Composition containing plant extracts intended to improve female fertility and / or treat female infertility

Abstract

The present invention relates to the field of health and nutrition, and in particular to dietary supplements. The invention relates to the use of a plant-based composition to improve female fertility and / or treat female infertility. Figure for the abstract: Fig. 1

Description

Title of the invention: Composition comprising plant extracts intended to improve female fertility and / or treat female sterility

[0001] The present invention relates to the field of health and nutrition and in particular to food supplements. The invention relates to the use of a plant-based composition for improving female fertility and / or treating female infertility.

[0002] State of the prior art

[0003] Infertility is a fairly common disease these days. It is divided into two categories: female infertility and male infertility. To be classified as infertile, the couple of childbearing age must have regular sexual intercourse without using contraception for more than a year without being able to conceive. Nowadays, the degree of infertility is increasing sharply, reaching an unprecedented level. It is evident that infertility has become a modern disease. Infertility can not only affect physical health, but can also cause family problems. This disease can cause family conflicts and disrupt a happy and fulfilling marriage. Therefore, a treatment based on plant extracts is described in the present application to solve this problem.

[0004] Chinese herbal medicine has the advantages of having only mild side effects, having significant efficacy and having a low cost, so there is an appeal for herbal remedies. In the treatment of infertility, Traditional Chinese Medicine (TCM) treats infertility by identifying the symptoms and using Chinese herbal medicine in a way that regulates the internal environment of the body and brings the hormone level to the normal range, thus promoting normal follicle development, ovulation and normal fertilization and pregnancy.

[0005] The applicant proposes a non-toxic and effective herbal treatment to solve this problem of female infertility.

[0006] Patent CN201210388218 discloses a Chinese medicine composition for counteracting the decline of ovarian function, mainly composed of (in % total weight of the composition) Cuscuta chinensis 30-70%, Lycium barbarum 10-50%, Morus alba 4-20%, Rehmannia glutinosa 10-20% and Carthamus tinctorius 1-10% which has a significant proliferative effect on isolated ovarian granulosa cells and can significantly increase the secretion of estrogen and progesterone in isolated ovarian cells, increase the anti-apoptotic effect of granulosa cells isolated ovarian cells and can reduce FSH, LH, and T levels in mice with polycystic ovary syndrome, and have a sex hormone modulating effect in mice with cyclophosphamide-induced premature ovarian failure. However, this patent does not mention the treatment of infertility, so its effect on infertility and / or fertility is unknown and not predictable. Statement of the invention

[0007] The technical problem solved by the present invention is to provide a herbal composition for use in the treatment of female infertility.

[0008] To achieve this objective, the present invention provides the following technical solution: a herbal composition, comprising the following raw materials or extracts of raw materials:

[0009] - between 30% and 50%, preferably between 34% and 48% by weight of Cuscuta chinensis,

[0010] - between 10% and 30%, preferably between 17% and 24% by weight of Lycium barbarum;

[0011] - between 10% and 30%, preferably between 15% and 22% by weight of Morus alba;

[0012] - between 5% and 20%, preferably between 10% and 15% by weight of Rehmannia glutinosa ; And

[0013] - Between 1% and 5%, preferably between 3% and 4% by weight of Carthamus tinctorius;

[0014] for its use in the treatment of female infertility.

[0015] According to one embodiment, the present invention relates to a plant-based composition consisting of active ingredients and pharmaceutically acceptable excipients. Thus, the pharmaceutically acceptable excipients may be chosen from: sucrose, dextrin, starch, lactose, mannitol, xylitol, chitosan, soluble starch, talc, water-soluble dextrin, sodium carboxymethylcellulose (CMS-Na), microcrystalline cellulose, micronized silica gel, hydroxypropyl starch, ethanol, hydroxypropylcellulose, polyethylene glycol, chitosan, alone or in combination.

[0016] The present invention relates to a composition for its use and administered orally. Thus, the dosage form of said composition may be in the form of an oral solution, capsules, granules, tablets, powder, gelatin capsules or soft capsules.

[0017] The present invention relates to a process for preparing herbal composition consisting of active ingredients and pharmaceutically acceptable excipients, wherein the active ingredient is obtainable by extraction, concentration and spray drying of said herbal compound.

