A composition for improving premature ovarian failure

Abstract

The application provides a composition for improving premature ovarian failure, and relates to the field of biological medicine.The composition is composed of the following crude drugs: tortoise shell glue, dendrobium officinale, myrtle, lamiophlomis rotata, sweet cinnamon, green calyx plum, chirita palmata, rhizoma scirpae, dried scallop, gordon euryale seed, radix paeoniae alba, zedoary, perilla frutescens, ginger; the composition is reasonably matched, and has the effects of nourishing yin and filling essence, promoting blood circulation to remove meridian obstruction, soothing liver and regulating qi, and invigorating spleen and transportation; the tortoise shell glue, chirita palmata and rhizoma scirpae in the composition are combined to realize the complementation of nourishing yin and warming yang and the mutual generation of qi and essence; the sweet cinnamon soothes liver and resolves depression and opens up depression to invigorate the spleen, and breaks through the limitation of traditional soothing liver and promoting blood circulation; most of the raw materials are food and medicine homologous products, are mild, safe and effective, and can be taken for a long time; pharmacological studies show that the composition can inhibit ovarian granulosa cell apoptosis, improve ovarian function, regulate the endocrine system and restore reproductive function, and has a significant curative effect on premature ovarian failure.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to a composition for improving premature ovarian failure. Background Technology

[0002] Premature ovarian failure (POF) refers to the decline of ovarian function in women before the age of 40, characterized primarily by elevated gonadotropin levels and decreased estrogen levels. Clinical manifestations include infrequent menstruation, amenorrhea, decreased fertility, and even infertility, severely impacting the patient's physical, mental, and reproductive health. Currently, hormone replacement therapy is the standard treatment. Estradiol / estradiol-dydrogesterone tablets, by mimicking the cyclical secretion of sex hormones, can effectively alleviate low estrogen-related symptoms and induce menstruation. However, long-term use of this therapy may increase the risk of endometrial lesions and metabolic disorders, and relapse is common after discontinuation, presenting significant limitations. Existing traditional hormone replacement therapies primarily aim to improve and alleviate symptoms, but are unlikely to achieve a fundamental restoration of ovarian function.

[0003] Traditional Chinese medicine (TCM) has a long history of treating gynecological diseases, demonstrating unique advantages in efficacy and safety. TCM categorizes premature ovarian failure (POF) under the concept of "blood depletion," believing its primary pathogenesis to be kidney essence deficiency, leading to the exhaustion of Tian Gui (a concept in TCM related to reproductive function) and imbalance of the Chong and Ren meridians, thereby affecting follicle development and reproductive function. Although TCM shows promising application prospects in gynecological diseases, it still faces challenges such as insufficient elucidation of its mechanisms of action and a lack of high-quality pharmacological and efficacy studies. Therefore, exploring gentle, safe, and effective treatment methods based on TCM's holistic approach and syndrome differentiation is of great significance for improving POF. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a composition for improving premature ovarian failure.

[0006] (II) Technical Solution

[0007] This invention is achieved through the following technical solution:

[0008] On one hand, the present invention provides a composition for improving premature ovarian failure, the composition being made from the following raw materials in parts by weight: 3-18 parts tortoise shell glue, 5-22 parts Dendrobium officinale, 8-20 parts Myrtle, 2-18 parts Lycopus lucidus, 1-15 parts Nardostachys jatamansi, 1-15 parts Prunus mume, 3-24 parts Panax quinquefolius, 3-25 parts Panax notoginseng, 5-35 parts dried scallop, 2-20 parts Euryale ferox, 6-25 parts Paeonia lactiflora, 1-12 parts Amomum villosum, 2-20 parts Perilla frutescens stem, and 1-10 parts fresh ginger.

[0009] Further, the composition is made from the following raw materials in parts by weight: 6-12 parts tortoise shell glue, 10-15 parts Dendrobium officinale, 10-15 parts Myrtle, 6-12 parts Lycopus lucidus, 3-6 parts Nardostachys jatamansi, 3-6 parts Prunus mume, 9-15 parts Panax ginseng, 9-12 parts Panax notoginseng, 10-25 parts dried scallop, 10-15 parts Euryale ferox, 10-15 parts Paeonia lactiflora, 3-6 parts Amomum villosum, 6-12 parts Perilla frutescens stem, and 3-6 parts fresh ginger.

