Composition comprising plant extracts for improving female fertility and / or treating female infertility

Abstract

The present invention relates to a composition based on plant extracts for improving female fertility and / or treating female infertility. It uses a mixture of extracts of Cuscuta chinensis, Lycium barbarum, Morus alba, Rehmannia glutinosa and Carthamus tinctorius. This composition is available in various forms, such as tablets, capsules or drinkable solutions, and the efficacy thereof has been demonstrated in studies carried out on mice and zebrafish. Toxicity tests indicate that this composition is non-toxic. It improves indicators of female fertility, such as ovarian coefficient, and reduces oocyte malformations.

Description

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

[0001] 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.

[0002] Prior state of the art

[0003] Infertility is a fairly common condition these days. It is divided into two categories: female infertility and male infertility. To be considered infertile, a couple of reproductive age must have regular unprotected sexual intercourse for more than a year without conceiving. Currently, the rate of infertility is rising sharply, reaching unprecedented levels. It is clear that infertility has become a modern condition. Infertility can not only affect physical health but can also cause family problems. This condition can lead to family conflicts and disrupt a happy and fulfilling marriage. Therefore, this application describes a treatment based on plant extracts to address this problem.

[0004] Chinese herbal medicine offers the advantages of minimal side effects, significant efficacy, and low cost, making herbal remedies appealing. In treating infertility, Traditional Chinese Medicine (TCM) addresses the issue by identifying symptoms and utilizing Chinese herbal remedies to regulate the body's internal environment and bring hormone levels into the normal range, thereby promoting normal follicle development, ovulation, 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 in ovarian function, primarily composed of (as a percentage by total weight of the composition) Cuscuta chinensis 30-70%, Lycium barbarum 10-50%, Morns alba 4-20%, Rehmannia glutinosa 10-20%, and Carthamus tinctorius 1-10%. This composition has a significant proliferative effect on isolated ovarian granulosa cells and can significantly increase estrogen and progesterone secretion in these cells, enhancing the anti-apoptotic effect of granulosa cells. Isolated ovarian cells may reduce FSH, LH, and T levels in mice with polycystic ovary syndrome, and may have a modulating effect on sex hormones in mice with premature ovarian failure caused by cyclophosphamide. However, this patent does not mention the treatment of infertility, so its effect on infertility and / or fertility is unknown and unpredictable. Description of the invention

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

[0008] To achieve this objective, the present invention provides the following technical solution: a plant-based 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 can be selected 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 use and administration by the oral route. Thus, the dosage form of said composition may be in the form of an oral solution, capsules, granules, tablets, powder, gelatin capsules or softgels.

[0017] The present invention relates to a method for preparing a herbal composition consisting of pharmaceutically acceptable active ingredients and excipients, the active ingredient being 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 It can significantly increase the ovarian coefficient (P<0.01) in infertile mice and significantly reduce the rate of oocyte malformations (P<0.01). The effect of the composition of the present invention is superior to that of Cuscuta chinensis alone. Furthermore, 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 can contribute to improving 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 micronucleus test in mouse bone marrow erythrocytes 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 older mice; it can increase the number of embryos developing to the blastocyst stage in vitro, thereby improving cell morphology; it can also stimulate early hatching of fish larvae and promote embryonic development. Furthermore, acute toxicity tests have shown that this composition is non-toxic.

[0021] The present invention offers a new clinical use for the herbal composition, thus expanding its scope of application.

[0022] Another object of the invention is the use of the herbal composition according to the invention to improve female fertility and / or treat female infertility.

[0023] According to one embodiment, said use according to the invention can increase the ovarian coefficient, reduce the rate of malformations of oocytes 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 upon reading the description of exemplary embodiments, given purely for illustrative purposes and in no way limiting, with reference to the attached drawings in which:

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

[0028] [Fig.2] illustrates the effect of the plant-based 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 group of young mice with treatment.

