded from the statistical analysis because he had not pressed the inactive lever during the test (Vitamin E / HS: n = 15)

In the light extinction test, the total number of lever presses for the rats of the five groups did not indicate any significant difference. The rats in the Vehicle, Vitamin E / heat stroke and EPCl / heat stroke groups press the active lever more than the inactive lever. Rats treated with EPC2 / heat stroke only tend to squeeze the active lever more than the inactive lever, while those treated with Vehicle / heat stroke do not distinguish between the active lever and the inactive lever.

Morris Water Labyrinth Test

In Trial 1, the analysis of variance showed a significant difference in latency to reach the platform in the five treatment groups. Dunnett's t test shows that latency to reach the platform in the vehicle / heat stroke rats is significantly lower than that of the other rats. In the other trials, the analysis of the variance did not show any significant heterogeneity of latency over time to reach the platform in the five groups.

Table 9

Morris Latency Water Labyrinth Test to reach Platform: s) (Mean ± SEM)

Day 20 Day 21

Group es Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6

(n = 16 / group)

Vehicle 48.81 29.56 27.38 18.50 14.38 12.12

± ± ± ± ± ±

7.43 5.41 4.45 2.94 3.04 2.25

29.69 35.56 21.00 13.94 14.19 12.50

Vehicle / HS ± ± ± ± ± ±

2.73 7.55 4.62 1.32 2.05 1.71

43.81 38.88 22.81 17.50 11.94 10.19

CPE 1 / HS ± ± ± ± ± ±

5.52 7.29 4.45 3.78 1.70 1.28

54.88 25.25 17.63 12.25 8.63 12.69

CPE 2 / HS ± ± ± ± ± ±

6.37 4.89 2.73 1.49 1.07 2.66

51.75 25.38 26.44 17.75 13.56 14.69

Vitamin E / HS ± ± ± ± ± ±

6.45 4.94 5.20 2.44 1.37 2.22

ANOVA F _(4,75) - 2.78 - (4.75) = 1-00 F (4.75) = 0.84 F _(4.75) = 1.14 F _(4, 75) = 1.47 F _{(4, 75)} = 0.60

Character p <0.05 NS NS NS NS NS significant

Test 3 is the reference test because the platform has been moved away from the labyrinth wall of water so that the rats do not reach it by chance.

The paired t-test showed that the rats in the vehicle group significantly improved their latency to reach the platform between trial 3 and trials 4, 5, and 6. The rats in the Vehicle / Heatstroke group did not did not improve their latency between trial 3 and trials 4, 5 and 6. The rats in the Vitamin E / heat stroke group did not improve their latency between trial 3 and test 4 and improved it. significantly between trial 3 and trial 5 and have tendency to improve between trial 3 and test 6. The EPCl / heat stroke rats did not improve their latency between trial 3 and test 4 and significantly improved it. between test 3 and tests 5 and β. EPC2 / heat stroke rats tend to improve their latency between Trial 3 and Trial 4, significantly improving it between Trial 3 and Trial 5, and tend to improve it. between test 3 and test 6.

Rats in the Vehicle / Heatstroke group showed no significant improvement throughout the test.

Table 10

Morris Water Maze Test: Comparing Tests with Each Other

Groups

Trial 3 vs. Trial 4 Trial 3 vs. Trial 5 Trial 3 vs. Trial 6

(n = 16 / group)

Vehicle t = 2.26 t = 2.54 t = 3.27 p <0.05 p <0.05 p <0.01

Vehicle / HS t ≈ 1.52 t = 1.38 t = 1.75

NS NS NS

CPE 1 / HS t = 1.15 t = 2.46 t = 2.72

NS p <0.05 p <0.05

CPE 2 / HS t = 1.95 t = 3.86 t = 2.08 p <0.08 p <0.005 p <0.06

Vitamin E / HS t = 1.57 t = 2.34 t = 2.11

NS p <0.05 p <0.06

3) Discussion and conclusion

Several studies have shown the antioxidant activity of flavonoids in humans by rapid absorption of epicatechins into serum after consumption of chocolate or infusion of green tea. In the presence of cocoa polyphenols, the oxidation of low density lipoproteins decreases. It has also been reported that catechin and epicatechin have been competitively absorbed into the gastrointestinal tract of rats. A large variation in the total phenol content in each type of chocolate and cocoa has also been described. _ _

