TW201600022A - Food compositions comprising tungsten (VI) salts - Google Patents

Abstract

The invention relates to food compositions comprising tungsten (VI) salts as well as the preparation thereof for favoring normal reproduction and fertility in a non-diabetic female mammal.

Description

Food composition including tungsten (VI) salt

The present invention relates to the use of tungsten (VI) salts for the preparation of food compositions and to such food compositions which are particularly useful for the normal reproduction and fertility of non-diabetic female mammals.

Fertility is a multi-stage process that is not completely natural and effective in mammals. In particular, factors such as age, eating habits or lifestyle adjust the success of this process. In this sense, for example, the pregnancy rate for women aged 20 to 24 is known to be about 86%, and it is reduced to 50% among women aged 35 to 32 [ Management of the infertile Woman by Helen A.Carcio; The Fertility Sourcebook by M.Sara Rosenthal, ASAS Summary of FAIR 2012] . Other factors, such as overweight or underweight, also cause delays in achieving a natural pregnancy or even impossible to achieve a natural pregnancy [ Fertil Steril 2013; 100:631-7 ]. It is estimated that approximately 66% of couples cannot naturally conceive during the first 3 months of unprotected sexual intercourse. This number is reduced to 15% of couples in the first year of unprotected sexual intercourse. The definition of infertility (WHO, ASRM, NICE) is not conceived after 12 months of regular, unprotected sexual intercourse. The term “low fertility” (especially in Europe) was also created to define the couples who caused any form or degree of fertility decline due to delay in conception [ Gnoth C et al. Definition and prevalence of subfertility and infertility, Human reproduction 2005; (5): 1144-1147 ].

The main causes of infertility among women include ovulation dysfunction and genital tract disease Change, oocyte quality degradation and follicle depletion inherent in aging. However, there is also a large proportion of women with unexplained infertility, also known as idiopathic infertility, one of the possible reasons for the defect in the implantation process.

For example, by some endocrine organs, such as pituitary gland, hypothalamic and thyroid hormone secretions regulate follicular development, ovulation, migration of immature eggs (oocytes) and subsequent implantation of fertilized and fertilized eggs On the wall of the uterus. In the different biological causes of female infertility, the reasons for the imbalance of hormone stimulation that regulates the entire process can also be pointed out.

Different treatments for female infertility are known, including, inter alia, the administration of pharmaceutical products for the treatment of hormonal problems including ovulation destruction. Similarly, it has been assumed that there are some beneficial effects based on the various treatments of the following: vitamin supplements, especially vitamin B, vitamin C, vitamin E and folic acid; mineral supplements such as selenium, zinc or iron or salts thereof; essential fatty acids (ω-3); and an extract from a plant, the plant such as a tree Vitex (chaste tree) (Vitex agnus-castus (Vitex agnus-castus)), Damiana (Damiana), licorice (licorice), red clover flowers (red clover flower), Chasteberry (chasteberry), black cohosh (black cohosh), Angelica (dong quai / Angelica sinensis), wild yam (wild yam) or sweet potatoes (sweet potato) (long pubescent yam (Dioscorea villosa)), false unicorn root (false unicorn root), green tea (green tea), nettle (nettles / Urtica dioica), wild oat (wild oats / Avena sativa), dandelion (dandelion) (Taraxacum officinale ( Taraxacum officinale )), etc., although the efficacy has not been clearly demonstrated in any of these treatments.

The proportion of pregnancy achieved by means of the aforementioned treatments has limitations. In this sense, for example, it has been observed that women who have irregular or no ovulation by means of administration of the stilbene citrate clomiphene citrate are allowed to recover ovulation at a high rate, but the pregnancy rate remains Lower, equal to or less than about 50%.

In vitro fertilization treatment is extremely effective in the oocyte fertilization step. However, the rate of embryo implantation on the uterine wall is low. This promotes multiple embryo transfer per in vitro fertilization cycle The result is a higher proportion of risky multiple pregnancies.

Finally, metabolic disorders such as diabetes or obesity are known to cause fertility limitations. Partial or complete recovery of blood glucose, insulinemia, and/or body weight of diabetic or obese female mice with impaired reproductive function is known to be accompanied by improvements in fertility.

When infertility is due to diabetes or insulin disorders, different pharmacological treatments (such as metformin or tungsten (VI) salts) or even lifestyle changes have been shown to improve diabetes or insulin disorders, such as insulin deficiency or insulin resistance, such as complete Or partially restore reproductive function.