[0010] Furthermore, the composition is made from the following raw materials in parts by weight: 10 parts tortoise shell glue, 12 parts Dendrobium officinale, 12 parts Myrtle, 8 parts Lycopus lucidus, 4 parts Nardostachys jatamansi, 4 parts Prunus mume, 12 parts Panax ginseng, 10 parts Panax notoginseng, 12 parts dried scallop, 12 parts Euryale ferox, 14 parts Paeonia lactiflora, 5 parts Amomum villosum, 8 parts Perilla frutescens stem, and 4 parts fresh ginger.

[0011] The compositions of this invention can be added to different pharmaceutical excipients to form any of the commonly used oral formulations.

[0012] Furthermore, the oral preparation can be any one of pills, powders, granules, oral liquids, tablets, capsules, or ointments.

[0013] The composition of this invention is prepared using pure traditional Chinese herbal medicines as raw materials. The sources and efficacy indications of the traditional Chinese herbal medicines are described below:

[0014] Tortoise shell glue: A solid glue made from the carapace and plastron of the tortoise *Chinemys reevesii* (Gray), a species of turtle in the family Testudinidae, through boiling and concentration. It is salty, sweet, and cool in nature, and enters the liver, kidney, and heart meridians. It has the effects of nourishing yin, replenishing blood, and stopping bleeding; it is used for yin deficiency and blood deficiency, consumptive fever with bone steaming, hematemesis, irritability and palpitations, kidney deficiency with lower back pain, metrorrhagia, and leukorrhea.

[0015] Dendrobium officinale: The dried stem of Dendrobium officinale, an orchid. Sweet and slightly cold in nature. It enters the stomach and kidney meridians. It has the effects of nourishing the stomach and promoting the production of body fluids, nourishing yin and clearing heat. It is used for febrile diseases with fluid depletion, dry mouth and thirst, stomach yin deficiency, poor appetite and dry retching, persistent low-grade fever after illness, yin deficiency with exuberant fire, steaming bone fever, blurred vision, and weakness of muscles and bones.

[0016] Myrtle: An evergreen shrub of the Myrtaceae family, its mature fruit is the Chinese medicinal herb. It is neutral in nature and sweet and astringent in taste, entering the liver and spleen meridians. It has the effects of nourishing blood and stopping bleeding, astringing the intestines and consolidating menstruation. It is used for blood deficiency and weakness, hematemesis, epistaxis, coughing up blood due to overwork, hematochezia, metrorrhagia, seminal emission, leukorrhea, dysentery, rectal prolapse, burns, and external bleeding.

[0017] Ze Lan: The dried aerial parts of *Lycopus lucidus* Turcz. var. *hirtus Regel*, a plant belonging to the genus *Lycopus* in the Lamiaceae family. It has a bitter and pungent taste, and is slightly warm in nature; it enters the liver and spleen meridians. It has the effects of promoting blood circulation and regulating menstruation, removing blood stasis and reducing swelling, and promoting diuresis and reducing edema. It is mainly used to treat irregular menstruation, amenorrhea, dysmenorrhea, postpartum abdominal pain due to blood stasis, carbuncles and boils, and edema with ascites.

[0018] Nardostachys jatamansi DC., a plant in the Valerianaceae family, is a dried root and rhizome. It has a pungent and sweet taste, and is warm in nature; it enters the spleen and stomach meridians. It has the effects of regulating qi and relieving pain, relieving stagnation and invigorating the spleen, and is mainly used to treat abdominal distension, loss of appetite, and vomiting; externally, it has the effects of removing dampness and reducing swelling, and is mainly used to treat beriberi and edema.

[0019] Green calyx plum: The flower of *Prunus mume* Sieb. et Zucc. var. *viridicalyx Makino*, a plant in the genus *Prunus* of the Rosaceae family. It tastes bitter and slightly sweet, and is neutral in nature. It has the effect of calming the liver and stomach, and is mainly used to treat chest and rib pain, stomach pain, indigestion, and neurasthenia.