71

This is why it seemed important to study the antioxidant activity of each specific extract depending on the production process and the origin of the cocoa. The preventive antioxidant effects of the two CPEs, administered orally for 14 days, were evaluated based on the overproduction of free radicals and cognitive impairments after exposure to heat stroke in male Wistar rats. The levels of free radicals in plasma and antioxidant effects of both products were determined in vitro after heat stroke on 16 ^th j our. The test of light extinction was made on ^18th day and our maze test of Morris water was made on ^20th day and ^21st our our j. These cognitive tests were used to evaluate the preventive effects of cocoa extracts on cognitive impairments induced by overproduction of free radicals as a result of exposure to heat stroke. Conclusion: These results suggest that vitamin E (200 mg / kg body weight / day), EPCI (22.9 mg / kg body weight / day) and PC2 (24.3 mg / kg body weight / day) ), administered orally for 14 consecutive days, protect rats from cognitive impairments induced by heat stroke (ie, learning discrimination in the extinction test and spatial learning in the body). Morris water labyrinth test).

To profitably utilize these two rich polyphenolic sources, it is necessary to enrich the cocoa powder or other chocolate products with EPCs. The antioxidant properties of EPC will be evaluated after incorporation into food products. EXAMPLE 10 Evaluation of the preventive effects of a 2-dose oral cocoa polyphenol extract on the development of chemotherapy-induced prostate tumors in adult male rats of the Wistar Unilever strain. A) Study report no.

The purpose of Study 1, below, is to evaluate the preventive effects of a daily oral EPC at a dose of 24 mg / kg body weight or 48 mg / kg body weight on the body. development of chemically induced prostate tumors and to evaluate the effects of EPC on cognitive performance in male Wistar Unilever strain rats.

1. Animal Protocol 75 male Wistar Unilever strain rats weighing 250 to 275 g are used.

Breeding conditions

Temperature: 22 + 1 ⁰ C, hygrometry: 50 + 10%, reverse cycle light / dark: 12h / 12 h. Treatment groups

5 groups of 15 rats are used: "control" group: no chemo-induction or treatment;

- "Vehicle" group: no chemo-induction and treatment with the placebo vehicle; group "Chemo-induction control": placebo treatment;

- "Chemo-induction + EPC 24" group: treatment with EPC at 24 mg / kg; - "Chemo-induction + EPC 48" group: treatment with EPC at 48 mg / kg.

Randomization of animals

Based on body weight at the time of OJ.

Induction of prostate tumors ^

The induction is done in 2 steps:

- initiation step = single intravenous injection of MNU at a dose of 35 mg / kg;

- activation step = one week later, subcutaneous implantation of a 2 cm long silastic tube capped at one end by a biological glue and containing 40 mg of testosterone propionate (PT).

The activation step was repeated 3 times at 2 month intervals. Substances studied

Two-dose cocoa polyphenol extract (CPE): 24 mg / kg body weight and 48 mg / kg body weight.

Route of administration

Oral gavage. Duration of treatment

For 38 weeks, Monday to Friday, with a break on Saturday and Sunday.

Beginning of treatment

2 weeks after induction of prostate tumors. Evaluation criteria

Body weight, food and water consumption, histopathological examination of the prostate.

Cognitive tests

Aversive light stimulus avoidance test (TESLA) performed 2, 4, 6 and 8 months after induction of prostate tumors and Morris water maze test performed 5 and 8 months after tumor induction. prostate .

Sacrifice of animals Six of the animals in each group were sacrificed 9 months after the induction of prostate tumors for histopathological examinations.