In the specific case of sodium tungstate, rats with diabetes and insulin deficiency induced by streptozotocin injection are known to be partially partially reversed after treatment with sodium tungstate (10 weeks). Restore circulating insulin levels. Subsequently, when female rats recovered from the diabetic portion were mated with healthy male rats, it was observed that in parallel with the recovery from the diabetic portion, it partially restored its reproductive ability. In particular, it was observed that the ratio of the birth of female mice to the number of positive curettages has recovered from the diabetic portion after treatment with sodium tungstate, increasing to 66%, which is lower than that of non-diabetic female mice. The ratio is 100%). (See J. Ballester et al., "Tungstate administration improves the sexual and reproductive function in female rats with streptozotocin-induced diabetes", Human Reproduction, 2007, Vol. 22, pp. 2128-2135). However, neither tungsten nor (VI) salts have been observed to have an effect on non-diabetic females.

Traces of tungsten are found in animals and plants. For example, tungsten content up to 100 mg/kg in plants has been described [ Gbaruko BC & Igwe JC Global Tungsten: Occurrence, Chemistry, Environmental and Health Exposure Issues. Journal of Environmental Research 2007; 1(1): 27-32 ].

In view of the foregoing, despite extensive research and progress in understanding and manipulating the reproductive processes of mammals and especially humans, many couples do not benefit from the available Different methods of increasing fertility because it is ineffective. Therefore, there is still a need to find novel alternatives that provide greater efficacy for improving natural reproductive efficiency and/or treating female infertility.

It has been found that administration of a tungsten (VI) salt or a solvate of the salt is effective for aiding the normal reproduction and fertility of non-diabetic female mammals. Despite the fact that sodium tungstate is known to partially reverse the reproductive dysfunction of diabetic female mice (because of its normalized blood glucose levels), it has not been described or suggested that sodium tungstate may be a function of female reproductive system in non-diabetic female mammals. Has an effect that contributes to normal reproduction and fertility.

Accordingly, a first aspect of the present invention relates to the use of a tungsten (VI) salt or a solvate thereof for the preparation of a food composition which contributes to the normal reproduction and fertility of a non-diabetic female mammal.

A second aspect of the invention relates to a food composition comprising a tungsten (VI) salt or a solvate thereof at a concentration greater than 100 mg/kg.

As indicated above, according to a first aspect, the invention relates to the use of a tungsten (VI) salt or a solvate thereof for the preparation and/or promotion of normal reproduction and fertility of a non-diabetic female mammal. Food composition.

In the context of the present invention, the term "food composition" will encompass any solid or liquid tungsten (VI) salt-enriched food product and any nutritional supplement or supplement containing at least one tungsten (VI) salt.

The term "helps normal reproduction and fertility" or "promotes normal reproduction and fertility" means promoting normal reproduction and fertility or improving normal reproduction and fertility (reducing the time to achieve pregnancy), "normal reproduction and fertility" Understood to follow the definitions provided above Status of fertility in pregnancy: * In the case of low fertility: Pregnancy within 12 months of regular, unprotected sexual intercourse; and * In case of infertility: 12 months rule, unprotected sexual intercourse Pregnancy is achieved after the time period. This article includes the following needs: recovery and / or promote ovulation; improve oocyte and embryo quality; increase fertilized eggs implanted in the uterine wall; regulate hypothalamus - pituitary - ovarian axis disorders, including polycystic ovary syndrome, Metabolic syndrome, hyperprolactinemia, endometriosis, hypothyroidism, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, cirrhosis, rheumatoid arthritis, celiac disease, chronic renal failure, Idiopathic etiology and eating disorders (such as anorexia nervosa and bulimia).

According to a particular embodiment, the food composition of the present invention contains a tungsten salt of at least 100 mg/kg total concentration (i.e., the sum of all tungsten (VI) salts contained in the composition); according to other specific embodiments, The total concentration is preferably at least 150 mg/kg, at least 200 mg/kg, at least 250 mg/kg, at least 300 mg/kg, at least 350 mg/kg, at least 400 mg/kg, at least 450 mg/kg, at least 500 mg/kg, at least 550 mg/kg. At least 600 mg/kg, at least 650 mg/kg, at least 700 mg/kg, at least 750 mg/kg, at least 800 mg/kg, at least 850 mg/kg, at least 900 mg/kg or at least 850 mg/kg.

According to another particular embodiment, the total concentration of tungsten (VI) salts is equal to or less than 1000 mg/kg.

In the context of the present invention, a female mammal can be any mammal, including but not limited to humans, mice, rats, rabbits, dogs, cats, guinea pigs, hamsters, cows, horses, pigs, Sheep, goats, etc.

