A composition comprising radix dipsaci, epimedium and red peony root and its use for the prevention and treatment of premature ovarian failure

Abstract

The application discloses a first composition containing radix dipsaci, epimedium and red peony root, which is composed of radix dipsaci, epimedium, red peony root, ligusticum wallichii, sparganium stoloniferum bunge, curcuma zedoary and anemarrhena asphodeloides. The mass ratio of the epimedium, the radix dipsaci, the red peony root, the ligusticum wallichii, the sparganium stoloniferum bunge, the curcuma zedoary and the anemarrhena asphodeloides is (1-10):(0.1-5):(0.1-5):(0.1-5):(0.1-5):(0.1-5):(0.1-5). In the application, the effective range of the traditional Chinese medicine formula is screened by CCK8 experiment by using the uniform design method. Then, the POF mouse model is constructed by intraperitoneal injection of cisplatin (CDDP), and the traditional Chinese medicine formula 2 (GSB-2) and the traditional Chinese medicine formula 9 (GSB-9) are given for intervention, so as to explore the protective effect of the traditional Chinese medicine formula 2 (GSB-2) and the traditional Chinese medicine formula9 (GSB-9) on the ovary of the POF mouse.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a composition comprising Dipsacus asper, Epimedium and Paeonia lactiflora, and its use in preventing and treating premature ovarian failure. Background Technology

[0002] Premature ovarian failure (POF) refers to a condition in women of premenopausal age characterized by elevated levels of gonadotropins (FSH and LH), decreased ovarian reserve, and symptoms primarily including amenorrhea, delayed menstruation, and infertility. Data shows that the global incidence of POF is [missing data].

[0003] The incidence rate in women with premature ovarian failure is 1.0%, while in China it ranges from 1.0% to 3.8%. Studies have found that premature ovarian failure not only affects female fertility but also increases the risk of cardiovascular disease, osteoporosis, and depression. Therefore, finding effective treatments for premature ovarian failure is crucial.

[0004] Traditional Chinese medicine believes that premature ovarian failure (POF) is caused by kidney deficiency, spleen weakness, liver stagnation and blood depletion, leading to malnourishment of the uterus and the Chong and Ren meridians. The prior art does not contain the composition of Dipsacus asper, Epimedium, and Paeonia lactiflora claimed in this invention, nor its use in the prevention and treatment of premature ovarian failure. Summary of the Invention

[0005] Based on this, the present invention provides a first composition comprising Dipsacus asper, Epimedium and Paeonia lactiflora, wherein the first composition is composed of Dipsacus asper, Epimedium and Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria and Anemarrhena asphodeloides;

[0006] The mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria and Anemarrhena asphodeloides is (1-10):(0.1-5):(0.1-5):(0.1-5):(0.1-5):(0.1-5):(0.1-5).

[0007] Furthermore, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria and Anemarrhena asphodeloides is (4-8):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5).

[0008] Furthermore, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria and Anemarrhena asphodeloides is (5-7):(0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2).

[0009] Furthermore, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria and Anemarrhena asphodeloides is approximately 6:1:1:1:1:1:1:1:1:1.

[0010] Furthermore, the weight of the epimedium ranges from 10 to 20g, for example, about 15g.

[0011] Furthermore, the mass of the Dipsacus asperoides ranges from 1 to 5g, for example, about 2.5g.

[0012] Furthermore, the weight range of the red peony is 1 to 5g, for example, about 2.5g.

[0013] Furthermore, the weight range of the chuanxiong is 1–5g, for example, about 2.5g.

[0014] Furthermore, the mass of the triangular prism ranges from 1 to 5g, for example, about 2.5g.

[0015] Furthermore, the weight of the turmeric ranges from 1 to 5g, for example, about 2.5g.

[0016] Furthermore, the weight range of Anemarrhena asphodeloides is 1–5g, for example, about 2.5g.

[0017] According to another aspect of the present invention, a second composition comprising Dipsacus asper, Epimedium and Paeonia lactiflora is provided, the second composition comprising Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas; wherein the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas is (4-25):(0.5-5):(0.5-5):(0.5-5):(0.5-5):(0.5-5):(0.5-5):(0.5-10):(0.1-1).

[0018] Furthermore, the mass ratio of the Epimedium, Dipsacus, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas is (10.5–22.5): (1–3): (1–3): (1–3): (1–3): (1–3): (1–3): (1–3): (1.2–5.7): approximately 0.3.

[0019] Furthermore, the mass ratio of the Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas is approximately 19.5: approximately 1.5: approximately 1.5: approximately 1.5: approximately 1.5: approximately 1.5: approximately 1.2: approximately 0.3.

[0020] Furthermore, the mass ratio of the Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas is approximately 22.5: approximately 1: approximately 1: approximately 1: approximately 1: approximately 1: approximately 1: approximately 1.2: approximately 0.3.

[0021] Furthermore, the mass ratio of the Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas is approximately 15:1.5:1.5:1.5:1.5:1.5:1.5:1.5:5.7:0.3.

[0022] Furthermore, the mass ratio of the Epimedium, Dipsacus, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas is approximately 10.5: approximately 3: approximately 3: approximately 3: approximately 3: approximately 3: approximately 3: approximately 1.2: approximately 0.3.

[0023] Furthermore, the mass ratio of the Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas is not approximately 6:1:1:1:1:1:1:1:1:1:8:10.

[0024] Furthermore, the first composition or the second composition further comprises one or more pharmaceutically acceptable excipients.

[0025] Furthermore, the excipient is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, encapsulating agents, flavoring agents, sustained-release agents, flow aids, lubricants, dispersants, plasticizers, opacifiers, and antioxidants.

[0026] Furthermore, the dosage form of the first composition or the second composition is a powder, tablet, pellet, capsule, film, lozenge, granule, injection, or oral liquid.

[0027] Furthermore, the first composition or the second composition further comprises one or more other drugs for the prevention and / or treatment of premature ovarian failure.

[0028] Furthermore, these other drugs include hormonal drugs and non-hormonal drugs.

[0029] Furthermore, these hormonal drugs include estrogen drugs, progestin drugs, and combined estrogen-progestin preparations.