Statistical analyzes Analysis of variance followed by unpaired t-test

(bilateral) for comparisons of animal body weights; Kruskal-Wallis test followed by a Mann-Mann test

Whitney ϋ for comparisons of food and water consumption and cognitive test performance.

2. Results

During the 9 months of the experiment, the body weight gain was significantly higher in the "control" and "vehicle" groups than in those subjected to chemoinduction treated orally with the vehicle or the vehicle. EPC at 24 or 48 mg / kg (see Figure 43).

With respect to body weight gain, no significant difference was observed between the "control" and "vehicle" groups. Similarly, no significant difference was observed between the animals in the chemo-induction groups treated orally with the vehicle or with the EPC at 24 or 48 mg / kg (Figure 43).

Between week 1 and week 14, no significant difference was detected between the 5 groups with respect to food consumption (Figure 44).

Between Week 15 and Week 39, food consumption was significantly higher in chemically-induced animals treated with either placebo or EPC at 24 or 48 mg / kg those of the "Witness" and "Vehicle" groups. "

Between Week 1 and Week 5, no significant difference was detected between the 5 groups with respect to water consumption (Figure 45).

Between Week 5 and Week 39, water consumption was significantly higher in the chemically induced animals treated with oral placebo or EPC at 24 or 48 mg / kg. in those of the "Witness" and "Vehicle" groups. "

Between Week 16 and Week 39, consumption ^

In the placebo - treated animals, water in the chemically induced and oral groups was significantly higher than in those receiving the EPC at 24 or 48 mg / kg. In the TESLA test and overall (ie during the 4 test and repeat test sessions), the average total number of levers was invariably higher in the animals in the chemo groups. -induction treated orally with EPC at 24 or 48 mg / kg (Figure 46). The lowest mean total number of levers was recorded in chemically induced animals treated with oral placebo.

In the first, second and third TESLA tests and at the test repetitions performed 2 (Figure 47), 4 (Figure 48) and 6 months (Figure 49) after the induction of prostate tumors, the numbers of leverage The highest active potency was measured in animals in chemically-induction groups treated orally with EPC at 24 or 48 mg / kg and those in the "Control" and "Vehicle" groups. In all cases, the numbers of active leverage support were significantly higher in the chemically induced groups treated orally with EPC at 24 or 48 mg / kg than in the other groups, and discrimination between the active lever and the inactive lever has been reported in these animals.

In the fourth TESLA test and at the test repetitions performed 8 months after the induction of prostate tumors, the animals (Figure 50) of the chemically induced groups treated orally with the EPC at 24 or 48 mg / day kg and those of the groups "Witness" and "Vehicle" have more often relied on the active lever than on the inactive lever. The numbers of active lever support were significantly higher in the chemically induced group treated with EPC at 24 mg / kg than in the other groups. groups. The number of supports on the highest inactive leverage was recorded in the group of chemically - induced animals treated with the placebo.

In the first Morris water maze test performed 5 months after induction of prostate tumors, the curves were similar in chemically induced groups treated orally with EPC at 24 or

48 mg / kg and in the "Control" and "Vehicle" groups during the 5 trials of the training session. The mean latency time was significantly higher in placebo-treated animals than in the other groups. In the test session, the mean latency times were significantly longer in animals in the chemo-induction groups treated with placebo or EPC at 24 or 48 mg / kg than in the "control" or "Vehicle. "(Figure 51).

In the second Morris water maze test performed 8 months after induction of prostate tumors, the curves were similar in chemically induced groups treated orally with EPC at 24 mg / kg. in the "Witness" and "Vehicle" groups during

5 trials of the learning session and the test session. The mean latency times were significantly higher in placebo or EPC-treated animals at 48 mg / kg than in the other groups, and the mean latency time was significantly longer. in animals in the chemo-induction group treated with placebo than in those in the chemo-induction group receiving the EPC at 48 mg / kg (Figure 52).