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

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

[0033] [Fig.3] illustrates the effect of the plant-based 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 group of aged mice with treatment.

[0036] Detailed description of specific implementation methods

[0037] The following detailed description is merely illustrative and is not intended to limit the described embodiments or their applications and uses. As used herein, the term "illustrative" means "serving as an example, instance, or illustration." All implementations described below are illustrative implementations provided to enable persons skilled in the art to manufacture 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 the 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

[0044] 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) Proceed with extraction using an 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) Put into a jar and sterilize to obtain a drinkable solution of the herbal composition.

[0046] Example 2: Preparation of granules for the herbal 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) Wash the raw materials with water to remove impurities; (2) Soak the materials in water at 25°C for 60 minutes; (3) Proceed with extraction using an ethanol solution at a concentration of 70% for 60 minutes; (4) Extract three times, filter and concentrate the decoction; (5) Add starch and spray dry; and (6) Granulate, pack and sterilize to obtain granules of the herbal composition.

[0049] Example 3: Preparation of capsules for the herbal composition

[0050] 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) Wash the raw materials with water to remove impurities; (2) Soak the raw materials in water at 25°C for 60 minutes; (3) Proceed with extraction using an ethanol solution at a concentration of 70% for 60 minutes; (4) Extract three times, filter and concentrate the decoction; (5) Add starch and spray dry; and (6) Granulate, fill capsules, pack and sterilize to obtain a capsule of the herbal composition.

[0052] Example 4: Preparation of herbal composition tablets

[0053] The raw materials of the formula are: 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) Wash the raw materials with water to remove impurities; (2) Soak the raw materials in water at 25°C for 60 minutes; (3) Proceed with extraction using an 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 tablets, 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 a powder with a controlled 20-mesh fineness. The 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 a pH of 6.3 and thoroughly stirred in a shaker. The mixture was then heated under reflux for 12 hours to completely dissolve the active ingredients in the ethanol solution. The residue was filtered to obtain approximately 5000 kg of the crude solution of the invention. The crude solution was spray-dried. After drying, approximately 58 kg of dry extract of the plants of the invention is 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] This example aims 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 current standards, well known to those skilled in the art. Thus, these tests yielded the following results:

[0059] - The LD50 (Lethal Dose 50) of the acute oral toxicity test for female and male mice of the herbal composition prepared according to the method of Example 5 was greater than 10000 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, when the composition of the present invention, prepared according to Example 5, was administered to mice at doses of 2 g / kg bw / day, 4 g / kg bw / day, and 8 g / kg bw / day for 28 days, the animals' general condition was good, with no abnormal changes in body weight, feed intake, organ weights, or organ coefficients (organ coefficient = weight of each organ / body weight); no abnormal changes were observed. This was observed during eye examinations; the results of urinary, hematological, and biochemical indicators showed that all indicators were within the normal range; pathological and histological examinations of the organs showed no difference compared to those of the control group. Histological examination of all organs revealed no pathological changes related to the test samples.

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

[0062] - The Ames test, the mouse bone marrow multicolor erythrocyte micronucleus test and the 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 demonstrate 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, demonstrated 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 the 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) proceed with extraction using 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 lyophilization.

[0067] The experimental animals were healthy, mature, and fertile white mice, which were reared with free access to drinking water and randomly divided into five groups after a 7-day acclimation period. Sixty animals from 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 render them infertile. Among these, a low-dose group, consisting of 15 mice, received 175 mg / kg of the herbal composition of the present invention; a high-dose group, consisting of 15 mice, received 700 mg / kg of the herbal composition of the present invention; the Cuscuta chinensis group, consisting of 15 mice, received 305.5 mg / kg of the Cuscuta chinensis sample; Finally, the model group, composed of 15 mice, received no treatment other than the Busulfan injection. 15 animals in the control group received an intra- A single peritoneal injection of DMSO at a concentration of 3.75 ml / kg was administered. After 30 days of regular feeding, researchers evaluated the effects of doses of the herbal composition of the present invention on the fertility of infertile mice, compared simultaneously to a 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 were used as indicators.