[0030] Furthermore, the estrogen-based drugs include 17-β estradiol, estradiol valerate, and conjugated estrogens.

[0031] Furthermore, the progestin drugs include micronized progesterone, dydrogesterone, and medroxyprogesterone acetate.

[0032] Furthermore, the estrogen-progestin combination preparation includes estradiol tablets / estradiol dydrogesterone tablets and estradiol valerate / estradiol valerate cyproterone acetate tablets.

[0033] Furthermore, the non-hormonal drugs include herbal drugs and phytoestrogens.

[0034] According to another aspect of the present invention, the use of the first composition or the second composition described above in the preparation of a medicament for the prevention and / or treatment of premature ovarian failure is provided.

[0035] Furthermore, the first composition or the second composition prevents and / or treats premature ovarian failure through one or more of the following mechanisms: promoting the proliferation of KGN cells damaged by chemotherapy drugs, improving estrous cycle disorders, increasing ovarian index, improving ovarian tissue pathological abnormalities, and improving abnormal serum biochemical indicators.

[0036] Furthermore, the chemotherapy drug is cisplatin.

[0037] Furthermore, this estrous cycle disorder includes prolonged and sustained interestrous periods.

[0038] Furthermore, the ovarian index is the percentage of bilateral ovarian wet weight to body weight.

[0039] Furthermore, the pathological abnormalities in this ovarian tissue included a decrease in the number of primordial follicles, a decrease in the number of growing follicles, a decrease in the number of mature follicles, and an increase in the number of atretic follicles.

[0040] Furthermore, the abnormal serum biochemical indicators included decreased estradiol levels, decreased anti-Miller hormone levels, increased follicle-stimulating hormone levels, and increased luteinizing hormone levels.

[0041] The beneficial effects of this invention are:

[0042] Cisplatin is one of the most widely used chemotherapy drugs, which can damage the ovarian structure and seriously affect women's physical and mental health. In this invention, the effective range of the traditional Chinese medicine formula was first screened using the CCK8 assay with a uniform design method. Then, combined with in vivo experiments, a POF mouse model was constructed by intraperitoneal injection of cisplatin (CDDP), and traditional Chinese medicine formulas 2 (GSB-2) and 9 (GSB-9) were administered to investigate the protective effects of traditional Chinese medicine formulas 2 (GSB-2) and 9 (GSB-9) on the ovaries of POF mice. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0044] Figure 1 This diagram illustrates the effect of different concentrations of CDDP on KGN cell survival. In this diagram, ns indicates no significant difference; **P<0.01; ***P<0.001.

[0045] Figure 2 This is a schematic diagram illustrating the effects of different traditional Chinese medicine formulations on CDDP-induced KGN cell viability. Among them, Figure 2 A~2H: These are schematic diagrams showing the effects of each GSB1-8 formulation on CDDP-induced KGN cell viability; ns indicates no significant difference. # This indicates a comparison with the control group. ## P<0.01; ### P<0.001; #### P<0.0001; * indicates comparison with CDDP group, *P<0.05; **P<0.01; ***P<0.001.

[0046] Figure 3 Images of vaginal cells in mice at different estrous cycles (HE staining ×100). 3A: proestrus, 3B: estrus, 3C: metestrus, 3D: diaestrus.

[0047] Figure 4 This is a schematic diagram showing the effects of traditional Chinese medicine formula group 2 (GSB-2) and traditional Chinese medicine formula group 9 (GSB-9) on the ovarian index of POF mice. Among them, compared with the control group, ****P<0.0001; compared with the CDDP group, ## P<0.01, # P<0.05.

[0048] Figure 5 Schematic diagram showing the effects of traditional Chinese medicine formula group 2 (GSB-2) and traditional Chinese medicine formula group 9 (GSB-9) on the morphology of ovarian tissue in POF mice (HE staining, ×100).

[0049] Figure 6 This is a schematic diagram showing the effects of traditional Chinese medicine formula group 2 (GSB-2) and traditional Chinese medicine formula group 9 (GSB-9) on the number of follicles at each stage in POF mice. Among them, compared with the control group, **P<0.01; compared with the control group, ***P<0.001; compared with the CDDP group, # P<0.05.

[0050] Figure 7 This is a schematic diagram showing the effects of traditional Chinese medicine formula group 2 (GSB-2) and traditional Chinese medicine formula group 9 (GSB-9) on serum E2 in POF mice. In the diagram, ns indicates no significant difference; ***P<0.001; **P<0.01.

[0051] Figure 8 This is a schematic diagram showing the effects of traditional Chinese medicine formula group 2 (GSB-2) and traditional Chinese medicine formula group 9 (GSB-9) on serum AMH in POF mice. *P<0.05.

[0052] Figure 9 This is a schematic diagram showing the effects of traditional Chinese medicine formula group 2 (GSB-2) and traditional Chinese medicine formula group 9 (GSB-9) on serum FSH in POF mice. ***P<0.001; **P<0.01; *P<0.05.

[0053] Figure 10 This is a schematic diagram showing the effects of traditional Chinese medicine formula group 2 (GSB-2) and traditional Chinese medicine formula group 9 (GSB-9) on serum LH in POF mice. ***P<0.001; **P<0.01; *P<0.05. Detailed Implementation

[0054] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0056] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0057] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0058] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0059] As described in the background section, the prior art does not contain the composition comprising Dipsacus asper, Epimedium, and Paeonia lactiflora claimed in this invention, nor its use in preventing and treating premature ovarian failure. To address the above-mentioned problem, this invention provides a first composition comprising Dipsacus asper, Epimedium, and Paeonia lactiflora, wherein the first composition is composed of Dipsacus asper, Epimedium, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, and Anemarrhena asphodeloides;

[0060] The mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria and Anemarrhena asphodeloides is (1-10):(0.1-5):(0.1-5):(0.1-5):(0.1-5):(0.1-5):(0.1-5).