[0020] Hand-shaped Gymnadenia: The dried tuber of *Gymnadenia conopsea* (L.) R. Br. or *Gymnadenia crassinervis Finet*, both belonging to the Orchidaceae family. It has a sweet taste and neutral properties, and enters the lung, spleen, and stomach meridians. It possesses the effects of relieving cough and asthma, tonifying the kidneys and spleen, regulating qi and blood, and relieving pain. It is mainly used to treat cough and asthma due to lung deficiency, emaciation due to overwork, weakness and soreness in the lower back and legs due to kidney deficiency, impotence, spermatorrhea, frequent urination, chronic diarrhea, blood loss, leukorrhea, insufficient lactation, and injuries from falls and blows.

[0021] Ren Tou Qi: The whole herb, including rhizomes, of *Herminium monorhis* (L.) R.Br., a plant in the Orchidaceae family. It has a sweet taste and warm properties; it enters the stomach and kidney meridians. It has the effects of tonifying the kidneys and spleen, regulating menstruation and promoting blood circulation, and detoxifying. It is mainly used to treat dizziness, insomnia, irritability, thirst, loss of appetite, irregular menstruation, and snake bites.

[0022] Dried scallops: The adductor muscle of three scallop species: *Chlamys farreri* (Jones et Preston), *Chlamys nobilis* (Reeve), and *Chlamys pica* (Reeve). They are sweet and salty in taste, and slightly warm in nature; they enter the spleen and kidney meridians. They have the effects of nourishing yin, nourishing blood, tonifying the kidneys, and regulating the middle jiao (digestive system). They are used for diabetes, frequent urination due to kidney deficiency, and loss of appetite.

[0023] Euryale ferox Salisb., a plant belonging to the genus Euryale in the family Nymphaeaceae. It has a sweet and astringent taste, and is neutral in nature; it enters the spleen and kidney meridians. It has the effects of tonifying the kidneys and securing essence, strengthening the spleen and stopping diarrhea, and eliminating dampness and stopping leukorrhea. It is mainly used to treat seminal emission, enuresis, frequent urination, chronic diarrhea due to spleen deficiency, leukorrhea, and vaginal discharge.

[0024] White peony root: The root of Paeonia lactiflora Pall., a plant belonging to the genus Paeonia in the family Ranunculaceae. It tastes bitter and sour, and is slightly cold in nature; it enters the liver and spleen meridians. It has the effects of nourishing blood and regulating menstruation, astringing yin and stopping sweating, softening the liver and relieving pain, and calming liver yang. It is used for blood deficiency and chlorosis, irregular menstruation, spontaneous sweating, night sweats, hypochondriac pain, abdominal pain, limb spasms, headache, and dizziness.

[0025] Amomum villosum Lour. (family Zingiberaceae), var. *xanthioides* TLWu et Senjen, or var. *hainanensis* TLWu are all species of Amomum villosum Lour. (family Zingiberaceae). It has a pungent taste and warm properties; it enters the spleen, stomach, and kidney meridians. It has the effects of resolving dampness and invigorating the stomach, warming the spleen and stopping diarrhea, regulating qi and calming the fetus. It is mainly used to treat dampness obstructing the middle jiao, epigastric fullness and lack of appetite, spleen and stomach deficiency and cold, vomiting and diarrhea, morning sickness, and threatened abortion.

[0026] Perilla stem: The dried stem of Perilla frutescens, a plant in the Lamiaceae family. It is pungent and warm in nature, and enters the lung and spleen meridians. It has the effects of regulating qi, relieving chest tightness, alleviating pain, and calming the fetus; it is mainly used to treat chest tightness, stomach pain, belching and vomiting, and threatened abortion.

[0027] Ginger: The fresh rhizome of *Zingiber officinale* Rosc., a plant belonging to the ginger family (Zingiberaceae). It has a pungent taste and slightly warm properties; it enters the lung, spleen, and stomach meridians. It has the effects of relieving exterior syndromes and dispersing cold, warming the middle jiao and stopping vomiting, resolving phlegm and relieving cough, and detoxifying fish and crab poisoning. It is mainly used for colds due to wind-cold, vomiting due to stomach cold, cough with cold phlegm, and fish and crab poisoning.