The following observations have been reported in histopathological examinations of the prostate in 6 animals in each group sacrificed 9 months after induction of prostate tumors. It has been observed: Focal hyperplasia in the 5 groups of rats;

1 diffuse hyperplasia in the "control" groups and

"Vehicle", 2 in the chemically-induced group treated with oral EPC at 24 mg / kg and 3 in the chemically-induction groups treated orally with placebo or EPC at 48 mg / kg;

1 prostatic adenoma in the group treated with oral chemotherapy by the EPC at 48 mg / kg and 2 in the group subjected to a chemo-induction treated orally with the placebo;

1 prostatic lymphoma in the chemically-induced group treated orally with EPC at 48 mg / kg and 2 in the group treated with oral chemo-induction by placebo. No adenoma or prostatic lymphoma was detected in the chemoinduced group treated with EPC at 24 mg / kg or in the "Control" and "Vehicle" groups.

3. Conclusion

No significant differences were detected with respect to body weight gains between the chemically induced groups treated orally with placebo or EPC at 24 or 48 mg / kg. Body weight gains were significantly higher in the "control" groups and

"Vehicle" than in chemically-induced ones treated orally with placebo or EPC at 24 or

48 mg / kg.

Food consumption was significantly higher between week 15 and week 39 in the chemically-induced groups treated with oral placebo or EPC at 24 or 48 mg / kg than in the

"Witness" and "Vehicle. "

Water consumption was significantly higher between week 5 and week 39 in the chemically induced groups treated orally with placebo or EPC at 24 or 48 mg / kg than in the "control" and "vehicle" groups. Between weeks 15 and 39, water intake was significantly higher in the placebo-treated chemoinduced group than in the other two chemically-induced groups treated with oral route by the EPC at 24 or 48 mg / kg.

In the aversive light stimulus (TESLA) avoidance test, the learning and memorizing performance of the rats in the chemically induced groups treated orally with EPC at 24 or 48 mg / kg significantly better than animals from other groups.

In the Morris Water Maze test, the learning and memorizing performance of rats in the chemically induced groups treated orally with EPC at 24 or 48 mg / kg was significantly better than that of the rats. Animals in the chemo-induction group treated orally with placebo. The storage performance of rats in the chemically-induction group treated orally with EPC at 24 mg / kg was comparable to those in the "Control" and "Vehicle" groups. "

The largest lesions detected 9 months after induction of prostate tumors, ie prostatic adenoma or lymphoma, were reported only in the chemically induced groups treated orally with placebo or by EPC at 48 mg / kg. Lesions of this type were not observed in the chemo-induction group treated with EPC at 24 mg / kg or in the "Control" and "Vehicle" groups.

At a dose of 24 mg / kg, EPC confers protection against chemically induced prostate tumors in the rat. Study Report 2: Continuation of Study Report No. 1 for 6 months

The aim of the following study n ° 2 is to prolong the study n ° 1 described above and to evaluate the preventive effects of a CPE administered daily at the dose of 24 mg / kg of body weight or of 48 mg / kg body weight on the development of chemically induced prostate tumors and to evaluate the effects of EPC on cognitive performance in male Wistar Unilever strain rats. 1. Animal Protocol

45 male Wistar Unilever strain rats weighing 250 to 275 g at the start of the experiment are used.

Breeding conditions Temperature: 22 ± 2 ° C, hygrometry: 50 + 10%, inverted light / dark cycle: 12h / 12 h.

Treatment groups 5 groups of 9 rats are used: "control" group: no chemo-induction or treatment;

- "Vehicle" group: no chemo-induction and treatment with the placebo vehicle; group "Chemo-induction control": placebo treatment; - "Chemo-induction + EPC 24" group: treatment with EPC at 24 mg / kg;

- "Chemo-induction + EPC 48" group: treatment with EPC at 48 mg / kg.