According to a particular embodiment, the female mammal is a human. According to another particular embodiment, the food composition will be suitable for a daily dose (hereinafter referred to as mg/kg/d) between 0.001 mg per kilogram of female mammal body weight and the maximum tolerated dose of the corresponding female mammal species. ) Cast tungsten salts.

According to another particular embodiment, the maximum daily dose will be 1000 mg/kg/d; according to additional specific embodiments, administration will be carried out at a dose of at least 0.001 mg/kg/d, at least 0.01 mg/kg/d, at least 0.1 Mg/kg/d, at least 0.5 mg/kg/d, at least 1 mg/kg/d, at least 10 mg/kg/d, at least 25 mg/kg/d, at least 50 mg/kg/d, at least 100 mg/kg/d, at least 200 mg/kg/d, at least 300 mg/kg/d, at least 400 mg/kg/d, at least 500 mg/kg/d, at least 600 mg/kg/d, at least 700 mg/kg/d, at least 800 mg/kg/d or at least 900 mg /kg/d.

Tungsten salts generally comprise a tungsten (VI) anion and a pharmaceutically or pharmaceutically acceptable cation. The cation is preferably an alkali metal or alkaline earth metal cation. The cation is more preferably selected from the group consisting of sodium, potassium, magnesium, calcium and zinc. According to a particular embodiment, the tungsten (VI) salt is a tungsten (VI) sodium salt.

The tungsten (VI) anion is further preferably selected from the list consisting of WO ₄ ^2- , HWO ₄ ^- , W ₂ O ₇ ^2- and HW ₂ O ₇ ^- . The anion is preferably WO ₄ ^2- .

According to a particular embodiment, the salt is in the form of a solvate; the solvate is preferably a hydrate, more specifically a dihydrate.

All of the features listed separately for different elements of the invention may be combined with each other, and all possible combinations are included within the scope of the invention. For example, according to a preferred embodiment, the solvate of the tungsten (VI) salt is a dihydrate and the cation is a sodium cation. Similarly, it is intended that the remaining possible combinations are included within the scope of the invention.

The food composition can be a liquid composition, according to the most commonly used term in society, that is, a beverage. In the context of the present invention, such liquid compositions include, but are not limited to, any beverage selected from the group consisting of animal or vegetable milk and any derivatives thereof, such as milkshakes, yogurt, kefir Kefir et al; juice and / or vegetable juice; still water or soda, or flavored or sweet water or drink (by means of nutritive sweeteners (sucrose, fructose, etc.) or artificial sweeteners); seasonings, Such as any salsa, salsa, seasoned ketchup, oil, vinegar or vinegar preparation; any type of alcoholic beverage; tea, coffee; and all types Refreshing drinks or refreshing drinks or refreshing drinks.

The food composition can also be a solid composition. Such solid compositions may, for example, be selected from, but not limited to, a group consisting of animal or vegetable milk derivatives such as cheese, butter, margarine and tofu; any type of bread, including fresh, packaged or frozen bread, Sliced bread, whole wheat bread, flavored bread, sweet bread, salted bread, etc.; pasta made from any grain flour (such as wheat or semolina flour) (macaroni, spaghetti, Noodles, etc.; baked goods, including cakes, biscuits, muffins, doughnuts, etc.; bulk or bagged brewing liquids, tea or coffee for preparing beverages; jelly, candy, including soft candy, preferably called "Soft fruit candy"; and any type of solid seasoning, such as oregano, salt, coriander, parsley, basil, etc.; or mixtures thereof.

Finally, the food composition may also be a nutraceutical, dietary or food supplement or supplement, and any of these terms are used in an equivalent manner in the context of the present invention. These terms are commonly used in oral consumption compositions containing the ingredients to be used in a supplemental diet, in the case of the present invention, tungsten (VI) salts/salts. It will never replace a conventional food or be the only component of a meal or diet. It can be found in different manifestations, such as tablets, pills, troches, capsules, soft gelatin capsules, gelatin capsules, wafers, foamed ingots, liquids (solutions, suspensions, syrups), granules The above-mentioned owners are included as specific embodiments within the scope of the present invention. Dietary or pharmaceutically acceptable excipients are readily apparent to those skilled in the art to obtain any of the foregoing forms of expression and are included within the scope of the invention.

According to a particular embodiment, any of the aforementioned food compositions (solid compositions, liquid compositions or nutritional supplements/supplements) comprises at least one nutritive sweetener, such as sucrose or fructose, in its composition. The intake of such nutritive sweeteners in diabetic patients must be controlled and limited, and therefore, food compositions comprising such nutritive sweeteners will be banned from diabetic mammals or at least the diet controlling diabetic mammals will be considered.