[0018] The present invention relates to the use of a herbal composition for the treatment of female infertility. Compared to the model group, this composition can significantly increase the ovarian coefficient (PcO.Ol) in infertile mice and significantly reduce the oocyte malformation rate (PcO.Ol). The effect of the composition of the present invention is superior to that of the plant Cuscuta chinensis alone. In addition, it can promote follicular development and oocyte maturation in aging ovaries, improve cell morphology, and increase the number of embryos developing into blastocysts during in vitro fertilization. Finally, it can promote the early development of zebrafish larvae. The results suggest that the specific combination of medicinal plants used in the composition may contribute to the improvement of female fertility.

[0019] The present invention relates to the use of a herbal composition in the treatment of female infertility, which has been subjected to safety toxicity tests, a 28-day oral toxicity test, an Ames test, a mouse bone marrow erythrocyte micronucleus test and a mouse sperm malformation test, all demonstrating the absence of toxic effect of this composition.

[0020] The herbal composition of the present invention can significantly increase the ovarian coefficient of infertile mice and significantly reduce the rate of oocyte malformations; it can promote follicle development and oocyte maturation in aged mice; it can increase the number of embryos developing to the blastocyst stage in vitro, thereby improving cell morphology; it can also stimulate the early hatching of fish larvae and promote embryonic development. In addition, acute toxicity tests have shown that this composition is non-toxic.

[0021] The present invention provides a novel clinical use for the herbal composition, thereby broadening its scope of application.

[0022] Another object of the invention is the use of the herbal composition according to the invention for improving female fertility and / or treating female sterility.

[0023] According to one embodiment, said use according to the invention can increase the ovarian coefficient, reduce the rate of oocyte malformations and improve reproductive capacity in a state of infertility.

[0024] According to one embodiment, said use according to the invention can improve the development of fertilized embryos in the aged state and improve the development of fertilized embryos in vivo.

[0025] According to one embodiment, said use according to the invention can improve cell morphology, increase the number of embryos developing into blastocysts and improve reproductive capacity during in vitro fertilization. Brief description of the drawings

[0026] The present invention will be better understood on reading the description of exemplary embodiments, given purely for informational purposes and in no way limiting, with reference to the appended drawings in which:

[0027] [Fig-1] illustrates the effect of the herbal composition of the present invention on embryonic development in a zebrafish model according to Example 8.

[0028] [Fig.2] illustrates the effect of the herbal composition of the present invention on the in vitro embryonic development capacity of aged female mice according to the results of Example 9 A (Superovulation, Mating, IVC (in vitro culture)).

[0029] Figure 2A illustrates the results of the control group of young mice.

[0030] Figure 2B illustrates the results of the young mouse group with treatment.

[0031] Figure 2C illustrates the results of the control group of aged mice.

[0032] Figure 2D illustrates the results of the aged mouse group with treatment.

[0033] [Fig.3] illustrates the effect of the herbal composition of the present invention on in vitro fertilization of oocytes and embryonic development in aged female mice according to the results of Example 9 B (Superovulation, Mating, IVC).

[0034] Figure 3A illustrates the results of the control group of aged mice.

[0035] Figure 3B illustrates the results of the aged mouse group with treatment.

[0036] Detailed description of particular embodiments

[0037] The following detailed description is merely illustrative and is not intended to limit the disclosed embodiments or the applications and uses thereof. As used herein, the term "illustrative" means "serving as an example, instance, or illustration." All implementations described below are illustrative implementations provided to enable those skilled in the art to make or use the embodiments of the disclosure and are not intended to limit the scope of the disclosure, which is defined by the claims.

[0038] The term "ovulation" is understood to be that process by which a woman releases oocytes into the uterus.

[0039] The term "embryo" refers to the early stages of growth of an organism, including human and non-human mammals, following fertilization up to the blastocyst stage. An embryo is characterized by totipotent cells, which are undifferentiated.

[0040] The term "blastocyst" is given to an embryo of an organism (including human and non-human mammals) that grows 5 to 6 days after fertilization.

[0041] The following examples illustrate the invention without, however, limiting its application.

