FR2964834A1 - Antioxidant composition, useful as food supplement and to prevent and/or treat infertility, protect sperm and prevent spontaneous abortion/embryonic malformations, comprises prickly pear extract, vitamins B6, B9 and B12 and zinc - Google Patents

Abstract

Antioxidant composition comprises an extract of prickly pear, vitamin B6, B9 and B12, and zinc. ACTIVITY : Anabolic; Antiinfertility; Anti-Abortive. MECHANISM OF ACTION : None given.

Description

Field of the Invention The present invention relates to the field of the treatment of infertility in men and women. In particular, the present invention relates to a dietary supplement applied to prevent and / or treat infertility in humans and, especially marital infertility related to oxidative stress. STATE OF THE ART The causes of infertility are sought at four levels: sperm quality, ovulation, female and male genital tract, and incompatibility between semen and the female genital environment. Numerous studies have demonstrated the decline in sperm quality of fertile men over the last twenty years, which would lead to an alteration of fertility in the years to come. The issue of marital infertility must raise awareness of the impact on male and female fertility of environmental factors and nutritional factors. In industrialized countries, 15% of couples are infertile whose causes are identified but a large proportion show no anomaly at the level of each of the two spouses and yet they fail to conceive a child: it is infertility unexplained.

Infertility can be caused by the action of toxic agents, pesticides (dioxin with estrogenic effect by blocking androgen receptors), heavy metals, tobacco (through free radicals), alcohol, pharmacological agents , X and gamma rays, hormones (absorbed by livestock and then consumed by humans), infection or inflammation. These factors are likely to lead to the onset of oxidative stress. Oxidative stress is, in this context, the intermediary in infertility. Oxidative stress is the imbalance between the production of free radicals and antioxidant defenses. Under normal conditions, free radicals are permanently produced in the human body and destroyed by antioxidant defenses. This process is harmful to the body and is a major cause of damage to cellular structures including lipids and membranes, proteins and DNA. It should be noted that the risk of lipoperoxidation exists even in normally fertile couples, hence the importance of remedying oxidative stress. Oxidative stress is a risk factor for a condition that occurs in about half of all cases in infertile men.

The possible consequences of oxidative stress on fertility are as follows: - Breaks at the level of chromosomes altering the genetic message (measured by the sperm DNA fragmentation index (DFI)), fragmentation of the DNA can be isolated or associated with a decondensation of chromatin (expressed by the DNA decondensation index (DSI)); - Peroxidation of polyunsaturated fatty acids of cell membranes (identified by the analysis of membrane phospholipids) resulting in impaired motility and ability to fuse sperm, a deficit in permeability and disorders in trade up to cell death. In humans, oxidative stress will affect: sperm concentration, sperm motility (asthenospermia, less than 40% motile sperm), sperm morphology (teratospermia, less than 30% normal spermatozoa).

Impairment of sperm production, motility, trajectory and integrity are identified by spermogram and spermocytogram. In women, oxidative stress can cause the same problems for the oocyte as for the spermatozoon (attack of DNA and polyunsaturated fatty acids of the membrane). In addition, the woman is exposed to specific risks, depending on the deficit in certain antioxidants. For example, selenium deficiency (which is associated with a decrease in glutathione peroxidase) could lead to miscarriage as a result of direct oxidative damage to membranes and fetal DNA.

Studies on the causes of infertility and their treatments have already been performed and it is known that antioxidants such as carnitine, zinc, vitamin E, coenzyme Q10 and vitamin B12 act on the motility of sperm. Zinc and vitamin B12 also act on sperm production. Vitamin C helps protect the gene pool of sperm. Glutathione and selenium are two essential elements for the antioxidant defense of sperm, glutathione also helps increase sperm motility. So far, these different treatments have been used in isolation.

The object of the invention is therefore to propose a new composition having an antioxidant action for preventing and / or treating infertility in humans, at least as effective as those currently on the market at the present time.