According to a particular embodiment, the non-diabetic female mammal is selected from the group consisting of needing to restore and/or promote ovulation, improve oocyte and embryo quality, increase fertilized egg implantation on the uterine wall, and regulate hypothalamic-pituitary-ovarian axis disorders. Group of female mammals, hypothalamic-pituitary-ovarian axis disorders including polycystic ovarian syndrome, metabolic syndrome, hyperprolactinemia, endometriosis, hypothyroidism, multiple sclerosis, Rheumatoid arthritis, lupus erythematosus, cirrhosis, rheumatoid arthritis, celiac disease, chronic renal failure, idiopathic etiology, and eating disorders such as anorexia nervosa and bulimia nervosa.

According to another specific embodiment, the non-diabetic female mammal is selected from the group consisting of female mammals having insulin resistance (also known as resistance to insulin or insulin); when insulin resistance is associated with hyperglycemia When it occurs, it can lead to the development of diabetes, but insulin resistance alone does not lead to diabetes. The female mammal having insulin resistance is preferably selected from the group consisting of female mammals having one or more of the following conditions: obesity or overweight, metabolic syndrome, pre-diabetes, polycystic ovary syndrome, hypertension, heart Disease, hyperlipidemia or dyslipidemia, thyroid hyperactivity, parathyroid hyperactivity, hyperleptinemia or leptin resistance, sedentary lifestyle, eating disorders, obstructive sleep apnea syndrome, fetal malnutrition, Puwei II Prader-Willi syndrome, Rabson-Mendenhall syndrome, fatty liver, leprechaunism, lesions associated with peroxisome secretion (eg, acromegaly), Pharmacological treatment (eg, glucocorticoids, thiazide diuretics, beta-blockers), insulin resistance caused by stress or early onset menarche [ Egas Béjar, Daniela et al. Insulinorresistencia/lnsulin resistance. Medicina (Guayaquil); 10(2): 159-166, April 2005; Graves, Thomas K.WHY ISN'T THIS INSULIN WORKING? Western Veterinary Conference 2013 (SA113); Ximena Gaete V. Adelanto de la pubertad en Chile y el mundo. Rev Chil Pediatr 77 (5); 456-465, 2006; Leszek Szablewski Glucose Homeostasis and Insulin Resistance (elSBN: 978-1- 60805-) Bentham e-book]. Many of these indications have been described to be unrelated to infertility, including pre- diabetes [ Acta Diab . Latina 4, 507, 1967 ].

Another aspect of the invention pertains to a food composition comprising a tungsten (VI) salt or salt or a solvate thereof at a concentration of at least 100 mg/kg. According to a particular embodiment, the tungsten (VI) salt or salt is included in the composition at a concentration of at least 150 mg/kg, at least 200 mg/kg, at least 250 mg/kg, at least 300 mg/kg, at least 350 mg/kg, at least 400 mg. /kg, at least 450 mg/kg, at least 500 mg/kg, at least 550 mg/kg, at least 600 mg/kg, at least 650 mg/kg, at least 700 mg/kg, at least 750 mg/kg, at least 800 mg/kg, at least 850 mg/kg, at least 900 mg /kg or at least 850mg/kg.

All of the definitions and preferred meanings provided above in relation to the first aspect of the invention apply to this second aspect of the invention. Thus, for example, the tungsten (VI) salt will comprise a tungsten (VI) anion and a pharmaceutically or pharmaceutically acceptable cation, preferably an alkali metal or alkaline earth metal cation, even more preferably sodium, potassium, magnesium and Calcium and zinc cations. In particular, the salt will be the tungsten (VI) sodium salt. The tungsten (VI) anion is again selected from the group consisting of WO ₄ ^2- , HWO ₄ ^- , W ₂ O ₇ ^2- and HW ₂ O ₇ ^- ions, preferably WO ₄ ^2- , and in the form of a solvate, The solvate will preferably be a hydrate, more specifically a dihydrate. The remaining specific embodiments provided above for the first aspect are also related to this second aspect.

The food composition of the second aspect of the invention may be a liquid composition or a beverage, a solid composition or a nutritional supplement or supplement (also known as a dietary or food supplement or supplement).

The liquid composition has been defined above in relation to the first aspect of the invention, and all of the preferred embodiments are also applicable to this second aspect of the invention.

Likewise, solid compositions have been defined above with respect to the first aspect of the invention, and all of the preferred embodiments are also applicable to this second aspect of the invention.