[0028] (III) Beneficial Effects

[0029] The formula uses tortoise shell glue, ginseng, and human head seven-seven as the principal herbs. Tortoise shell glue, being a substance rich in blood and flesh, can directly enter the lower burner to replenish kidney essence, serving as a key herb for nourishing blood and replenishing essence. It can nourish the ovaries and provide the material basis for restoring "Tiankui" (a concept in Traditional Chinese Medicine), thus replenishing essence deficiency. Human head seven-seven warms the kidneys and replenishes essence, thus replenishing yang deficiency. Ginseng tonifies qi, consolidates essence, benefits the kidneys, and calms the mind, representing qi. The combination of these three herbs achieves the effects of nourishing yin, tonifying yang, and mutually generating qi and essence. Cornus officinalis tonifies the liver and kidneys, assisting the principal herbs in warming the kidneys and nourishing yin. Euryale ferox consolidates the kidneys, astringes essence, strengthens the spleen, and stops leukorrhea. When used with other yin-tonifying herbs, it can especially help replenish essence. The astringent properties of Cornus officinalis and Euryale ferox can assist the principal herbs in preventing the loss of essence and qi from the lower burner. Dendrobium officinale nourishes stomach yin to replenish the source of life and benefits kidney yin to strengthen the innate foundation. Amomum villosum regulates qi, warms the middle jiao, and eliminates dampness to invigorate the spleen. Both can balance the heavy and purely yin nature of the principal ingredient, tortoise shell glue, which can be cloying and harmful to the stomach. Therefore, Cornus officinalis, Euryale ferox, Dendrobium officinale, and Amomum villosum are used as assistant ingredients. Myrtle and Lycopus lucidus invigorate blood, regulate menstruation, and unblock meridians. The combination of these two herbs is warming and does not harm the body's vital energy. Together, they unblock the blood vessels of the Chong and Ren meridians and the uterus, clearing the way for the circulation of essence and blood and the recovery of ovarian function, so that essence and blood can nourish the ovaries. Green plum blossom, Nardostachys jatamansi, and Perilla frutescens are aromatic and effective remedies for depression. They can soothe the liver, regulate qi, relieve depression, and calm the mind. The combination of these three herbs can open up the channels of qi and blood circulation, assisting the assistant ingredients in invigorating blood and removing blood stasis. At the same time, they can also promote the flow of qi throughout the body and resolve the causes of liver stagnation. White peony root nourishes blood and softens the liver, which can help tortoise shell glue transform into yin through its sour and sweet properties, while balancing the warm and dryness of the first seven days of the lunar calendar. Ginger can also help Amomum villosum warm the middle and harmonize the stomach, and can harmonize the various medicines, making it the guiding medicine in the prescription.

[0030] This study established a mouse model of premature ovarian failure (POF) to evaluate the in vivo and in vitro efficacy of this composition. In vitro results showed that the composition inhibited ovarian granulosa cell apoptosis, reduced the expression levels of pro-apoptotic proteins Bax and Cleavedcaspase-3 / caspase-3, and increased the expression of the anti-apoptotic protein Bcl-2. In vivo results showed that the drug intervention significantly restored the estrous cycle in mice and significantly increased the ovarian proportion in the model group, suggesting that the composition can restore normal ovulation function and has repairing and nourishing effects on ovarian tissue. Simultaneously, the composition increased serum E2 and AMH levels and decreased FSH levels in the model group mice, indicating that it can improve the endocrine status and ovarian reserve function of mice to a certain extent. Furthermore, mating experiments further verified the positive effect of the composition on the recovery of reproductive function in the POF model mice. These results indicate that the composition exerts its therapeutic effect through the synergistic action of multiple components, providing new experimental evidence and reference for the application of traditional Chinese medicine compound formulas in the field of reproductive health. Attached Figure Description

[0031] Figure 1 The results of cell viability testing were compared with those of the control group. 1 P<0.05, 2 P<0.01; compared with the model group, 3 P<0.05, 4P<0.01; compared with the composition, 5 P<0.05, 6 P<0.01.

[0032] Figure 2 Regarding cell apoptosis, compared with the control group, 1 P<0.05, 2 P<0.01; compared with the model group, 3 P<0.05, 4 P<0.01; compared with the composition, 5 P<0.05, 6 P<0.01.

[0033] Figure 3 The expression levels of apoptosis-related markers Bcl-2, BaX, and Cleaved-caspase-3 / caspase-3 proteins were compared with those of the control group. 1 P<0.05, 2 P<0.01; compared with the model group, 3 P<0.05, 4 P<0.01; compared with the composition, 5 P<0.05, 6 P<0.01.

[0034] Figure 4 The estrous cycle of each group of animals.