DESCRIPTION OF THE INVENTION For this purpose, the present invention relates to an antioxidant composition comprising as active ingredients: an extract of prickly pears, vitamins B6, B9, B12, and zinc. The present applicant has determined that the use of conventional antioxidants such as vitamins A, C, E and selenium, has an inconsistent impact on both sperm survival and motility, fragmentation index of DNA (DFI), and on the analysis of membrane phospholipids. Thus, the mere fact of using antioxidants is not enough to make an effective composition to prevent and / or treat infertility in humans. The present invention thus relates to a very particular selection of antioxidants capable of preventing problems of infertility, miscarriage or embryonic malformations. The present applicant has in fact surprisingly discovered that the mixture of the abovementioned active compounds, namely an extract of prickly pears, vitamins B6, B9 and B12 and zinc, has a synergistic antioxidant activity in order to contribute to the protection of cellular constituents against oxidative damage, including reducing the fragmentation and decondensation of sperm DNA in humans. He discovered that prickly pear extract combined with the action of vitamins B6, B12, B9 and zinc reduced the risk of DNA fragmentation and reduced homocysteine levels via methylation, and a role in the metabolism and sperm formation in humans. In addition, zinc plays a role in testicular development, especially in the transformation of testosterone into DHT (dihydrotestosterone, active form of testosterone). Therefore, the composition according to the present invention does not intervene, unlike existing treatments, only on the mobility of sperm, but also acts on the protection of sperm membranes, protection of genetic background, mobility and trajectory of sperm and the supply of energy. Overall protection of spermatozoa is thus obtained.

Preferably, the prickly pear extract comprises, by weight relative to the total weight of the prickly pear extract, from 0.10% to 25%, preferably from 8% to 20% of betalaines, from , 10% to 25%, preferably 8% to 15% quercetin and 0.10% to 20%, preferably 6% to 15% terpenes. According to the present applicant, betalaines would indeed reduce the risk of DNA fragmentation and lipid peroxidation, as would quercetin, which would also protect the quality of sperm in humans, as well as the early stages of embryogenesis and therefore help to fight against miscarriage and malformations of the embryo in women. Quercetin would also have a protective effect on the germ line. Terpenes trigger the mechanism of protein repair HSPs. The process for obtaining an extract of prickly pearls enriched with betalaines, quercetin and terpene according to the invention comprises the step of carrying out an extraction with a polar solvent from fruit extract of Opuntia ficus indica in order to recover betalaines, quercetin and terpenes.

By extract is meant a composition obtained from a plant by a physical and / or chemical operation, in particular at least a part of the plant with at least one solvent. This process comprises in particular the following steps: prickly pear is placed in a polar solvent, then an extraction step is performed and the resulting extract is recovered. The polar solvent is preferably a solvent having a dipole moment other than zero and in particular greater than 1.5. The polar solvent is advantageously a protic solvent. Alcohols such as methanol, ethanol, propanol, butanol and pentanol, water as osmosis water, distilled, permuted are suitable polar solvent for carrying out the process. The solvent may furthermore comprise an additive such as an antioxidant agent, a preserving agent, one or more enzymes, in particular intended to degrade the plant walls, for example to facilitate the extraction, an anti-foaming agent, an acid or else a base, especially to modify the isoelectric point and promote precipitation. The extraction step can be done by: decoction, maceration, infusion, digestion, percolation, leaching, hot water entrainment, such as distillation or hydrodistillation, microwave assisted solvent, and ion exchange column . The decoction comprises the following steps: - the boiling of a mixture comprising prickly pear fruits in a polar solvent as defined above; and the maintenance of this boiling for a particular duration, in particular from 1 minute to 2 hours, in particular approximately 20 minutes, then optionally a return to ambient temperature. By "maceration" is meant a method of cold soaking the fruits of prickly pear in the polar solvent for a particular period of time in order to extract the soluble compounds therefrom. In particular, the maceration step has a duration ranging from 1 hour to 15 days, and preferably 1 day. In particular, it is at room temperature, ie 10 to 25 ° C. The infusion is a process of pouring the polar solvent preferably at boiling temperature onto the prickly pears fruits and then allowing the mixture to infuse for a period of one minute to two hours. Digestion is a process of heating a mixture comprising the prickly pear fruit in the polar solvent at a temperature below the boiling point, for example in the case of water at 10 to 89 ° C, and 25 to 70 ° C.