Similarly, a nutritional supplement or supplement (or diet or food supplement or supplement) has been defined above with respect to the first aspect of the invention, and all of the preferred embodiments are also applicable to this second aspect of the invention. .

The composition preferably comprises a nutritive sweetener such as sucrose or fructose.

A series of non-limiting, illustrative examples of the invention are included below.

Instance Example 1. Description of animal model (IRS2-/-female mouse) The mouse model used to determine the activity of tungsten (VI) salts for normal reproduction and fertility is the Irs2 knockout mouse IRS2 ^-/- [ Burks et al., "IRS-2 pathways integrate female reproduction and energy homeostasis" , Nature, 2000, Vol. 407, pp. 377-382 ]. The Irs2 gene deletion translates into a distinct amphoteric profile of fertility and carbohydrate metabolism.

Male mice of this model have insulin resistance and severe hyperglycemia that begin at an early age. In contrast, female mice maintained relatively normal blood glucose at early age and developed mild insulin resistance, which was maintained until the age of onset (4-5 months). IRS2 ^-/- female mice exhibited low follicular development and continued anovulation at early age (about 10 weeks of age), with the absence of an estrous cycle in most mice. The pregnancy rate of IRS2 ^-/- female mice was 9% compared to the 100% ratio of IRS ^wt (IRS-2 ^+/+ wild type) female mice. Assuming that female mice of these ages maintain normal blood glucose and that they only develop mild insulin resistance, the deep disorder of fertility is not a direct result of abnormal glucose metabolism.

Example 2. Ovulation, Implantation, and Pregnancy Studies A. Animals

IRS2 ^-/- female mice aged between 6 weeks and 8 weeks.

Six "wild-type" (IRS2 ^wt ) male mice aged between 6 and 8 weeks.

Female and male mice were housed separately under normal conditions, ie 12 h light/dark cycle and controlled temperature and humidity. The animals were fed ad libitum (also known as arbitrarily) on a standard feeding diet.

B. Method Pretreatment stage

After the acclimation period, IRS2 ^-/- female mice were housed in groups of 4-6 mice per cage. Animals were dosed with tungstate-free drinking water during the pre-treatment phase (2 weeks).

Treatment stage

To the drinking water (optional) by means of a solution of 2 mg/ml sodium tungstate dihydrate (commercially available from Carlo Erba) in distilled water after the pre-treatment phase (day 0 of treatment) and up to 4 weeks before the sacrifice of the animals With sodium tungstate. The daily dose of sodium tungstate ingested by the mice is about 180 mg/kg body weight.

mating

After the first three weeks of treatment, IRS2 ^-/- female mice were continuously housed in pairs in cages with IRS2 ^wt male mice.

Mice were observed daily to look for signs of pregnancy or birth.

After 4 weeks, male mice were exchanged between cages and the male mice were kept therein for another 4 weeks.

The administration of tungstate was maintained for 8 weeks, in which males and female mice were housed together. After these 8 weeks, treatment was withdrawn and males were housed with female mice for an additional 4 weeks.

sacrifice

After this time, female mice sacrificed and performed a biopsy to look for signs of pregnancy.

C. Results Results of ovulation studies

Six randomly selected female mice on days -8, -5, -2, -1, 7, and 8 during the pre-treatment period and during the first 3 weeks of treatment On the 14th, 15th, and 22nd day, a vaginal smear was performed to determine the stage of the estrous cycle.

A saline solution between 1 ml and 2 ml was introduced into the vagina of the mouse by Pasteur pipette. Vaginal secretions are collected by the same pipette and spread over the slide. After air drying, it was fixed and stained by Papanicolau technique.

The Papanicolau technique involves staining a vaginal smear fixed on a slide in the following manner: -10 times immersed in 50% v/v alcohol; - immersed in Harris hematoxylin solution for 3 minutes; - rinsed with running water; - 10 times immersed in acidic alcohol (1% hydrochloric acid); - rinsed with running water; -10 times immersed in 95% v/v alcohol; - immersed in OG-6 solution for 30 seconds; -10 times immersed in 96% v/v alcohol Medium; - immersed in eosin solution for 1 minute; -10 times immersed in 96% v/v alcohol; -10 times immersed in 86% v/v alcohol; and -10 times immersed in xylene

The formulations were analyzed in a single-blind manner by trained personnel to eliminate observer bias.