Induction of prostate tumors The induction is done in 2 stages for 9 months before the realization of the present study:

- initiation step = single intravenous injection of MNU at a dose of 35 mg / kg; - activation step = one week later, subcutaneous implantation of a 2 cm long silastic tube capped at one end by a biological glue and containing 40 mg of testosterone propionate (PT). The activation step was repeated 3 times at 2 month intervals.

Substances studied,

Two-dose cocoa polyphenol extract (CPE): 24 mg / kg body weight and 48 mg / kg body weight. Route of administration

Oral gavage.

Duration of treatment

For 26 weeks, Monday to Friday, with a break on Saturday and Sunday. Beginning of treatment

2 weeks after induction of prostate tumors.

Evaluation criteria

Body weight, food and water consumption, dopamine, catecholamine and creatinine levels in the urine, histopathological examination of the prostate.

Cognitive tests

Aversive light stimulus avoidance test (TESLA) and Morris water maze test 11 and 13 months after induction of prostate tumors. Statistical analyzes

Analysis of variance followed by unpaired t-test

(bilateral) for body weight comparisons of rats; Kruskal-Wallis test followed by a Mann-Whitney U test for comparisons of food and water consumption and cognitive test performance.

2. Results

During the first 4 months of the experiment, body weight gain in the "Control" and "Vehicle" groups was significantly higher than in those of the three groups. chemically induced with the vehicle group or EPC at 24 or 48 rαg / kg (see Figure 53).

With respect to body weight gain, no significant difference was observed between the "control" and "vehicle" groups. Similarly, no significant differences were observed between the animals in the chemo-induction groups treated orally with the vehicle or with the EPC at 24 or 48 mg / kg (Figure 53).

The food intake of the chemically induced groups, treated orally with the vehicle or with the EPC at 24 or 48 mg / kg, is significantly greater than that of the "control" and "vehicle" groups (Figure 54).

The water consumption of the animals in the chemically induced groups treated orally with EPC at 24 or 48 mg / kg is no different from those of the "control" and "vehicle" groups. Water intake in animals from chemically-induction groups treated orally with vehicle was significantly greater than animals in chemically-induction groups treated orally with EPC at 24 or above. 48 mg / kg and the "Control" and "Vehicle" groups (Figure 55).

In the TESLA assay and test replicates performed 11 months after the induction of prostate tumors, the animals in the chemoinduction groups treated orally with EPC at 24 or 48 mg / kg and control groups "And" vehicle "have the highest level of activity in terms of leverage support and higher active leverage support than inactive leverage. The numbers of total leverage support for rats were greater in the chemically induced groups treated orally with EPC at 24 mg / kg and were lower in the chemically treated groups. induction treated orally by a vehicle. Groups subject to OZ

Oral chemo-induction with EPC at 24 mg / kg exhibits a higher number of active levers than inactive levers and chemically induced groups of animals treated orally with the vehicle has a greater number of supports on the idle levers.

In the TESLA test and test replicates, performed 13 months after the induction of prostate tumors, animals in the chemically-induction groups treated orally with EPC at 24 or 48 mg / kg and control groups "And" vehicle "have the highest level of activity in terms of leverage support and higher active leverage support than inactive leverage. In all cases, animals in the chemo-induction groups treated orally with EPC at 24 mg / kg or 48 mg / kg had a significantly higher number of supports on active levers than animals. other groups and they make a difference between active and inactive leverage. Chemically induced groups treated orally with the vehicle have a greater number of supports on the inactive levers.

For the Morris water maze test conducted 11 months after induction of prostate tumors, the curves were similar in the chemically induced groups treated orally with EPC at 24 mg / kg. in the "Witness" and "Vehicle" groups during

5 tests of the learning session and the test session

(Figure 59). The mean latency time was significantly higher in chemically-induction animals treated orally with EPC at 48 mg / kg than in the other groups. Mean latency times were significantly longer in the animals in the chemically-induction groups treated orally with the vehicle than in those in the chemically-induced groups. OJ

induction treated orally with EPC at 48 mg / kg (Figure 59).