[0035] Figure 5 The ovarian index of each group of animals was compared with that of the control group. 1 P<0.05, 2 P<0.01; compared with the model group, 3 P<0.05, 4 P<0.01; compared with the high-dose group of the composition, 5 P<0.05, 6 P<0.01.

[0036] Figure 6 The levels of E2, AMH, and FSH in the serum of each group of animals were compared with those in the control group. 1 P<0.05, 2 P<0.01; compared with the model group, 3 P<0.05, 4 P<0.01; compared with the high-dose group of the composition, 5 P<0.05, 6 P<0.01.

[0037] Figure 7 For the pregnancy and parturition status of animals in each group, compared with the control group, 1P<0.05, 2 P<0.01; compared with the model group, 3 P<0.05, 4 P<0.01; compared with the high-dose group of the composition, 5 P<0.05, 6 P<0.01. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. 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.

[0039] Example 1

[0040] A composition for improving premature ovarian failure, said composition being made from the following ingredients in the indicated weights: 3g tortoise shell glue, 5g Dendrobium officinale, 8g Myrtle, 2g Lycopus lucidus, 1g Nardostachys jatamansi, 1g Prunus mume, 3g Panax quinquefolius, 3g Panax notoginseng, 5g dried scallop, 2g Euryale ferox, 6g Paeonia lactiflora, 1g Amomum villosum, 2g Perilla frutescens stem, and 1g fresh ginger.

[0041] Example 2

[0042] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in the indicated weights: 18g of tortoise shell glue, 22g of Dendrobium officinale, 20g of Myrtle, 18g of Lycopus lucidus, 15g of Nardostachys jatamansi, 15g of Prunus mume, 24g of Codonopsis pilosula, 25g of Panax notoginseng, 35g of dried scallop, 20g of Euryale ferox, 25g of Paeonia lactiflora, 12g of Amomum villosum, 20g of Perilla frutescens stem, and 10g of fresh ginger.

[0043] Example 3

[0044] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in the indicated weights: 6g of tortoise shell glue, 10g of Dendrobium officinale, 10g of Myrtle, 6g of Lycopus lucidus, 3g of Nardostachys jatamansi, 3g of Prunus mume, 9g of Panax ginseng, 9g of Panax notoginseng, 10g of dried scallop, 10g of Euryale ferox, 10g of Paeonia lactiflora, 3g of Amomum villosum, 6g of Perilla frutescens stem, and 3g of fresh ginger.

[0045] Example 4

[0046] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials by weight:

[0047] 12g of tortoise shell glue, 15g of Dendrobium officinale, 15g of Myrtle, 12g of Lycopus lucidus, 6g of Nardostachys jatamansi, 6g of Prunus mume, 15g of Codonopsis pilosula, 12g of Panax notoginseng, 25g of dried scallop, 15g of Euryale ferox, 15g of Paeonia lactiflora, 6g of Amomum villosum, 12g of Perilla frutescens stem, and 6g of fresh ginger.

[0048] Example 5

[0049] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials by weight:

[0050] 8g of tortoise shell glue, 12g of Dendrobium officinale, 12g of Myrtle, 8g of Lycopus lucidus, 4g of Nardostachys jatamansi, 4g of Prunus mume, 10g of Codonopsis pilosula, 10g of Panax notoginseng, 12g of dried scallop, 12g of Euryale ferox, 12g of Paeonia lactiflora, 4g of Amomum villosum, 10g of Perilla frutescens stem, and 4g of fresh ginger.

[0051] Example 6

[0052] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials by weight:

[0053] 10g of tortoise shell glue, 14g of Dendrobium officinale, 14g of Myrtle, 10g of Lycopus lucidus, 5g of Nardostachys jatamansi, 5g of Prunus mume, 12g of Codonopsis pilosula, 12g of Panax notoginseng, 15g of dried scallop, 14g of Euryale ferox, 14g of Paeonia lactiflora, 5g of Amomum villosum, 8g of Perilla frutescens stem, and 5g of fresh ginger.

[0054] Example 7

[0055] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials by weight:

[0056] 16g of tortoise shell glue, 18g of Dendrobium officinale, 16g of Myrtle, 16g of Lycopus lucidus, 12g of Nardostachys jatamansi, 12g of Prunus mume, 18g of Codonopsis pilosula, 18g of Panax notoginseng, 30g of dried scallop, 18g of Euryale ferox, 20g of Paeonia lactiflora, 8g of Amomum villosum, 18g of Perilla frutescens stem, and 8g of fresh ginger.