[0042] Examples

[0043] Example 1: Preparation of an oral solution of the herbal composition The raw materials of the formulation are as follows: 120 kg of Cuscuta chinensis, 60 kg of Lycium barbarum, 50 kg of Morus alba, 35 kg of Rehmannia glutinosa and 10 kg of Carthamus tinctorius.

[0045] Preparation method and steps: (1) Wash the raw materials with water to remove impurities; (2) Soak the raw materials in water at 25°C for 60 minutes; (3) Extract with ethanol solution at a concentration of 70% for 60 minutes; (4) Extract three times, filter and concentrate the decoction; (5) Add honey or potassium sorbate; and (6) Jar and sterilize to obtain an oral solution of the herbal composition.

[0046] Example 2: Preparation of the granules of the plant-based composition

[0047] The raw materials of the formulation are as follows: 120 kg of Cuscuta chinensis, 60 kg of Lycium barbarum, 50 kg of Morus alba, 35 kg of Rehmannia glutinosa and 10 kg of Carthamus tinctorius.

[0048] Preparation method and steps: (1) washing the raw materials with water to remove impurities; (2) soaking the materials in water at 25°C for 60 minutes; (3) extracting with the ethanol solution at a concentration of 70% for 60 minutes; (4) extracting three times, filtering and concentrating the decoction; (5) adding starch and spray drying; and (6) granulating, packaging and sterilizing to obtain granules of the herbal composition.

[0049] Example 3: Preparation of capsules of the herbal composition The raw materials of the formulation are as follows: 120 kg of Cuscuta chinensis, 60 kg of Lycium barbarum, 50 kg of Morus alba, 35 kg of Rehmannia glutinosa and 10 kg of Carthamus tinctorius.

[0051] Preparation method and steps: (1) washing the raw materials with water to remove impurities; (2) soaking the raw materials in water at 25°C for 60 minutes; (3) extracting with ethanol solution at a concentration of 70% for 60 minutes; (4) extracting three times, filtering and concentrating the decoction; (5) adding starch and spray drying; and (6) granulating, filling capsules, packaging and sterilizing to obtain a capsule of the herbal composition.

[0052] Example 4: Preparation of tablets of the herbal composition

[0053] The raw materials of the formula are as follows: 120 kg of Cuscuta chinensis, 60 kg of Lycium barbarum, 50 kg of Morus alba, 35 kg of Rehmannia glutinosa and 10 kg of Carthamus tinctorius.

[0054] Preparation method and steps: (1) washing the raw materials with water to remove impurities; (2) soaking the raw materials in water at 25°C for 60 minutes; (3) Extracting with ethanol solution at a 70% concentration for 60 minutes; (4) extract three times, filter and concentrate the decoction; (5) add starch and spray dry; and (6) granulate, press into tablet, pack and sterilize to obtain tablets of the herbal composition.

[0055] Example 5: Preparation of plant extracts for the preparation of a composition of the present invention

[0056] 150 kg of Cuscuta chinensis, 180 kg of Lycium barbarum, 50 kg of Morus alba, 90 kg of Rehmannia glutinosa and 16 kg of Carthamus tinctorius were ground in a pulverizer to obtain their powder form with a controlled mesh size of 20 mesh. The above powders of Cuscuta chinensis, Lycium barbarum, Morus alba, Rehmannia glutinosa and Carthamus tinctorius were mixed with 5000 kg of ethanol solution at a concentration of 75% and pH 6.3 and stirred well in a stirrer, then heated under reflux for 12 hours to completely dissolve the active ingredients in the ethanol solution. The residue was filtered to obtain about 5000 kg of crude solution of the invention. The above crude solution is spray dried. After drying, approximately 58 kg of dry extract of the plants of the invention are obtained, and the dry extract is finally processed to obtain the final product in the form of tablets, capsules, granules or powder.