In the second Morris water maze test, performed 13 months after induction of prostate tumors, the curves were similar in chemically induced groups treated orally with EPC at 24 mg / kg. in _"groups" Witness "and" vehicle "during the 5 tests of the learning session and the testing session. Mean latency times were significantly higher in chemically-induced animals treated with the vehicle or EPC at 48 mg / kg orally than in the other groups. No difference was observed between the mean latency times in the chemically-induction animals treated orally with the vehicle or with the EPC at 48 mg / kg (Figure 60).

For the two urine dopamine, catecholamine and creatinine levels measured at 9 and 12 months after induction of prostate tumors, the groups of chemoinduced animals treated orally with or 48 mg / kg showed dopamine levels similar to those of the "control" and "vehicle" groups. The chemically induced groups treated orally with the vehicle have significantly lower dopamine levels (FIGS. 61 and 62). 3. Conclusion

No significant difference was detected during the first 4 months of the experiment with respect to body weight gains between the chemically induced groups treated orally by the vehicle or the 24-hour EPC. or 48 mg / kg. However, body weight gains were significantly higher in the "Control" and "Vehicle" groups than in chemically-induced chemoinduced vehicle or EPC groups at 24 or 48 mg / kg. During the first 4 months of the experiment, food consumption was significantly higher in the chemically-induced groups treated orally with vehicle or EPC at 24 or 48 mg / kg than in the "Witness" and "Vehicle".

During the first 4 months of the experiment, no significant difference was found in water intake in rats between chemically induced groups treated orally with EPC at 24 or 24 hours. 48 mg / kg and the "control" and "vehicle" groups. Water consumption was significantly higher in the vehicle-treated chemoinduction group than in the other chemically-induced groups treated orally with 24 or 48 mg EPC. / kg and the groups "Witness" and "Vehicle" during the same period.

In both aversive light stimulus (TESLA) avoidance tests, the learning and memorizing performance of rats in chemically induced groups treated orally with EPC at 24 or 48 mg / kg were significantly better than animals from the other groups.

In both Morris water maze experiments, the learning and memorizing performance of the rats in chemically induced groups treated orally with EPC at 24 mg / kg was significantly better than that of chemically induced groups treated orally with vehicle or EPC at 48 mg / kg.

Levels of dopamine, catecholamine and creatinine levels in the urine of chemically-induced rats treated with oral CPE at 24 or 48 mg / kg are comparable to those seen in the control and vehicle groups. ». EXAMPLE 11 Effects of a Polyphenol Cocoa Extract (CPE) as a Preventive Treatment on the Development of Chemically Induced Prostate Tumors, on Cognitive Performance and on Dopamine in Rats

1.Materials and methods

Animals :

45 male Wistar-Unilever rats, 250-275 g,

Controlled conditions of temperature (22 ± 2 ° C), humidity (50 ± 10%), inverted light cycle of 12 hours (light 9:00 to 9:00 pm), food and drink ad libitum. Treatments:

Polyphenyl Cocoa Extract (CPE) administered daily orally at doses of 24 or 48 mg / kg for 10 months * or 23 months **, Monday to Friday.

Dose Treatment Group

Not induced and untreated - -

Not induced - EPC 24 * EPC * 24 mg / kg, P .0. *

Chemo-induced + Vehicle Vehicle -

Chemo-induced + EPC 24 ** EPC ** 24 mg / kg, P. 0. **

Chemo-induced + EPC 48 ** EPC ** 48 mg / kg, P. 0. **

Tracking parameters:

- Daily control of animal survival,

- Weighing animals 3 times a week,

- Followed every 2 weeks of food and drink intake,

- Urine sampling for the dopamine assay performed 21 months after the induction of prostatic tumors.