[0061] In this invention's traditional Chinese medicine compound formula, Epimedium tonifies the kidneys, warms Yang, dispels wind, and eliminates dampness, serving as the principal herb; Paeonia lactiflora and Dipsacus asper tonify the liver and kidneys, and strengthen tendons and bones, serving as assistant herbs. Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa, and calcined oyster shell are all adjuvant herbs. Calcined oyster shell benefits the kidneys and subdues Yang; Rehmannia glutinosa and Anemarrhena asphodeloides nourish Yin and tonify the kidneys; Ligusticum chuanxiong, Sparganium stoloniferum, and Curcuma zedoaria invigorate blood, remove blood stasis, and unblock the meridians. The combined effects of these herbs tonify the kidneys, soothe the liver, nourish blood, and invigorate blood circulation. Treatment primarily focuses on tonifying the kidneys, soothing the liver, strengthening the spleen, replenishing Qi and nourishing blood, and regulating the Chong and Ren meridians to improve ovarian function.

[0062] In this invention, when a mass ratio, mass, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1 to 10” is disclosed, the described range should be interpreted as including the ranges “1 to 10”, “1 to 8”, “1 to 6”, “1 to 4”, “1 to 2”, “2 to 10”, “2 to 8”, “2 to 6”, “2 to 4”, “4 to 10”, “4 to 8”, “4 to 6”, “6 to 10”, “6 to 8”, “8 to 10”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0063] In a preferred embodiment, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria and Anemarrhena asphodeloides is (4-8):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5).

[0064] In a preferred embodiment, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria and Anemarrhena asphodeloides is (5-7):(0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2).

[0065] In a preferred embodiment, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria and Anemarrhena asphodeloides is about 6: about 1: about 1: about 1: about 1: about 1: about 1: about 1.

[0066] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3; "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0067] In a preferred embodiment, the weight of the epimedium ranges from 10 to 20 g, for example, about 15 g.

[0068] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75.

[0069] In a preferred embodiment, the mass of the *Dipsacus asper* ranges from 1 to 5 g, for example, about 2.5 g.

[0070] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.5" includes ±5% of 2.5, or from 2.375 to 2.625.

[0071] In a preferred embodiment, the weight of the peony root ranges from 1 to 5 g, for example, about 2.5 g.

[0072] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.5" includes ±5% of 2.5, or from 2.375 to 2.625.

[0073] In a preferred embodiment, the mass of the Ligusticum chuanxiong ranges from 1 to 5 g, for example, about 2.5 g.

[0074] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.5" includes ±5% of 2.5, or from 2.375 to 2.625.

[0075] In a preferred embodiment, the mass of the triangular prism ranges from 1 to 5 g, for example, about 2.5 g.

[0076] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.5" includes ±5% of 2.5, or from 2.375 to 2.625.

[0077] In a preferred embodiment, the weight of the turmeric ranges from 1 to 5 g, for example, about 2.5 g.

[0078] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.5" includes ±5% of 2.5, or from 2.375 to 2.625.

[0079] In a preferred embodiment, the mass of the Anemarrhena asphodeloides ranges from 1 to 5 g, for example, about 2.5 g.

[0080] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 2.5" includes ±5% of 2.5, or from 2.375 to 2.625.

[0081] According to another aspect of the present invention, a second composition comprising Dipsacus asper, Epimedium and Paeonia lactiflora is provided, the second composition comprising Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas; wherein the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas is (4-25):(0.5-5):(0.5-5):(0.5-5):(0.5-5):(0.5-5):(0.5-5):(0.5-10):(0.1-1).

[0082] In a preferred embodiment, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas is (10.5-22.5):(1-3):(1-3):(1-3):(1-3):(1-3):(1-3):(1.2-5.7):approximately 0.3.

[0083] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.3" includes ±5% of 0.3, or from 0.285 to 0.315.

[0084] In a preferred embodiment, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas is about 19.5: about 1.5: about 1.5: about 1.5: about 1.5: about 1.5: about 1.2: about 0.3.

[0085] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 19.5" includes ±5% of 19.5, or from 18.525 to 20.475; "about 1.5" includes ±5% of 1.5, or from 1.425 to 1.575; "about 1.2" includes ±5% of 1.2, or from 1.14 to 1.26; and "about 0.3" includes ±5% of 0.3, or from 0.285 to 0.315.

[0086] In a preferred embodiment, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas is about 22.5: about 1: about 1: about 1: about 1: about 1: about 1: about 1.2: about 0.3.

[0087] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 22.5" includes ±5% of 22.5, or from 21.375 to 23.625; "about 1" includes ±5% of 1, or from 0.95 to 1.05; "about 1.2" includes ±5% of 1.2, or from 1.14 to 1.26; and "about 0.3" includes ±5% of 0.3, or from 0.285 to 0.315.

[0088] In a preferred embodiment, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas is about 15: about 1.5: about 1.5: about 1.5: about 1.5: about 1.5: about 1.5: about 5.7: about 0.3.

[0089] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75; "about 1.5" includes ±5% of 1.5, or from 1.425 to 1.575; "about 5.7" includes ±5% of 5.7, or from 5.415 to 5.985; and "about 0.3" includes ±5% of 0.3, or from 0.285 to 0.315.

[0090] In a preferred embodiment, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas is about 10.5: about 3: about 3: about 3: about 3: about 3: about 3: about 1.2: about 0.3.

[0091] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10.5" includes ±5% of 10.5, or from 9.975 to 11.025; "about 3" includes ±5% of 3, or from 2.85 to 3.15; "about 1.2" includes ±5% of 1.2, or from 1.14 to 1.26; and "about 0.3" includes ±5% of 0.3, or from 0.285 to 0.315.

[0092] In a preferred embodiment, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa and Ostrea gigas is not about 6: about 1: about 1: about 1: about 1: about 1: about 1: about 1: about 8: about 10.

[0093] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6" includes ±5% of 6, or from 5.7 to 6.3; "about 1" includes ±5% of 1, or from 0.95 to 1.05; "about 8" includes ±5% of 8, or from 7.6 to 8.4; and "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0094] In a preferred embodiment, the first composition or the second composition further comprises one or more pharmaceutically acceptable excipients.