[0057] Example 6: Toxicological safety test of the herbal composition prepared according to the method of Example 5

[0058] The purpose of this example is to demonstrate the safety and non-toxicity of the compositions according to the invention by means of the acute oral toxicity test, the 28-day oral toxicity test, the calculation of the NOAEL (No Observed Adverse Effect Level), the Ames test, the mouse bone marrow multicolor erythrocyte micronucleus test and the chromosomal aberration test. All these tests were carried out according to the standards in force, well known to those skilled in the art. Thus, these tests give the following results:

[0059] - The LD50 (Lethal Dose 50) of the acute oral toxicity test for female mice and males of the herbal composition prepared according to the method of Example 5 was greater than 10,000 mg / kg body weight (bw), and the sample was of practically non-toxic quality according to the acute toxicity dose classification standard;

[0060] - The results of the 28-day oral toxicity test showed that the composition of the present invention was practically non-toxic. Indeed, the composition of the present invention prepared according to Example 5 administered to mice at doses of 2 g / kg bw / day (d), 4 g / kg bw / d and 8 g / kg bw / d for 28 days, the general condition of the animals was good without any abnormal changes in body weight, feeding amount, organ weight or organ coefficients (organ coefficient = weight of each organ / body weight); no abnormal changes were found in the eye examination; the results of urinary, hematological and biochemical indicators showed that all indicators were within the normal range; pathological and histological examinations of the organs did not show any difference from those of the control group. Histological examination of all organs did not reveal any pathological changes related to the test samples.

[0061] - According to the 28-day oral toxicity test, no harmful effects were observed for the composition of the invention at doses of up to 8g / kg bw / day (equivalent to 100 times the recommended dose in humans);

[0062] - The Ames test, the multi-colored erythrocyte micronucleus test of the bone marrow mouse bone marrow and mouse sperm aberration test performed on the herbal composition of the present invention were all negative.

[0063] Example 7: Study of the effect of the herbal composition of the present invention (prepared according to the method of Example 2) on infertile female mice

[0064] This example aims to show the effect of the herbal composition of the present invention in vivo in an infertile mouse model. Indeed, the herbal composition of the present invention formulated at different concentrations has shown improved fertility in the Busulfan-induced model. In this model, Busulfan is a chemotherapeutic agent that renders mice sterile and is widely used to create infertility models (Abbas Ahmadi et al. Veterinary Research Forum. 2017; 8 (4) 281-286).

[0065] Materials and methods:

[0066] Preparation of Cuscuta chinensis sample: (1) wash 120 kg of Cuscuta chinensis with water to remove impurities; (2) soak Cuscuta chinensis in water at 25°C for 60 minutes; (3) extract with ethanol solution at a concentration of 70% for 60 minutes; (4) extract three times, filter and concentrate the decoction; and (5) dry into powder by freeze-drying.

[0067] The experimental animals were healthy, mature, and fertile white mice, which were raised with free access to drinking water, and randomly divided into five groups after a 7-day adaptation period. 60 animals in the experimental groups received a single intraperitoneal injection of Busulfan at a concentration of 30 mg / kg (solvent containing 3.75 ml / kg of DMSO (Dimethyl sulfoxide)) to be rendered infertile. Among them, a low-dose group, composed of 15 mice, received 175 mg / kg of the herbal composition of the present invention; a high-dose group, composed of 15 mice, received 700 mg / kg of the herbal composition of the present invention; the Cuscuta chinensis group, composed of 15 mice, received 305.5 mg / kg of the Cuscuta chinensis sample; Finally, the model group, composed of 15 mice, received no other treatment than the injection of Busulfan. 15 animals in the control group received an intravenous injection single peritoneal DMSO at a concentration of 3.75 ml / kg. After 30 days of regular feeding, the researchers evaluated the effects of the doses of the herbal composition of the present invention on the fertility of infertile mice, simultaneously compared to the group of 15 mice that received the Cuscuta chinensis sample. For this evaluation, the ovarian coefficient, the total number of eggs and the rate of oocyte malformation are used as indicators.

[0068] Statistical analysis: All data were analyzed using Student's one-way ANOVA (Analysis of Variance). Differences with p-values ​​<0.05 were considered significant.

[0069] Ovarian coefficient = (ovarian weight / body weight) * 1000

[0070] Oocyte malformation rate = (total number of ovulated eggs - normal stage Mil cells) / total number of ovulated eggs

[0071] Results: The results are presented in Table 1 below.

[0072] Table 1. Effect of the composition of the present invention on the parameters of re production of female mice injected with Busulfan.