[0068] Statistical analysis: All data were analyzed using one-way ANOVA (Analysis of Variance) and Student's t-test. 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 Mil-stage 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 reproductive parameters of female mice that received an injection of Busulfan.

[0073] [Tables 1]

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

[0075] As shown by the reproductive data, the herbal composition of the present invention, at both high and low doses, exhibits a higher ovarian coefficient (P<0.01) and a lower rate of oocyte malformation (P<0.01) compared to 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 in Example 2) can restore the Busulfan-induced infertility to a state 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 comparator 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 for determining the effect of the plant-based composition of the present invention on embryonic development.

[0077] Materials and Methods:

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

[0079] Transgenic Tg zebrafish (vas: EGEP (Enhanced Green Fluorescent Protein)) are reproduced by natural pair mating. Fluorescent zebrafish are randomly selected on the first day after fertilization and divided into four groups: a control group (treated with embryo culture medium), a group with a low concentration of the plant-based composition of the present invention (prepared according to the method of Example 5), a group with a medium concentration of the composition (prepared according to the method of Example 5), and a group with a high concentration of the composition (prepared according to the method of Example 5). Approximately 20 embryos are immersed in each group. After one day of immersion (2 days post-fertilization), approximately 10 zebrafish per group are randomly selected and photographed after fixation with low-melting-point adhesive.

[0080] Results :

[0081] As shown in [Fig. 1], after administering different concentrations of the plant-based composition of the present invention to TG transgenic zebrafish (vas:EGFP), a significant increase in the hatching rate of zebrafish larvae at 2 days post-fertilization was observed, indicating that the composition can stimulate early larval hatching 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 demonstrate that the plant-based composition of the present invention has a beneficial effect on oocyte maturation and on fe- in vitro conditioned 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 ground mouse food; the mixture is homogenized to prepare mouse food containing said composition. To ensure the administered dose, and considering 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 addition to consuming said composition.

[0087] After continuous administration for 4 weeks, an embryo development experiment was conducted 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 an intraperitoneal injection of PMSG (pregnant mare serum gonadotropin), followed by an intraperitoneal injection of hCG (human chorionic gonadotropin) 48 hours later. Female mice are then mated with normal male mice. Fertilized embryos are collected two days after mating and cultured in KSOM medium to the blastocyst stage. Embryonic developmental status (such as blastocyst count) is then measured and compared between the different groups.

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

[0091] Superovulation is induced in mice by an intraperitoneal injection of PMSG, followed by an intraperitoneal injection of hCG 48 hours later. Oocytes are collected 13 to 16 hours after the hCG injection and fertilized in vitro (IVF) in HTF (human tubal fluid). Sperm are removed 4 to 6 hours after fertilization, and the embryos are then cultured in KSOM medium and observed. Embryonic developmental statuses (such as fertilization rate and blastocyst rate) are then measured and compared between the 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 to 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 improves the ability to de- development of fertilized embryos in aged female mice.

[0096] 2) Compared to control groups, the blastocyst rate was higher in the groups of young and old mice administered with the composition of the present invention, indicating that said composition improves the blastocyst development capacity 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 fertilization rate of oocytes between the group of aged mice administered with the composition of the present invention and the control group.

[0099] It was noted in particular that the blastocyst rate was higher in the group of older mice administered with the composition of the present invention, which indicates that said composition improves the blastocyst development capacity in embryos resulting from in vitro fertilization, thereby increasing the reproductive capacity of older 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, thereby improving 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, thereby increasing reproductive capacity.

Claims

AMENDED CLAIMS received by the International Bureau on April 15, 2025 (15.04.2025)

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 its use in the treatment of female infertility.

2. Composition for its 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 selected 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. A method 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, and then by spraying said ingredients.

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

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

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

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