Samples are identified in the following stages: estrus, pre-estrus, estrus, late estrus, unresponsive or non-evaluable. The periodicity of the four phases of the 4-day to 6-day cycle indicates the normal estrous cycle, and the absence of this periodicity and persistence in the estrus, estrus, or estrus phase indicates that there is no estrous cycle.

In the 10 female mice subjected to the study, Table 1 summarizes the various stages of the estrous cycle in 6 of these IRS2 ^-/- female mice undergoing vaginal smears.

The stages of the estrus cycle are as follows: A: no estrus; D: estrus; P: estrus; E: estrus; and M: estrus.

The stages of the estrous cycle found in the vaginal smears of the IRS2 ^-/- female mice of Table 1 show that all mice were in the pre-emotional (P) or estrus (D) phase during the pre-treatment period, ie There is no estrous cycle.

However, after the start of administration of tungstate, it was observed that the IRS2 ^-/- female mice were in the later stages of the estrous cycle, that is, the estrus (E) and late estrus (M) phases, indicating a return to the normal estrous cycle.

These results indicate that administration of a tungsten (VI) salt allows rapid recovery (on the seventh day of treatment) 100% analysis of the estrous cycle of infertile non-diabetic IRS2 ^-/- female mice.

Implantation and pregnancy study results

After the hybridization period of the method of Part B, the female mice sacrificed and performed a biopsy to look for signs of pregnancy.

Table 2 summarizes the age at the time of treatment (whether or not there is a pregnancy) and the number of embryos per female mouse.

The results in Table 2 show that the pregnancy rate of TISS ^- treated IRS2 ^-/- female mice increased to 80% when the pregnancy rate of untreated IRS2 ^-/- female mice was 9%.

Moreover, these results also show that the average of the pups/implanted embryos of each pregnant female mouse is about 5, which can be considered to be comparable to the number of pups in female mice.

Thus, the results of Tables 1 and 2 illustrate that tungsten (VI) salts are effective treatments for restoring ovulation and/or increasing oocyte implantation. Therefore, administration of a tungsten (VI) salt as defined in the present invention is effective for treating infertility in a non-diabetic female mammal.

Example 3. Blood sugar and body weight studies A. Animals

Six IRS2 ^-/- female mice between the ages of 6 and 8 weeks.

Female mice were housed under normal conditions, ie 12 h light/dark cycle and controlled temperature and humidity. The animals were fed ad libitum (also known as arbitrarily) on a standard feeding diet.

B. Method Treatment stage

After the acclimation period, in the pre-treatment phase (day 0 of treatment) and after 12 days, the solution of 2 mg/ml sodium tungstate dihydrate (commercially available from Carlo Erba) in distilled water was poured into drinking water (optional). With sodium tungstate.

C. Results

Body weight was monitored on days 0, 2, 5, 7, 9, and 12 of the treatment period, and blood was drawn from the tail vein by means of a glucose sensor (AccuTrend glucose sensing) , Roche, Mannheim, Germany) Blood glucose was measured 6 hours after fasting on each of the previous days.

Table 3 summarizes the blood glucose levels expressed in mg/dl, and Table 4 summarizes the body weight of the mice studied in grams.

The results in Tables 3 and 4 show that no changes in body weight or blood glucose were observed 12 days prior to treatment during the administration of sodium tungstate, and female mice on day 7 of treatment tested during the same time period successfully reconstituted ovulation (see Table 1).

Thus, the results of Tables 1 through 4 illustrate that tungsten (VI) salts are therapeutically effective in restoring ovulation and/or increasing oocyte implantation, regardless of changes in body weight and carbohydrate metabolism. Thus, it is demonstrated that administration of a tungsten (VI) salt as defined in the present invention or a composition containing such a tungsten (VI) salt has a direct effect on the female reproductive system and thus contributes to the normality of non-diabetic female mammals. Reproductive and fertility are effective.

Example 4. Endometrial Embryo Adhesion Study A. Model

The "in vitro" human-human embryo adhesion model consisting of HEC1-A endometrial cell line and JEG-3 trophectoderm cell line was used to determine the effect of sodium tungstate on endometrial receptivity.

JEG-3 cell lines that mimic the trophectoderm cells of human embryos are cells that grow in a single layer using low adhesion plates in the laboratory and are capable of forming spheroids that mimic human embryos; this is the most widely used for in vitro embryos. One of the cell lines for adhesion analysis.

B. Method

Cell lines were purchased commercially (American Type Culture Collection (ATCC); Rockville, MD, USA) for endometrial embryo adhesion experiments. It was thawed and expanded for 4 passages to obtain enough cells for all analyses.