[0073] [Tables 1] Group -3 Total number of ova Low dose group (175 mg / kg) 1.47+0.15** 32.0+2.8** 42.8+1.5** High dose group (700 mg / kg) 1.60+0.59** 36.3+5.6** 52.5+8.7** Cuscuta chinensis group (305.5 mg / kg) 0.59+0.05 20.3+1.5** 87.3+5.2 Control group 1.56+0.39** 32.1+5.0** 40.9+7.0** Model group 0.63+0.05AA 15.0+0.9AA 83.6+10.6AA

[0074] *P<0.05, **P<0.01 vs Model Group. AP<0.05, AAP<0.01 vs Control Group.

[0075] As shown in the reproduction data, the herbal composition of the present invention, both at high and low doses, has a higher ovarian coefficient (P<0.01) and a lower oocyte malformation rate (P<0.01) compared with the model group and these indicators are comparable to the normal control group. This also implies that the herbal composition (prepared according to the method of Example 2) can restore the infertility state caused by Busulfan to be comparable to normal. In contrast, the comparative group of Cuscuta chinensis showed no statistically significant difference compared to the control group in terms of ovarian coefficient and oocyte malformation rate. This indicates that this comparative group does not improve reproductive capacity in infertile mice. Therefore, the herbal composition of the present invention, thanks to its specific formulation, demonstrates the ability to improve reproductive capacity in infertile female mice.

[0076] Example 8: Zebrafish model to determine the effect of composition on herbal basis of the present invention on embryonic development.

[0077] Materials and Methods:

[0078] Zebrafish are reared in fish water at 28°C (water quality: 200 mg of sea salt added to 1 L of reverse osmosis purified water, conductivity 450-550 qS / cm; pH 6.5-8.5; hardness 50-100 mg / L CaCO3).

[0079] Transgenic zebrafish Tg (vas: EGFP (Enhanced Green Fluorescent Protein)) are bred by natural mating in pairs. The fluorescent zebrafish are randomly selected on the first day after fertilization and divided into four groups: control group (treated with embryo culture medium), low concentration group of the herbal composition of the present invention (prepared according to the method of Example 5), medium concentration group of the composition (prepared according to the method of Example 5) and high concentration group of the composition (prepared according to the method of Example 5); about 20 embryos are immersed in each group. After one day of immersion (2 days post fertilization), about 10 zebrafish per group are randomly selected and photographed after fixing with low-melting glue.

[0080] Results:

[0081] As shown in [Fig.l], after giving different concentrations of the herbal composition of the present invention to TG (vas:EGFP) transgenic zebrafish, a significant increase in the hatching rate of zebrafish larvae at 2 days post-fertilization was observed, indicating that the composition can stimulate early hatching of larvae and promote embryonic development.

[0082] Reference: Prostaglandin E2 promotes embryonic vascular development and maturation in zebrafish, Ugwuagbo KC Biology Open (2019) 8, bio039768. doi: 10.1242 / bio.039768.

[0083] Example 9: Effect of the herbal composition of the present invention (prepared according to the method of Example 2) on oocyte maturation and in vitro fertilization in aged female mice.

[0084] This example aims to show that the herbal composition of the present invention has a beneficial effect on oocyte maturation and on fe- in vitro condation in aged female mice.

[0085] Materials and Methods:

[0086] Powder of the composition of the present invention is added (at a dose of 3.2 mg / day) to the mouse food, ground into powder; the mixture is homogenized to prepare the mouse food containing said composition. To ensure the administered dose, and taking into account the daily food consumption of ICR mice of 4 to 8 g / day, the food is prepared by adding 3.2 mg of the composition of the present invention to 2 g of mouse food, thus ensuring that the mice maintain a normal diet in parallel with the intake of said composition.

[0087] After continuous administration for 4 weeks, an embryonic development experiment was carried out to study the effect of the herbal composition of the present invention on the reproductive capacity of aged female mice.

[0088] Part a: Superovulation, Mating, IVC (in vitro culture):

[0089] Superovulation is induced in mice by intraperitoneal injection of PMSG (Pregnant Mare Serum Gonadotropin), followed by intraperitoneal injection of hCG (Human Chorionic Gonadotropin) 48 hours later. The female mice are then mated with normal male mice. Fertilized embryos are collected two days after mating and then cultured in KSOM medium until the blastocyst stage. The developmental states of the embryos (such as the blastocyst rate) are then measured and compared between the different groups.