[0095] In a preferred embodiment, the excipient is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, encapsulating agents, flavoring agents, sustained-release agents, flow aids, lubricants, dispersants, plasticizers, light-blocking agents, and antioxidants.

[0096] In a preferred embodiment, the dosage form of the first composition or the second composition is a powder, tablet, pellet, capsule, film, lozenge, granule, injection, or oral liquid.

[0097] In a preferred embodiment, the first composition or the second composition further comprises one or more other drugs for the prevention and / or treatment of premature ovarian failure.

[0098] In a preferred embodiment, the other drugs include hormonal drugs and non-hormonal drugs.

[0099] In a preferred embodiment, the hormonal drug includes estrogen drugs, progesterone drugs, and combined estrogen-progesterone preparations.

[0100] In a preferred embodiment, the estrogen-like drug includes 17-β estradiol, estradiol valerate, and conjugated estrogen.

[0101] In a preferred embodiment, the progestin drug includes micronized progesterone, dydrogesterone, and medroxyprogesterone acetate.

[0102] In a preferred embodiment, the estrogen-progestin combination preparation comprises estradiol tablets / estradiol dydrogesterone tablets and estradiol valerate / estradiol valerate cyproterone valerate tablets.

[0103] In a preferred embodiment, the non-hormonal drug includes plant-based drugs and phytoestrogens.

[0104] According to another aspect of the present invention, the use of the first composition or the second composition described above in the preparation of a medicament for the prevention and / or treatment of premature ovarian failure is provided.

[0105] In a preferred embodiment, the first composition or the second composition prevents and / or treats premature ovarian failure through one or more of the following mechanisms: promoting the proliferation of KGN cells damaged by chemotherapy drugs, improving estrous cycle disorders, increasing ovarian index, improving ovarian tissue pathological abnormalities, and improving abnormal serum biochemical indicators.

[0106] In a preferred embodiment, the chemotherapy drug is cisplatin.

[0107] In a preferred embodiment, the estrous cycle disorder includes prolonged interestrous period and prolonged interestrous period.

[0108] In a preferred embodiment, the ovarian index is the percentage of the ratio of bilateral ovarian wet weight to body weight.

[0109] In a preferred embodiment, the ovarian histopathological abnormality includes a decrease in the number of primordial follicles, a decrease in the number of growing follicles, a decrease in the number of mature follicles, and an increase in the number of atretic follicles.

[0110] In a preferred embodiment, the abnormal serum biochemical indicators include decreased estradiol levels, decreased anti-Miller hormone levels, increased follicle-stimulating hormone levels, and increased luteinizing hormone levels.

[0111] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0112] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0113] The above-mentioned features of the present invention, or the features mentioned in the examples, can be combined arbitrarily. All the features disclosed in the specification of this patent can be used in any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0114] Examples

[0115] 1. Experimental materials

[0116] 1.1. Experimental cells and animals

[0117] Human ovarian granulosa cell line (KGN cells) was purchased from Wuhan Procell Life Science & Technology Co., Ltd.

[0118] SPF grade female ICR mice, 6-8 weeks old, with a body weight of 25-28g, a total of 40, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the animal license number: SCXK (Beijing) 2021-0006. All animals were housed in the SPF Laboratory Animal Center of China Academy of Chinese Medical Sciences, in a barrier environment, at room temperature of (24.5±0.5)°C, with humidity of (55±5)%, 12h day-night cycle, free access to food and water, and were subjected to intervention and modeling after 1 week of adaptive feeding.

[0119] 1.2. Experimental instruments

[0120] XSE 105DU electronic analytical balance (Mettler Toledo, Switzerland); KQ-250B numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); Axio Scope A1 biological microscope (Carl Zeiss Suzhou Co., Ltd.); Multiskan FC microplate reader (Thermo Scientific, USA); Revolution ECHO motorized intelligent all-in-one upright and inverted microscopic imaging system (Echo, USA).

[0121] 1.3. Experimental reagents

[0122] Eight medicinal herbs (Epimedium, Dipsacus, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa, and calcined oyster shell) were all purchased from Beijing Tongrentang Suzhou Street Pharmaceutical Co., Ltd., and were identified as genuine products by Researcher Zhu Jingjing of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences; DMEM / F12 basal culture medium, penicillin-streptomycin solution, fetal bovine serum, and phosphate buffer (Wuhan Pronosei Life Science Technology Co., Ltd., catalog numbers PM150312, PB180120, 164210-50, and PB180327 respectively); 0.25% trypsin (GIBCO, USA). Company (product number 25200072); CCK-8 (Beijing Lanjieke Technology Co., Ltd., product number BS350B); Cisplatin (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., batch number: HY-17394); Methylene blue staining solution (purchased from Solarbio Science & Technology Co., Ltd., batch number: G1303); Enzyme-linked immunosorbent assay (ELISA) kits for mouse serum AMH, E2, FSH, and LH (batch numbers: H324-1-2, H102-1-2, H101-1-2, H206-1-2) were purchased from Nanjing Jiancheng Biotechnology Co., Ltd.

[0123] 2. Experimental Methods

[0124] 2.1 Drug combination and dosage ratio

[0125] This formula consists of eight Chinese herbs (Epimedium, Dipsacus, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa, and calcined oyster shell). Referring to the prescribed dosage ranges for these eight herbs in the 2020 edition of the Chinese Pharmacopoeia and combining this with clinical practice, the dosage ranges for each herb were determined. These eight herbs were treated as eight factors, and a four-level design was implemented. The experiment used a U8 (8... 4 The drugs were arranged in a uniform design table, and the specific proportions of each dosage ratio group are shown in Table 1.

[0126] Table 1U8(8) 4 Uniform Design Table

[0127]

[0128] To eliminate the interference of dose-effect relationship on efficacy, while keeping the drug ratio of each group unchanged, the amount of raw drugs in each group was adjusted to the same dose (30g). The adjusted drug amounts of each group are shown in Tables 2 and 3.