By "percolation" or "leaching" is meant a process of passing the polar solvent through the fruits of prickly pear. The distillation can be carried out for example by VMHD (pulsed vacuum microwave hydrodistillation). During this extraction step, the polar solvent is brought to a pressure greater than atmospheric pressure. In particular when the solvent is water, it may be in the subcritical state, that is to say that the water is at a temperature between 100 and 374 ° C. and at a pressure between 1 and 218 bars. Under these conditions, the water is in liquid form.

At the end of the extraction step, the polar solvent is brought to a temperature ranging from ambient temperature (25 ° C.) to a temperature of 300 ° C. above the boiling point of the solvent used. and preferably at the boiling point of the solvent used. Thus, the polar solvent is at least partially reduced or completely eliminated, in particular by evaporation, by sublimation, and preferably under reduced pressure. The term "boiling point of the solvent" means the highest temperature that the solvent can reach before evaporating, in gaseous form, freely. This temperature is calculated at atmospheric pressure of 1 atmosphere (101.3 kPa).

The extraction step of the process according to the invention may be continuous or discontinuous. The method according to the invention may comprise a step involving heating, in particular of a duration ranging from 1 second to 15 days, and in particular from 10 seconds to 8 hours.

In a further step, the extract recovered after extraction can be sterilized, in particular by heating with or without pressure and / or by passage over a membrane. This prickly pear fruit extract can also be disinfected, in particular by heating with or without pressure or by means of a disinfecting agent. Finally, this extract is usually dried. This drying can be carried out by lyophilization and / or by atomization. This polar solvent extraction process thus makes it possible to obtain a fig tree fruit extract of betalaines, quercetin and terpenes.

Thus, this method makes it possible to obtain a prickly pear extract that is particularly suitable for the antioxidant composition according to the invention. In addition, the antioxidant composition also has the following characteristic: selenium from 1% by weight relative to the weight of said composition, superoxide oxidase (SOD) from 1% by weight relative to the weight of said composition and vitamin C from 10% by weight relative to the weight of said composition, are excluded from said antioxidant composition In fact, low-dose vitamin C makes it possible to protect the DNA from free radicals and to protect the hydroxylases essential for the production of energy. However, at higher doses, like selenium and SOD, it would increase the decondensation of DNA with the risk of failure of fertilization in both men and women. Advantageously, the composition further comprises carnitine or one of their derivatives, and coenzyme Q10. The present applicant has then shown that when, in addition to the prickly pear extract, vitamins B6, B9, B12, and zinc, carnitine such as L-carnitine and coenzyme Q10 are used, an increase of Substantial motility and sperm production and a decrease in DFI are obtained in humans. Therefore, better protection of the DNA is obtained. Advantageously, the composition also comprises a w6 long-chain polyunsaturated fatty acid, oligo-proanthocyanidines (OPC), zeaxanthin or a mixture thereof. The addition of polyunsaturated fatty acids (PUFAs) including long chain (omega 6) and / or OPC, such as pine OPCs and zeaxanthin (carotenoid) further improves the protection of DNA. It has been shown that pine OPCs and zeaxanthin have an action on singlet oxygen, a key player in the oxidation of spermatozoa proteins. According to one characteristic of the invention, the oligo-proanthocyanidines are derived from pine extract and the long-chain polyunsaturated fatty acid in w6 is chosen from: γ-linolenic acid, di-homo-γ acid -linolenic or arachidonic acid. Preferably, the composition further comprises magnesium in salt or complex form, vitamin E, cysteine or a derivative thereof, arginine or a derivative thereof, tyrosine or a derivative thereof, or one of their combinations. Magnesium plays a role in the energy of spermatozoa, it would also help fight against sperm asthenia. Vitamin E would protect the lipid membrane against lipoperoxidation and thus provide membrane stabilization. In particular, the composition comprises a polyphenol-rich extract selected from: a rosemary extract, a green tea extract, a lemon balm extract, a basil extract, a thyme extract, a pericarp extract and citrus fruit, a berry extract, orthosiphon extract, camu camu extract, grape extract, olive fruit, pine bark, or a mixture thereof. Preferably, the composition comprises, besides zeaxanthin, other carotenoids chosen from: astaxanthin, lycopene, lutein, F3-carotene, or a mixture thereof. According to another characteristic of the invention, the zeaxanthin resulting from the tagete extract, the astaxanthin is derived from the Hæmatococcus pluvialis extract, the lycopene is derived from the tomato extract, the F3-carotene is derived from from an extract of Dunaliella. The present applicant has indeed found that the antioxidant composition according to the invention (extract of prickly pears, Vit B6, B9, B12 and zinc), associated with polyphenols extracts of green tea leaves (Thea sinensis), olives (Olea europaea), pine bark (Pinus pinaster), pomegranate (Punica granatum), flavonoids from the extract of pericarp and Citrus pulp (Citrus aurantium and Citrus limonium), lycopene extract of tomato pulp (Solanum lycopersicum) and other carotenoids, such as astaxanthin and zeaxanthin, would reduce the fragmentation index of DNA as well as the decondensation index of DNA to improve the quality of spermatozoa in humans. The active ingredients may also be associated with others selected from: vitamin D and other vitamins, alpha-lipoic acid, and other trace elements and minerals, amino acids, glutathione, and other enzymes. Another object of the present invention relates to a dietary supplement comprising the antioxidant composition as described above. By definition, a food supplement is a foodstuff whose purpose is to supplement a normal diet and which constitutes a concentrated source of nutrients or other substances having a nutritional or physiological effect, alone or in combination, marketed in the form of doses. By dose is meant all conventional forms: capsules, lozenges, tablets, ampoules, or sachets.