HEC1-A cells were plated in 24-well plates and cultured in McCoy 5A medium supplemented with 10% fetal bovine serum and 0.1% antibiotic (fungizone and penicillin) until 90% confluence was reached. After confluence, add sodium tungstate (final concentration 10 μM ), and Withaferin A (as a control to prevent embryonic adhesion) or medium (baseline adhesion data) without any additional The component lasts for 24 hours.

JEG-3 cells were plated in low adhesion plates using medium supplemented with 10% fetal bovine serum and 0.1% antibiotic (amphoteric acid B and penicillin) in Eagle's minimal essential medium (EMEM). Twenty-four hours before the adhesion analysis, JEG-3 trophoblast spheroids were formed by this culture. To this end, JEG-3 cells in the medium described above were suspended in an Erlenmeyer flask at a concentration of 6 x 10 ⁵ cells / 6 ml with stirring. The resulting spheroids were collected for adhesion analysis.

For the adhesion analysis, the medium was replaced with the fresh medium without the therapeutic agent of the study 24 hours after the HEC-1A cells were cultured with the medium supplemented with sodium tungstate, guanosine A or without any therapeutic agent. Adding nourishing ectodermal spheroids to a single layer of HEC-1A cells On the top, 6 to 10 spheroids are placed in each well. The adhesion of the spheroids was measured after 60 minutes, and the spheroids floating in the medium as the non-adhering spheroids and the spheroids which did not float as the adhering spheroids were counted. The inspection was carried out by a flip-chip microscope (Nikon Diaphot 300; Nikon Corporation, Tokyo, Japan). The analysis was performed in triplicate.

C. Results

The results obtained are shown in Table 5. A higher proportion of embryo adhesion was observed relative to the untreated condition of the HEC-1A strain treated with sodium tungstate. No adhesion was observed in the cells treated with the negative control (C-) Cyclosporin A.

Specific compositions of the invention

The food composition according to the invention is prepared below in an illustrative manner. It is in no way to be construed as limiting the scope of the invention.

Example 5

Prepare a tungsten salt-enriched salad dressing. Na ₂ WO ₄ (100 mg/kg) in an amount of 25 mg was added to 250 grams of commercial lees and mechanically stirred for 30 minutes.

Example 6

A seasoned ketchup-type salsa rich in tungsten salt is prepared. To this end, 170 mg of CaWO _{4 was} added to 200 grams of commercially available flavored tomato sauce and mechanically stirred for 1 hour.

Example 7

Preparation of tungsten salt-enriched milk. To this end, 1 kg of whole milk was supplied, 150 mg of ZnWO _{4 was} added and it was mechanically stirred for 20 minutes. It is recommended to squish the package immediately before consumption.

Example 8

Prepare a tungsten salt-enriched juice. 500 mg of MgWO _{4 was} added to 1 kg of commercially available juice and mechanically stirred for 10 minutes. It is recommended to squish the package immediately before consumption.

Example 9

A coated tablet having the following composition was prepared: PH200 microcrystalline cellulose (diluent/smoothing agent) 250.00 mg

Anhydrous gelatinous cerium oxide (smoothing agent/adsorbent) 3.00mg

Magnesium stearate (lubricant) 5.00mg

Talc (lubricant) 7.00mg

Opadry® white (Opadry® and -1-7000 White) (*) (coating film) 8.00mg

Sodium tungstate dihydrate 200.00mg

(*) a mixture of hydroxypropyl methylcellulose, polyethylene glycol 6000 and titanium dioxide (E-171)

Example 10

A foamed ingot having the following composition was prepared: sorbitol, aspartame, sucralose, and xylitol (sweetener) 0.025 mg/10 mg

Calcium carbonate 0.350mg/10mg

Citric acid (acidifier) 0.650mg/10mg

Acidic sodium carbonate (acidity regulator) 0.350mg/10mg

Orange Flavor 0.25mg/10mg

Tungsten salt

Example 11

A gelatin capsule having the following composition was prepared: Nifedipine 6 mg

Sanxianjiao 5mg

Orange Flavor 0.3mg

Citric acid 0.4mg

Gelucire 44/14 90mg

PH10 microcrystalline cellulose (diluent) 92.00 mg / capsule

Sodium tungstate dihydrate 100.00 mg / capsule

Claims (34)