[0129] Table 2 Drug composition of each group in the uniformity experiment

[0130]

[0131]

[0132] Table 3 Specific Drug Composition

[0133]

[0134] Weigh each herb according to the proportions specified in Table 3. Crush the oyster shell into fine or very fine powder; add 4 to 20 times the amount of water to the remaining herbal herbs such as Epimedium and decoct twice, 2 hours each time; combine the decoctions, filter, concentrate the filtrate to an appropriate amount, dry, crush into fine powder, add the oyster shell powder, mix well, and the herbal compound powder is obtained.

[0135] 2.2 Cell Culture

[0136] KGN cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin in a 37°C incubator with 5% CO2 and saturated humidity. The medium was changed every other day, and cell growth was observed under an inverted microscope. When the cells grew and merged into a dense monolayer (coverage of 70%-80%), they were routinely digested with 0.25% trypsin, centrifuged at 1200 rpm for 3 min, and passaged every 2-3 days at a ratio of 1:3 or 1:4.

[0137] 2.3 Screening of cisplatin concentration using the CCK8 method

[0138] KGN cells in the logarithmic growth phase were selected and used at a concentration of 1×10⁻⁶. 3 Cells were seeded at a density of 100 μL of culture medium per well in 96-well plates. After 24 h of incubation, blank, control, and Tripterygium wilfordii glycoside groups were established. Cisplatin groups: 100 μL of Tripterygium wilfordii glycoside solution at concentrations of 1.25, 2.5, 5, 10, and 20 μM were added to each well. Blank and control groups: 100 μL of complete culture medium was added to each well. Five control wells were included in each group. The plates were incubated at 37°C in a 5% CO2 incubator for another 24 h. Afterward, the culture was terminated, the supernatant in the wells was aspirated, and 100 μL of culture medium containing 10% CCK8 solution was added to each well. The absorbance of each well was measured at 450 nm using a microplate reader. The results were recorded, and cell viability was calculated: Cell viability % = (Experimental group A - Blank group A) / (Control group A - Blank group A) × 100%.

[0139] 2.4 Effects of different traditional Chinese medicine formulations on the survival rate of cisplatin-damaged KGN cells

[0140] KGN cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C under 5% CO2 conditions. Regular observation was performed, and when the cells reached 85%–90% confluence, they were digested with 0.25% trypsin at a concentration of 1 × 10⁻⁶. 3Cells were seeded at a density of 100 μL of culture medium per well in 96-well plates. After 24 h of incubation, blank control, control, model, and different traditional Chinese medicine (TCM) formulation groups were established. Blank and control groups: 100 μL of complete culture medium was added to each well. Model group: 100 μL of 5 μM cisplatin solution was added to each well. Different TCM formulation groups were established by adding different amounts of deionized water to the TCM powder to create two concentrations: 0.1 μg / mL and 1 μg / mL. 100 μL of a mixture of the different TCM formulation solution and 5 μM cisplatin solution was added to each well. The plates were incubated at 37°C in a 5% CO2 incubator for 24 h. Cell culture was then stopped, the supernatant was aspirated, and 100 μL of culture medium containing 10% CCK8 solution was added to each well. The absorbance of each well was measured at 450 nm using a microplate reader, the results were recorded, and cell viability was calculated.

[0141] 2.5 Animal grouping, model establishment, and drug administration

[0142] Forty mice were randomly divided into three groups (n=10 per group): a normal control group, a model group, a traditional Chinese medicine (TCM) formula group 2 (GSB-2), and a TCM formula group 9 (GSB-9). Mice in the model group were intraperitoneally injected with cisplatin 2 mg / kg to induce POF (prolapse and reflux) for 7 consecutive days. Mice in the TCM formula groups 2 (GSB-2) and 9 (GSB-9) were administered a clinically equivalent dose of the extract (prepared by weighing 0.5 g of the TCM compound powder, adding 10 ml of deionized water, and sonicating to dissolve, ultimately preparing a suspension with a concentration of 50 mg / ml) via gavage in the morning, and intraperitoneally injected with cisplatin 2 mg / kg in the afternoon, for 7 consecutive days. After model establishment, mice in the TCM formula groups 2 (GSB-2) and 9 (GSB-9) were continuously administered the extract via gavage at a dose of 0.39 g / kg once daily for 21 consecutive days. Mice in the normal group and the model group were administered an equal volume of distilled water by gavage once daily for 21 consecutive days.

[0143] 2.6. Vaginal exfoliative cytology and determination of the estrous cycle

[0144] Every morning at 8:00 AM, vaginal exfoliated cell smears were observed on mice in each group. The mouse tail was fixed to fully expose the vaginal opening, and the dilation of the vaginal opening and surrounding secretions were observed. A small-headed cotton swab soaked in physiological saline was inserted into the mouse vagina and rotated moderately to a depth of about 4 mm. The swab was then evenly smeared on a glass slide, dried, and fixed with 95% ethanol for 30 min. After staining with methylene blue, the morphology of the exfoliated cells was observed under an optical microscope.

[0145] 2.7 Calculation of Mouse Ovarian Index

[0146] Bilateral ovaries of mice were harvested, excess adipose tissue was removed, and excess water was aspirated. The wet weight of the ovaries was measured using a precision balance, and the corresponding ovarian index was calculated. Ovarian index = bilateral ovarian wet weight (mg) / mouse body weight before sacrifice (g) × 100%.

[0147] 2.8. HE staining to observe morphological changes in ovarian tissue and count follicles at each stage.

[0148] Ovarian tissue was randomly collected from 6 mice in each group, fixed in 4% paraformaldehyde for 48 hours, and then subjected to routine dehydration, clearing, paraffin embedding, and embedding to prepare 5μm paraffin sections. Some paraffin sections were stained with hematoxylin and eosin (HE), and after mounting, the pathological changes of normally developing follicles and atretic follicles at each stage of development were observed under a microscope. Five fields of view were randomly selected from each section to count primordial follicles, growing follicles, mature follicles, and atretic follicles.