The various constituents of the food supplement can be administered simultaneously (in the same tablet for example) or as combination products for separate use or spread over time. In particular, dietary supplements are formulated to be administered orally. According to one characteristic of the invention, the food supplements are in the form of an aqueous solution or suspension in a glass, stick or beverage ampoule, dropper bottle, spray, soft capsule, or in the dry state of naked or coated tablets, dragees, capsules, capsules, powder, effervescent tablets, granules, sticks, lozenges. When the food supplements are in the form of a soft capsule or capsule, the envelope of these soft capsules or capsules may contain in particular animal gelatin such as fish gelatin, glycerin, or a source material plant such as a cellulose or starch derivative, or a vegetable protein. When the food supplements are in capsule, tablet, or granule form, the mixture of active ingredients may be attached to a powdery carrier such as silica, cellulose, and maltodextrin. Dietary supplements may comprise any suitable vehicle or excipient, acceptable from a nutraceutical point of view, as well as conventional additives, known to those skilled in the art, such as fillers, fluidizers, natural extracts, vitamins, minerals, trace elements, amino acids, fatty acids, anti-caking agents, natural oils, flavors, colorants, acidifiers, sweeteners, etc.

The fillers are advantageously microcrystalline cellulose, maltodextrin potato or corn. The thickener used preferably is potato starch, hydroxypropyl methylcellulose, citrus pectin, guar gum, carob, agar-agar, konjac, hydrogenated oils, beeswax.

The fluidizers may be magnesium silicate, magnesium stearate, colloidal silica. Anti-caking agents are those usually used in the food industry, such as magnesium stearate, colloidal silica.

The vitamins are selected from, among others, vitamins C, E, B6, B1, B2, B3, B5, B8. As an acid, citric acid may be used in the composition of the present invention.

The aromas that can be used are varied, such as aroma of coffee, lemon, apple, peach, chocolate, vanilla, strawberry. The sweeteners used are, inter alia, inulin, xylitol, aspartame, glucose syrup, fructo-oligosaccharide syrup, maltitol powder or syrup, acesulfame potassium, fructooligosaccharide, sucralose, neohesperidin, steviosides and sodium cyclamate. Fatty acids can also be added to the dietary supplement, such as omega 3, galactolipids, but also minerals: chromium, boron, magnesium, calcium, iron, molybdenum and amino acids, such as tryptophan, leucine, arginine and glycine.