A use of a tungsten (VI) salt or a solvate thereof for the preparation of a food composition which contributes to the normal reproduction and fertility of a non-diabetic female mammal. The use of claim 1, wherein the total concentration of the tungsten salts in the composition is equal to or greater than 100 mg/kg. The use of any of claims 1 and 2, wherein the mammal is a human. The use of claim 3, wherein the composition is administered in a daily dose of between 0.001 mg and 1000 mg of tungsten (VI) salt per kilogram of body weight of the female mammal. The use of any one of claims 1 to 4, wherein the salt comprises a tungsten (VI) anion and a pharmaceutically or pharmaceutically acceptable cation. The use of claim 5, wherein the cation is an alkali metal or alkaline earth metal cation. The use of any one of claims 5 or 6, wherein the cation is selected from the group consisting of sodium, potassium, magnesium, calcium, and zinc. The use according to any of the preceding claims, wherein the tungsten (VI) salt is a tungsten (VI) sodium salt. The use of any one of claims 5 to 8, wherein the tungsten (VI) anion is selected from the group consisting of WO ₄ ^2- , HWO ₄ ^- , W ₂ O ₇ ^2- and HW ₂ O ₇ ^- ions. The use of claim 9, wherein the anion is WO ₄ ^2- . The use according to any of the preceding claims, wherein the solvate is a dihydrate. The use of claim 6 to 11, wherein the solvate of the tungsten (VI) salt is a dihydrate and the cation is a sodium cation. The use of any of the preceding claims, wherein the composition is a liquid composition or a beverage. The use of claim 13, wherein the liquid composition or beverage is free from the group consisting of animal or vegetable milk and derivatives thereof, such as milkshakes, yogurt, and kefir; juice and/or Or vegetable juice; still water, soda and seasoning or sweet water; seasonings; alcoholic beverages; tea; coffee; and refreshing drinks or refreshing drinks. The use of any one of claims 1 to 12, wherein the composition is a solid composition. The use of claim 15, wherein the solid composition is selected from the group consisting of animal or vegetable milk derivatives such as cheese, butter, margarine and tofu; flour; bread; pasta; baked goods, including cakes and biscuits; Soft fruit candy; brewing liquid, tea or coffee; seasoning. The use of any of claims 1 to 12, wherein the food composition is a nutritional supplement or supplement. The use of claim 17, wherein it comprises at least one pharmaceutically acceptable excipient or carrier presented in the form of a pill, a lozenge, a tablet, a capsule, a powder, a wafer, Foamed powder or foamed ingot, solution, suspension, syrup or granules. The use of any of the preceding claims, wherein the composition comprises sucrose. The use according to any one of the preceding claims, wherein the non-diabetic female mammal is selected from the group consisting of needing to restore and/or promote ovulation, improve oocyte or embryo quality, increase fertilized egg implantation on the uterine wall, and regulate hypothalamus - In mammals with pituitary axis disorders, hypothalamic-pituitary axis disorders include polycystic ovarian syndrome, metabolic syndrome, hyperprolactinemia, endometriosis, hypothyroidism, multiple sclerosis, rheumatoid Arthritis, lupus erythematosus, cirrhosis, rheumatoid arthritis, celiac disease, chronic renal failure, idiopathic etiology, and eating disorders such as anorexia nervosa and bulimia nervosa. A food composition, characterized in that it comprises a concentration of greater than or equal to 100mg / kg of tungsten (VI) salt or solvate thereof. The composition of claim 21, wherein the salt comprises a tungsten (VI) anion and a pharmaceutically or pharmaceutically acceptable cation. The composition of claim 22, wherein the cation is an alkali metal or alkaline earth metal cation. The composition of any one of claims 22 and 23, wherein the cation is selected from the group consisting of sodium, potassium, magnesium, and calcium and zinc. The composition of any one of claims 21 to 24, wherein the tungsten (VI) salt is a tungsten (VI) sodium salt. The composition of any one of claims 22 to 25, wherein the tungsten (VI) anion is selected from the group consisting of WO ₄ ^2- , HWO ₄ ^- , W ₂ O ₇ ^2- and HW ₂ O ₇ ^- ions. The composition of claim 26, wherein the anion is WO ₄ ^2- . The composition of any one of claims 21 to 27, wherein the solvate is a dihydrate. The composition of any one of claims 21 to 28, wherein the solvate of the tungsten (VI) salt is a dihydrate and the cation is a sodium cation. The composition of any one of claims 21 to 29, wherein it is a liquid composition or a beverage, a solid composition or a nutritional supplement or supplement. The composition of claim 30, wherein it is a liquid composition or beverage as defined in claim 14. The composition of claim 30, wherein it is a solid composition as defined in claim 16. The composition of claim 30, wherein the food composition is a nutritional supplement or supplement as defined in claim 18. The composition of any one of claims 21 to 33, wherein it comprises sucrose.