[0149] 2.9 ELISA method for detecting E2, AMH, FSH, and LH levels in mouse serum

[0150] Venous blood was collected from 10 mice in each group via enucleation, allowed to stand for 30 minutes, and then infused at 3500 rpm. -1 After centrifugation for 15 minutes, the serum was collected and stored at -80°C. Serum parameters of each group of mice were measured according to the ELISA kit instructions. The absorbance of each well at 450 nm was observed and recorded using a microplate reader, and the levels of E2, AMH, FSH, and LH were calculated using a standard curve.

[0151] 2.10 Statistical Analysis

[0152] The obtained data were analyzed using GraphPad Prism 10.1.2 software. Between-group comparisons were performed using one-way ANOVA. For further pairwise comparisons, the least significant difference (LSD) test was used for homogeneous variances, and the Tamhane test was used for unequal variances. A p-value < 0.05 was considered statistically significant.

[0153] 3. Experimental Results

[0154] 3.1 Determination of cisplatin concentration

[0155] Depend on Figure 1 It can be seen that 2.5–20 μM cisplatin can significantly inhibit the viability of KGN cells (P<0.01 or P<0.001), and the higher the concentration, the lower the cell survival rate. Therefore, a cisplatin concentration of 5 μM and an action time of 24 h were selected as the modeling conditions for subsequent experiments.

[0156] 3.2. The compositions of each group promoted the proliferation of CDDP-induced KGN cells.

[0157] Compared with the normal group, compositions 2 and 6 (0.1 and 1 μg / mL) significantly promoted CDDP-induced KGN cell proliferation (P<0.001, P<0.01, or P<0.05) (results are shown in the figure). Figure 2 (As shown). In the various proportions of the traditional Chinese medicine formulas, the dosage ratio of Epimedium was 3-19.5, the total dosage ratio of Paeonia lactiflora, Curcuma zedoaria, Sparganium stoloniferum, Anemarrhena asphodeloides, Dipsacus asper, and Ligusticum chuanxiong was 9-24, the dosage ratio of Rehmannia glutinosa was 0-1.5, and the dosage ratio of Ostrea gigas was 0-0.3, which significantly improved the survival rate of CDDP-induced KGN cells (P<0.05). Assuming that X1 is Epimedium, X2 is Paeonia lactiflora, Curcuma zedoaria, Sparganium stoloniferum, Anemarrhena asphodeloides, Dipsacus asper, and Ligusticum chuanxiong, X3 is Rehmannia glutinosa, and X4 is Ostrea gigas, in the two groups of traditional Chinese medicine formulas (GSB-2), X1:X2:X3:X4 = 19.5:9:1.2:0.3 is within the effective dosage ratio range, which can significantly improve the survival rate of CDDP-induced KGN cells (P<0.001 or P<0.01). In the 9 groups of traditional Chinese medicine formulas (GSB-9), the ratio of X1:X2:X3:X4 = 15:15:0:0 is within the effective dosage ratio range, suggesting that it can significantly improve the survival rate of CDDP-induced KGN cells.

[0158] 3.3 Effects of Traditional Chinese Medicine Formula Group 2 (GSB-2) and Traditional Chinese Medicine Formula Group 9 (GSB-9) on the estrous cycle of POF model mice

[0159] The estrous cycle reflects ovarian function in mice. A normal mouse estrous cycle is stable at 4-5 days. Prolonged or prolonged estrous intervals indicate a successful establishment of an ovarian injury model. Microscopic examination reveals predominantly sheet-like anucleated keratinized epithelial cells during estrus; predominantly nucleated oval epithelial cells during proestrus; a large number of leukocytes during estrus; and the presence of all three cell types during mesoderm. Methylene blue staining of vaginal smears during each estrous cycle is as follows. Figure 3 As shown.

[0160] The estrous cycle disorder rate (%) = number of mice with estrous cycles disorder in each group / total number of mice in each group × 100%. Compared with the normal group, the estrous cycle disorder rate in the model group was significantly increased (P<0.01); compared with the model group, the estrous cycle disorder rate in the herbal formula group 2 (GSB-2) and the herbal formula group 9 (GSB-9) was significantly decreased (P<0.05). The results are shown in Table 4.

[0161] Table 4. Effects of herbal formula group 2 (GSB-2) and herbal formula group 9 (GSB-9) on the estrous cycle disorder rate in POF mice. n=6)

[0162]

[0163] Among them, compared with the normal group 1) P<0.01; compared with the model group 2) P<0.05.

[0164] 3.4 Effects of Traditional Chinese Medicine Formula Group 2 (GSB-2) and Traditional Chinese Medicine Formula Group 9 (GSB-9) on Ovarian Index in POF Mice

[0165] Compared with the control group, the ovarian index in the model group was decreased (P<0.0001); compared with the model group, the ovarian index in the traditional Chinese medicine formula 2 group (GSB-2) was significantly increased (P<0.01), and the ovarian index in the traditional Chinese medicine formula 9 group (GSB-9) was significantly increased (P<0.05); the results are as follows. Figure 4 As shown.

[0166] 3.5. Effects of Traditional Chinese Medicine Formula Group 2 (GSB-2) and Traditional Chinese Medicine Formula Group 9 (GSB-9) on the pathological morphology of mouse ovarian tissue and comparison of the number of follicles at each stage.

[0167] HE staining results showed that the ovaries of normal mice had no abnormalities in morphology or structure, no pathological damage, and the ovarian medulla was full and plump. The cortex contained multiple primordial follicles and follicles of various stages of growth, with granulosa cells arranged neatly. Compared with the normal group, the number of primordial follicles, follicles of growth, and mature follicles in the ovarian tissue of the model group mice was significantly reduced (P<0.001 or P<0.01), while the number of atretic follicles was significantly increased (P<0.05). Compared with the model group, the number of primordial follicles, follicles of growth, and mature follicles in the ovarian tissue of mice in the herbal formula group 2 (GSB-2) was significantly increased, the ovarian granulosa cells were neatly arranged, and the number of atretic follicles was significantly reduced (P<0.05). Compared with the model group, the number of primordial follicles, follicles of growth, and mature follicles in the ovarian tissue of mice in the herbal formula group 9 (GSB-9) was significantly increased, the ovarian granulosa cells were neatly arranged, and the number of atretic follicles was significantly reduced (P<0.05). Figure 5 and Figure 6 As shown.