All these compounds are in no way limiting, pharmaceutically and / or food acceptable agents that can be added to the composition according to the present invention. Preferably, the food supplements are preferably administered to humans, one to four times per day, per dose comprising from 10 mg to 3 g, preferably from 20 mg to 2 g and even more advantageously from 100 mg to 900 mg. mg of the mixture of assets for a period of 5 days to 3 years. The dietary supplements according to the invention make it possible to protect spermatozoa in humans and oocytes in women. The supplements according to the invention make it possible to prevent oxidative damage (on membranes, on DNA) on the embryo and thus reduce the risk of miscarriages. They also prevent the risk of early vascular damage at implantation (prevent elevation of homocysteine) and thus also allow, through this second mechanism, to reduce the risk of miscarriages, including miscarriages early. Thus, said supplements advantageously make it possible to reduce the risk of early miscarriages and the risk of embryonic malformations, in particular malformations: of the neural tube; -vésicales; - heart. Thus, the subject of the present invention is also an antioxidant composition as described above or a dietary supplement as described above, applied to prevent and / or treat infertility in humans, to ensure protection of spermatozoa in man, prevent miscarriages or embryonic malformations in pregnant women. Nonlimiting examples of embodiment The following examples are intended to illustrate the invention without in any way limiting its scope. Example 1: dietary supplement recommended for humans when the fragmentation of the sperm DNA is greater than 20% or is associated with the elevation of the chromatin decondensation The dietary supplement according to the invention preferably corresponds to the composition weight: For 2 tablets of 869.03 mg, per tablet: Marine Magnesium 80 - 300 mg, Acerola Berry Extract (Vitamin C) 5 - 40 mg, Prickly Pear Extract 20 - 200 mg, Leaf Extract of green tea 20 - 70 mg, zinc citrate 10 - 30 mg, Dunaliella extract (beta-carotene) 10 - 30 mg, olive fruit extract 20 - 50 mg, extract of pericarp and citrus fruit (bioflavonoids ) 10 - 40 mg, vitamin E 3 - 10 mg, pyridoxine hydrochloride (vitamin B6) 0.5 - 1.5 mg, folic acid (vitamin B9) 0.05 - 1 mg, cyanocobalamin (vitamin B12) 0.00030 - 0.000120 mg, tomato extract (lycopene) 0.5 - 1.5 mg.

For this particular tablet, the recommended dose is two tablets a day. Example 2: dietary supplement recommended for humans when the fragmentation of the sperm DNA is greater than 40% The dietary supplement according to the invention preferably corresponds to the following weight composition: For 1 tablet of 850 mg per tablet: L-cysteine 50 - 250 mg, prickly pear extract 50 - 200 mg, L-carnitine 40 - 150 mg, L-tyrosine 40 - 150 mg, tagete extract (zeaxanthin) 60 - 120 mg, Haematococcus pluvialis extract (astaxanthin) 30 - 90 mg, green tea leaf extract (polyphenols) 40 - 80 mg, zinc citrate 10 - 30 mg, beta-carotene 2 - 5 mg, olive fruit extract 25 - 50 mg, extract acerola berry (vitamin C) 10 - 50 mg, extract of pericarp and citrus fruit (bioflavonoids) 10 - 30 mg, coenzyme Q10 10 - 40 mg, extract of maritime pine bark (OPC) 10 - 50 mg, vitamin E 3 - 10 mg, pyridoxine hydrochloride (vitamin B6) 1 - 3 mg, tomato extract (lycopene) 0.5 - 2 mg, folic acid (vi B9) 0.04-1 mg, cyanocobalamin (vitamin B12) 0.00020 - 0.000120 mg, For a 500 mg capsule, per capsule: shark liver oil (alkylglycerols) 200 - 500 mg. For this particular tablet and capsule, the recommended dose is one tablet and one capsule per day.

Example 3: dietary supplement recommended for humans when the decondensation of the DNA is isolated The dietary supplement according to the invention preferably corresponds to the following composition by weight: For 1 tablet of 500 mg per tablet: extract of prickly pears 50 - 300 mg, zinc citrate 10 - 30 mg, pyridoxine hydrochloride (vitamin B6) 0.5 - 1.5 mg, folic acid (vitamin B9) 0.05 - 1 mg, cyanocobalamin (vitamin B12) 0.00030 - 0.000120 mg. For this particular tablet, the recommended dose is one tablet per day.