Cognitive tests:

TESLA: performed 21 months after the induction of prostatic tumors, Morris pool: 21 months after induction of prostatic tumors. Sacrifice of animals:

Sacrifice all surviving animals as soon as there are fewer than 5 for all treatment groups. 2 .Results

The survival of the animals is very different according to the groups 23 months after the induction of prostatic tumors or states "uninduced and untreated" group: 6 out of 9 surviving rats, "non-induced + EPC 24" group: 7 out of 9 surviving rats "chemo-induced + vehicle" group: 1 out of 9 rats, - "chemo-induced + EPC 24" group: 4 out of 9 surviving rats, "chemo-induced + EPC 48" group: 1 out of 9 surviving rats,

Regarding weight gain, no difference between the different groups, is not noted. Similarly for food and drink intake.

3. Conclusions Survival of animals:

The survival rate of the animals of the "chemo-induced + EPC 24" group is higher than that of the animals from the other 2 groups of chemically-induced rats, but it is lower than that of the 2 groups of uninduced rats,

. The survival rate of animals in the "non-induced + EPC 24" group is slightly higher than that of animals in the "uninduced and untreated" group.

The EPC administered at a dose of 24 mg / kg before or without induction of prostatic tumors makes it possible to increase the survival time of the rats.

TESLA (see Figures 63 and 64) . The chemically induced or non-chemically induced rats treated with EPC at 24 or 48 mg / kg showed greater activity on the levers than the rats of the 2 "chemoinduced + vehicle" and "uninduced and untreated" groups. And a positive discrimination between the active lever and the inactive lever for the two test and retest sessions,. Rats in the "chemo-induced + vehicle" group have the lowest activity and negative discrimination between the active lever and the inactive lever. The chemically induced or non-chemically induced rats treated with EPC have better learning and memorizing abilities than rats from other groups.

Morris (see Figure 65) The rats of the chemo-induced or untreated groups with the EPC at 24 mg / kg learn and memorize better than the rats of the other groups,

. The rats of the "chemo-induced + vehicle" and "non-induced and untreated" groups present a great difficulty of learning and spatial memorization. Spatial learning and storage of chemically induced or non - chemically induced groups treated with EPC at 24 or 48 mg / kg was better than that of rats in the "chemoinduced + vehicle" and "uninduced and uninduced" groups. treaty" . Dopamine determination (see Figure 66). The urinary dopamine level of the rats in the "chemo-induced + EPC 24" and "chemo-induced + EPC 48" groups is higher than that of the "chemo-induced + vehicle" and "non-induced and untreated" groups. and it is comparable to that observed for the group of rats "not induced + EPC 24",

The urinary dopamine level of rats in the "uninduced and untreated" group gradually decreases over time and is similar to that of rats in the "chemo-induced + vehicle" group. OO

EPC administered at 24 or 48 mg / kg maintains a high level of urine dopamine in rats with and without prostate tumor induction, which is higher than in untreated and untreated rats.

Overall assessment of pathological analyzes performed - Group 1 (chemo-induced + vehicle): 10 tumors out of 13 samples analyzed => 76, 9% of tumor incidence

Group 2 (chemically induced + EPC 24): 0 tumors out of 6 samples analyzed => 0, 0% of tumor incidence

Group 3 (chemo-induced + EPC 48): 5 tumors out of 12 samples analyzed => 41, 7% of tumor incidence

Group 4 (non-induced + vehicle): 0 tumor on 4 samples analyzed => 0, 0% of tumor incidence - Group 5 (uninduced and untreated): 0 tumor on 6 samples analyzed => 0, 0% tumor incidence

EXAMPLE 12 Antioxidant Activity of the Extract According to the Invention

The objective of this study is to evaluate the antioxidant effects of dark chocolate enriched with a cocoa polyphenol extract, administered daily for 4 weeks to healthy young people (18-30 years).

Protocol:

Sujs: The subjects are healthy volunteers, young and Caucasian sportsmen, practicing 4 to 6 hours of sport a week, aged 18 to 30, and weighing between 60 and 80 kg.