[0168] 3.6 Effects of Traditional Chinese Medicine Formula Group 2 (GSB-2) and Traditional Chinese Medicine Formula Group 9 (GSB-9) on serum E2, AMH, FSH, and LH in POF model mice

[0169] Compared with the control group, the serum E2 level in the model group mice was significantly decreased (P<0.001), the AMH level was also significantly decreased (P<0.05), and the FSH and LH levels were significantly increased (P<0.001). Compared with the model group, the serum E2 and AMH levels in the traditional Chinese medicine formula 2 group (GSB-2) mice were significantly increased (P<0.01 or P<0.05), and the FSH and LH levels were significantly decreased (P<0.01 or P<0.05). Compared with the model group, the serum E2 level in the traditional Chinese medicine formula 9 group (GSB-9) mice did not change significantly, the AMH level was significantly increased (P<0.05), and the FSH and LH levels were significantly decreased (P<0.05). The results are as follows. Figure 7 , Figure 8 , Figure 9 and Figure 10 As shown.

[0170] 4. Experimental Conclusions

[0171] 4.1. The effective range of traditional Chinese medicine prescriptions was determined using the uniform design method.

[0172] The traditional Chinese medicine compound consists of eight herbs. These eight herbs are considered as eight factors, and a four-level experimental design was implemented. The experiment used a U8(8) model. 4 The formulas were arranged using a uniform design table. Based on the results of the CCK8 experiment with different formulation ratios, it was found that when the dosage ratio of Epimedium was 3–19.5, the total dosage ratio of Paeonia lactiflora, Curcuma zedoaria, Sparganium stoloniferum, Anemarrhena asphodeloides, Dipsacus asper, and Ligusticum chuanxiong was 9–24, the dosage ratio of Rehmannia glutinosa was 0–1.5, and the dosage ratio of Ostrea gigas was 0–0.3, the survival rate of CDDP-induced KGN cells could be effectively improved (P<0.05).

[0173] 4.2 The combined in vitro and in vivo experiments demonstrated the effects of traditional Chinese medicine formulation 2 (GSB-2) on ovarian function.

[0174] This study used a human ovarian granulosa cell (KGN) model induced by incubation with 5 μM cisplatin (CDDP) for 24 h in vitro. KGN cells are currently the most commonly used cell line for studying changes in ovarian function. The CCK-8 assay results showed that co-incubation of KGN cells with traditional Chinese medicine formula 2 (GSB-2) for 24 h significantly improved cell survival rate (P<0.05), indicating that it has a protective effect on ovarian function.

[0175] In vivo experiments were conducted to establish a POF mouse model by intraperitoneal injection of 2 mg / kg CDDP for 7 consecutive days. Simultaneously with modeling, mice were administered a clinically equivalent dose of the extract via gavage daily at 0.39 g / kg. After modeling, this clinically equivalent dose of the extract was administered via gavage daily for 21 consecutive days. The results showed that treatment with the clinically equivalent dose of 0.39 g / kg of the herbal formula 2 (GSB-2) significantly increased the ovarian index in POF model mice (P<0.05), significantly increased serum E2 and AMH levels (P<0.05), significantly decreased FSH and LH levels (P<0.05), significantly increased the number of follicles at all stages (P<0.05), and significantly decreased the number of atretic follicles (P<0.05). These findings suggest that the herbal formula 2 (GSB-2) can reduce serum FSH and LH levels, increase AMH and E2 levels, and alleviate ovarian pathological damage in POF model mice, thereby treating the POF model mouse disease.

[0176] 4.3 In vivo experiments to demonstrate the effects of traditional Chinese medicine formula 9 (GSB-9) on ovarian function

[0177] In vivo experiments were conducted to establish a POF mouse model by intraperitoneal injection of 2 mg / kg CDDP for 7 consecutive days. Simultaneously with model establishment, mice were administered a clinically equivalent dose of traditional Chinese medicine formula 9 (GSB-9) at 0.39 g / kg via gavage daily. After model establishment, this dose was continued daily for 21 days. Results showed that treatment with a clinically equivalent dose of 0.39 g / kg of traditional Chinese medicine formula 9 (GSB-9) significantly increased the ovarian index in POF model mice (P<0.05), while serum E2 levels did not significantly increase. AMH levels significantly increased (P<0.05), FSH and LH levels significantly decreased (P<0.05), the number of follicles at all stages significantly increased (P<0.05), and the number of atretic follicles significantly decreased (P<0.05). The results suggest that the traditional Chinese medicine formula 9 (GSB-9) can reduce serum FSH and LH levels, increase AMH levels, and alleviate ovarian pathological damage in POF mice, thereby treating POF mice.

[0178] 4.4 The therapeutic effects of traditional Chinese medicine formula 2 (GSB-2) and traditional Chinese medicine formula 9 (GSB-9) on premature ovarian failure.

[0179] Assuming X1 is Epimedium, X2 is Paeonia lactiflora, Curcuma zedoaria, Sparganium stoloniferum, Anemarrhena asphodeloides, Dipsacus asper, and Ligusticum chuanxiong, X3 is Rehmannia glutinosa, and X4 is Ostrea gigas, the ratio of X1:X2:X3:X4 in herbal formula 2 (GSB-2) is 19.5:9:1.2:0.3, which is within the effective dose range and can effectively improve the survival rate of CDDP-induced KGN cells (P<0.05). In herbal formula 9 (GSB-9), the ratio of X1:X2:X3:X4 is 15:15:0:0, which is within the effective dose range and can significantly improve the survival rate of CDDP-induced KGN cells. Further pharmacodynamic studies were conducted on POF mice using herbal formulas 2 (GSB-2) and 9 (GSB-9). The pharmacodynamic studies also showed that herbal formulas 2 (GSB-2) and 9 (GSB-9) have therapeutic effects on premature ovarian failure.