[0090] Part b: Superovulation, IVF, IVC:

[0091] Superovulation is induced in mice by intraperitoneal injection of PMSG, followed by intraperitoneal injection of hCG 48 hours later. Oocytes are collected 13 to 16 hours after hCG injection and are fertilized in vitro (IVF) in HTF (Human Tubal Fluid) medium. 4 to 6 hours after fertilization, sperm are removed and embryos are then cultured in KSOM medium and observed. The developmental states of the embryos (such as fertilization rate and blastocyst rate) are then measured and compared between different groups.

[0092] Results:

[0093] Part a:

[0094] The results for part a are illustrated in [Fig.2]. Thus, in the natural mating and in vitro culture experiment, the following results were observed:

[0095] 1) Compared with the control group of aged mice, the number of fertilized embryos in the group of aged mice administered with the composition of the present invention was higher, indicating that said composition can improve the ability to de- development of fertilized embryos in aged female mice.

[0096] 2) Compared to the control groups, the blastocyst rate was higher in the groups of young mice and old mice administered with the composition of the present invention, indicating that said composition can improve the development capacity of blastocysts in fertilized embryos.

[0097] Part b:

[0098] The results for part b are illustrated in [Fig.3]. Thus, the results of the IVF experiment showed that there was no significant difference in the oocyte fertilization rate between the group of aged mice administered with the composition of the present invention and the control group.

[0099] It was notably noted that the blastocyst rate was higher in the group of elderly mice administered with the composition of the present invention, which indicates that said composition makes it possible to improve the capacity for development of blastocysts in embryos resulting from in vitro fertilization, thus increasing the reproductive capacity of elderly female mice.

[0100] This example demonstrates that the composition of the present invention makes it possible, on the one hand, in the case of natural mating, to increase the number of fertilized embryos as well as to increase the rate of blastocysts, making it possible to improve the development capacity of embryos resulting from in vivo fertilization and, on the other hand, to increase the development capacity of blastocysts in embryos resulting from in vitro fertilization, making it possible to increase the reproductive capacity.

Claims

Claims

1. Herbal composition, comprising the following raw materials or extracts of raw materials: - between 30% and 50%, preferably between 34% and 48% by weight of Cuscuta chinensis, - between 10% and 30%, preferably between 17% and 24% by weight of Lycium barbarum; - between 10% and 30%, preferably between 15% and 22% by weight of Morus alba; - between 5% and 20%, preferably between 10% and 15% by weight of Rehmannia glutinosa; and - Between 1% and 5%, preferably between 3% and 4% by weight of Carthamus tinctorius; for use in the treatment of female infertility.

2. A composition for use according to claim 1, further comprising a pharmaceutically acceptable excipient.

3. Composition for its use according to claim 2, characterized in that said pharmaceutically acceptable excipient comprises at least one element chosen from: sucrose, dextrin, starch, lactose, mannitol, xylitol, chitosan, soluble starch, talc, water-soluble dextrin, sodium carboxymethylcellulose (CMS-Na), microcrystalline cellulose, micronized silica gel, hydroxypropyl starch, ethanol, hydroxypropylcellulose, polyethylene glycol, chitosan, alone or in combination.

4. Composition for use according to any one of claims 1 to 3, characterized in that it is in a form suitable for oral administration.

5. Composition according to claim 4, characterized in that the dosage form is in the form of an oral solution, capsules, granules, tablets, powder, gelatin capsules or soft capsules.

6. Process for preparing the composition according to any one of claims 1 to 5, characterized in that the active ingredients are obtained by extraction, concentration and drying, then by spraying said ingredients.

7. Composition for use according to any one of claims 1 to 5, for improving female fertility.

8. Composition for use according to claim 7, for improving the fertility of women suffering from infertility problems related at ovulation.

9. Composition for use according to claim 7, for improving the development of embryos resulting from fertilization from aging ovaries and improving the development of embryos resulting from in vivo fertilization.

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