Example 4: dietary supplement recommended for humans in the event of asthenia of spermatozoa (less than 40% of motile spermatozoa) The dietary supplement according to the invention preferably corresponds to the following weight composition: For 2 tablets of 800 mg, per tablet: Marine Magnesium 80 - 300 mg, Prickly Pear Extract 20 - 200 mg, Pomegranate Fruit Extract 20 - 200 mg, Zinc Citrate 10 - 30 mg, Vitamin E 3 - 10 mg, Pyridoxine Hydrochloride (Vitamin B6) 0.5 - 2 mg, folic acid (vitamin B9) 0.05 -1 mg, cyanocobalamin (vitamin B12) 0.00030 - 0.000120 mg, N-acetyl cysteine 20 - 200 mg, L-arginine 50 - 500 mg , L-carnitine 70 - 140 mg, coenzyme Q10 10 - 30 mg. For this particular tablet, the recommended dose is two tablets a day. Example 5: dietary supplement recommended for the woman during conception The dietary supplement according to the invention preferably corresponds to the following weight composition: For 1 tablet of 800 mg: extract of prickly pear 50 - 250 mg, zinc gluconate 10 - 30 mg, pyridoxine hydrochloride (vitamin B6) 1 - 3 mg, folic acid (vitamin B9) 0.05 - 1 mg, cyanocobalamin (vitamin B12) 0.00030 - 0.000120 mg. For this particular tablet, the recommended dosage is one tablet per tablet. Although the invention has been described in connection with a particular embodiment, it is quite obvious that it is in no way limited and that it includes all technical equivalents of the means described and their combinations if they fall within the scope of the invention. day. 15 20

Claims (12)

REVENDICATIONS1. Antioxidant composition comprising as active ingredients: an extract of prickly pear, vitamins B6, B9, B12 and zinc. Antioxidant composition according to claim 1, wherein the prickly pear extract comprises, by weight relative to the total weight of the prickly pear extract, from 0.10% to 25% of betalaines, 0.10% to 15% quercetin and 0.10% to 20% terpenes. 3. Antioxidant composition according to one of the preceding claims, wherein selenium from 1% by weight relative to the weight of said composition, superoxide oxidase (SOD) from 1% by weight relative to the weight of said composition and vitamin C from 10% by weight relative to the weight of said composition, are excluded from said antioxidant composition 4. Antioxidant composition according to one of the preceding claims, further comprising carnitine or a derivative thereof, and coenzyme Q10. 5. An antioxidant composition according to one of the preceding claims, further comprising a polyunsaturated fatty acid long chain w6, oligo-proanthocyanidines, zeaxanthin or a mixture thereof. 6. Antioxidant composition according to claim 3, wherein the oligo-proanthocyanidines is derived from the pine extract and the long-chain polyunsaturated fatty acid in w6 is chosen from: γ-linolenic acid, the acid di-homo-ylinolenic or arachidonic acid. 7. Antioxidant composition according to one of the preceding claims, further comprising magnesium in salt or complex form, vitamin E, cysteine or a derivative thereof, arginine or a derivative thereof , tyrosine or a derivative thereof, or a combination thereof. 8. Antioxidant composition according to one of the preceding claims, further comprising a polyphenol-rich extract selected from: a rosemary extract, a green tea extract, a lemon balm extract, a basil extract, a thyme extract , an extract of pericarp and citrus fruit, a berry extract, an orthosiphon extract, a camu camu extract, a grape extract, olive fruit, pine bark, or a mixture thereof. 9. anti-oxidant composition according to one of the preceding claims, further comprising carotenoids selected from: astaxanthin, lycopene, lutein, F3-carotene, or a mixture thereof. 10. An antioxidant composition according to claim 5 or 9, wherein the zeaxanthin is derived from the tagete extract, the astaxanthin is derived from the Haematococcus pluvialis extract, the lycopene is derived from the tomato extract , F3-carotene is derived from an extract of Dunaliella. 11. Food supplement comprising the antioxidant composition according to one of claims 1 to 10. 12. Antioxidant composition according to one of claims 1 to 10 or dietary supplement according to claim 11, applied to prevent and / or treat infertility, provide protection of sperm, prevent miscarriage or embryonic malformations.