[0180] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A first composition comprising Dipsacus asper, Epimedium, and Paeonia lactiflora, characterized in that, The first composition consists of Dipsacus asper, Epimedium, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria and Anemarrhena asphodeloides; The mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria and Anemarrhena asphodeloides is (1-10):(0.1-5):(0.1-5):(0.1-5):(0.1-5):(0.1-5):(0.1-5).

2. The first composition according to claim 1, characterized in that, The mass ratio of the Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, and Anemarrhena asphodeloides is (4-8):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5):(0.5-1.5). Preferably, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, and Anemarrhena asphodeloides is (5-7):(0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2). More preferably, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria and Anemarrhena asphodeloides is about 6: about 1: about 1: about 1: about 1: about 1: about 1: about 1.

3. The first composition according to claim 1, characterized in that, The weight range of the Epimedium is 10-20g, for example, about 15g; Preferably, the mass of the *Dipsacus asper* is in the range of 1–5 g, for example, about 2.5 g; Preferably, the weight range of the red peony root is 1-5g, for example, about 2.5g; Preferably, the mass of the chuanxiong rhizome ranges from 1 to 5 g, for example, about 2.5 g; Preferably, the mass of the triangular prism ranges from 1 to 5 g, for example, about 2.5 g; Preferably, the weight of the turmeric ranges from 1 to 5g, for example, about 2.5g; Preferably, the mass of the Anemarrhena asphodeloides is in the range of 1 to 5 g, for example, about 2.5 g.

4. A second composition comprising Dipsacus asper, Epimedium, and Paeonia lactiflora, characterized in that, The second composition comprises Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa, and Ostrea gigas; wherein the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa, and Ostrea gigas is (4-25):(0.5-5):(0.5-5):(0.5-5):(0.5-5):(0.5-5):(0.5-5):(0.5-10):(0.1-1).

5. The second composition according to claim 4, characterized in that, The mass ratio of the Epimedium, Dipsacus, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa, and Ostrea gigas is (10.5–22.5):(1–3):(1–3):(1–3):(1–3):(1–3):(1–3):(1–3):(1.2–5.7):approximately 0.3; Preferably, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa, and Ostrea gigas is approximately 19.5: approximately 1.5: approximately 1.5: approximately 1.5: approximately 1.5: approximately 1.5: approximately 1.2: approximately 0.3; Preferably, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa, and Ostrea gigas is approximately 22.5: approximately 1: approximately 1: approximately 1: approximately 1: approximately 1: approximately 1: approximately 1.2: approximately 0.3; Preferably, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa, and Ostrea gigas is approximately 15: approximately 1.5: approximately 1.5: approximately 1.5: approximately 1.5: approximately 1.5: approximately 1.5: approximately 5.7: approximately 0.3; Preferably, the mass ratio of Epimedium, Dipsacus asper, Paeonia lactiflora, Ligusticum chuanxiong, Sparganium stoloniferum, Curcuma zedoaria, Anemarrhena asphodeloides, Rehmannia glutinosa, and Ostrea gigas is approximately 10.5: approximately 3: approximately 3: approximately 3: approximately 3: approximately 3: approximately 3: approximately 1.2: approximately 0.3; More preferably, the mass ratio of the Epimedium, the Dipsacus, the Paeonia lactiflora, the Ligusticum chuanxiong, the Sparganium, the Curcuma zedoaria, the Anemarrhena asphodeloides, the Rehmannia glutinosa and the oyster shell is not about 6: about 1: about 1: about 1: about 1: about 1: about 1: about 1: about 8: about 10.

6. The first composition according to any one of claims 1 to 3 or the second composition according to claim 4 or 5, characterized in that, The first composition or the second composition further comprises one or more pharmaceutically acceptable excipients; Preferably, the excipients are selected from one or more of the following: diluents, wetting agents, adhesives, disintegrants, encapsulating agents, flavoring agents, sustained-release agents, flow aids, lubricants, dispersants, plasticizers, light-blocking agents, and antioxidants.

7. The first composition according to any one of claims 1 to 3 or the second composition according to claim 4 or 5, characterized in that, The dosage form of the first composition or the second composition is powder, tablet, pellet, capsule, film, lozenge, granule, injection or oral liquid.

8. The first composition according to any one of claims 1 to 3 or the second composition according to claim 4 or 5, characterized in that, The first composition or the second composition further comprises one or more other drugs for the prevention and / or treatment of premature ovarian failure; Preferably, the other drugs include hormonal drugs and non-hormonal drugs; More preferably, the hormone drugs include estrogen drugs, progestin drugs, and estrogen-progestin combination preparations; Preferably, the estrogen-like drug includes 17-β estradiol, estradiol valerate, and conjugated estrogens; Preferably, the progestin drug includes micronized progesterone, dydrogesterone, and medroxyprogesterone acetate; Preferably, the estrogen-progestin combination preparation includes estradiol tablets / estradiol dydrogesterone tablets and estradiol valerate / estradiol valerate cyproterone acetate tablets; More preferably, the non-hormonal drugs include plant-based drugs and phytoestrogens.

9. Use of the first composition according to claim 1, 2, 3, 6, 7 or 8, or the second composition according to any one of claims 4 to 8, in the preparation of a pharmaceutical remedy for the prevention and / or treatment of premature ovarian failure.

10. The use according to claim 9, characterized in that, The first composition or the second composition has the effect of preventing and / or treating premature ovarian failure through one or more of the following mechanisms: promoting the proliferation of KGN cells damaged by chemotherapy drugs, improving estrous cycle disorders, increasing ovarian index, improving ovarian tissue pathological abnormalities, and improving serum biochemical abnormalities; Preferably, the chemotherapy drug is cisplatin; Preferably, the estrous cycle disorder includes prolonged interestrous period and prolonged interestrous period; Preferably, the ovarian index is the percentage of the ratio of bilateral ovarian wet weight to body weight; Preferably, the ovarian histopathological abnormalities include a decrease in the number of primordial follicles, a decrease in the number of growing follicles, a decrease in the number of mature follicles, and an increase in the number of atretic follicles; Preferably, the abnormal serum biochemical indicators include decreased estradiol levels, decreased anti-Miller hormone levels, increased follicle-stimulating hormone levels, and increased luteinizing hormone levels.

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