Their eating habits remain unchanged. They are also non-smokers and do not have a history of cardiovascular, respiratory, neuromuscular or joint disorders. Volunteers keep a notebook indicating the foods eaten during

1 study.

Products: dark chocolate as placebo (cocoa mass origin Ivory Coast 64%, sugar 26, 5%, cocoa butter 9%, emulsifier: soy lecithin, total fat: 43%) - dark chocolate cocoa origin Ivory Coast 64%), enriched with 102, 10 mg of vitamin E. dark chocolate (cocoa paste origin Tanzania 67, 5%, sugar 23, 5%, cocoa butter 9%, natural vanilla, total fat: 43%), enriched with 47, 93mg polyphenol extract of cocoa.

Dose: a bar of chocolate at noon, at the end of the meal.

Result: The group ingested the dark chocolate polyphenol extract supplement showed a significant increase in total cholesterol, LDL cholesterol and beta-carotene, while vitamin E and malondialdehyde significantly decreased between day 0 and day 28, after 4 weeks of treatment by oral administration.

This result indicates a significant antioxidant activity of dark chocolate enriched with polyphenolic extract, compared to non-enriched dark chocolate.

Claims

claims

1. Use of a polyphenolic cocoa extract for the preparation of a medicament for the treatment of

1 hyperplasia of the prostate.

2. Use according to claim 1, characterized in that the treatment relates to benign prostatic hyperplasia.

3. Use according to claim 1, characterized in that the treatment relates to hyperplasia of the prostate, resulting from prostate cancer.

4. Use according to claims 1 to 3, characterized in that the treatment is preventive.

5. Use according to claims 1 to 4, characterized in that the treatment is for curative purposes.

6. Use according to any one of the preceding claims, characterized in that the medicament is administered orally, intravenously or subcutaneously injected.

7. Use according to claim 4, characterized in that the drug is administered at a dose of 15 to 30 mg / kg / day orally.

8. Use according to claim 5, characterized in that the drug is administered at a dose of 40 to 60 mg / kg / day orally.

9. Use according to any one of the preceding claims, characterized in that the cocoa polyphenol extract has the following composition: 20 to 50% by weight of epicatechin equivalent of polyphenols, of which 55% to 80% of flavonoids,

3 to 15% by weight of lipids, of which 1 to 15% of β-sitosterol,

5 to 20% by weight of xanthines.

Polyphenol cocoa extract having the following composition:

20 to 50% by weight of epicatechin equivalent of polyphenols, of which 55% to 80% of flavonoids, 3 to 15% by weight of lipids including 1 to 15% of β-sitosterol, 5 to 20% by weight of xanthines.

11. Extract according to claim 10 for use as a medicament.

12. Use of the polyphenol cocoa extract according to claim 10 for the preparation of a medicament for preventing and / or treating prostate cancer.

13. Use of the polyphenol cocoa extract of claim 10 for the preparation of a medicament for alleviating the symptoms of prostate cancer.

14. Use of the cocoa polyphenol extract according to claim 10 for selectively inhibiting the proliferation of human prostatic tumor cells.

15. Use according to any one of claims 12 to 14, wherein the extract is administered in a mammal at doses ranging from 15 to 60 mg / kg / day, preferably ranging from 20 to 40 mg / kg.

16. Use of the cocoa polyphenol extract according to claim 10, for the preparation of a medicament for preventing and / or treating cognitive disorders.

17. Use according to claim 16 for improving the learning, storage and orientation capabilities in the space of a mammal.

18. Use of the extract according to claim 10, for the preparation of a medicament for preventing the overproduction of free radicals following a heat stroke.

The use of claim 18, wherein the drug is administered at a dose of Img / kg / day to 100mg / kg / day, before heat stroke or after heat stroke.

20. Use of the extract according to claim 10 for the preparation of a medicament for stimulating the production of dopamine.

21. Use according to claim 20, for the treatment of the symptoms of Parkinson's disease.

22. Use of the extract according to claim 10 for preventing oxidative stress.