[0057] Example 8

[0058] The difference between this embodiment and Embodiment 1 is that the traditional Chinese medicine preparation is made from the following raw materials in the indicated weights: 10g of tortoise shell glue, 12g of Dendrobium officinale, 12g of Myrtle, 8g of Lycopus lucidus, 4g of Nardostachys jatamansi, 4g of Prunus mume, 12g of Codonopsis pilosula, 10g of Panax notoginseng, 12g of dried scallop, 12g of Euryale ferox, 14g of Paeonia lactiflora, 5g of Amomum villosum, 8g of Perilla frutescens stem, and 4g of fresh ginger.

[0059] Experimental Example 1

[0060] 1.1 Experimental Drugs

[0061] Composition: 10g tortoise shell glue, 12g Dendrobium officinale, 12g Myrtle, 8g Lycopus lucidus, 4g Nardostachys jatamansi, 4g Prunus mume, 12g Panax quinquefolius, 10g Panax notoginseng, 12g dried scallop, 12g Euryale ferox, 14g Paeonia lactiflora, 5g Amomum villosum, 8g Perilla frutescens stem, 4g fresh ginger.

[0062] Comparative Example 1: 10g tortoise shell glue, 12g myrtle, 8g Lycopus lucidus, 12g ginseng, 10g human head herb, 12g dried scallop, 14g white peony root, 5g amomum villosum, 4g ginger.

[0063] Comparative Example 2: 10g of tortoise shell glue, 12g of Dendrobium officinale, 12g of Myrtle, 8g of Lycopus lucidus, 4g of Nardostachys jatamansi, 4g of Prunus mume, 12g of dried scallop, 12g of Euryale ferox, 14g of Paeonia lactiflora, 5g of Amomum villosum, 8g of Perilla frutescens stem, and 4g of fresh ginger.

[0064] Comparative Example 3: Dendrobium officinale 12g, Myrtle 12g, Lycopus lucidus 8g, Nardostachys jatamansi 4g, Prunus mume 4g, Codonopsis pilosula 12g, Panax notoginseng 10g, Scallop 12g, Euryale ferox 12g, Paeonia lactiflora 14g, Amomum villosum 5g, Perilla frutescens 8g, Ginger 4g.

[0065] Take the above-mentioned medicinal materials (except for tortoise shell glue), add water to completely cover the medicinal materials, soak for 30 minutes, then decoct for 30 minutes. Then add the melted tortoise shell glue to the decoction (this step can be omitted if there is no tortoise shell glue in the prescription), mix well, and concentrate to a raw herb content of 1g / ml for later use.

[0066] Preparation of drug-containing serum for each group: Male SD rats aged 6-8 weeks were randomly divided into 5 groups of 3 rats each. The rats were administered the drug via gavage. The gavage doses for the combined drug group, comparative example 1, comparative example 2, and comparative example 3 were 55 g / kg / d, 39 g / kg / d, 47 g / kg / d, and 50 g / kg / d, respectively. The control group was administered the same volume of physiological saline via gavage once daily for 7 consecutive days. One hour after the last gavage administration, the animals were anesthetized, blood was collected from the abdominal aorta, serum was separated, and the serum was inactivated and labeled at 56℃ for later use.

[0067] 1.2 Experimental Cells

[0068] Since ovarian granulosa cell apoptosis is the direct cause of follicular atresia, which in turn affects normal ovarian function and leads to premature ovarian failure, the formula screening experiment used the KGN cell line derived from human ovarian granulosa cells.

[0069] 1.3 Cell modeling and drug administration

[0070] Cells were cultured normally for 12 hours after plating. The normal group was given standard culture medium, while the experimental group was given 200 μM acrolein (a metabolite of cyclophosphamide). At the same time, 10% of the corresponding drug-containing serum was added to each group (the drug concentration was determined by previous basic experimental research). Cells were cultured for 24 hours and then tested.

[0071] 1.4 Detection Indicators

[0072] 1.4.1 Cell viability assay

[0073] KGN cells were used at 5 × 10 3 Cells were cultured at a density of 96-well plates. Cell modeling and drug treatment were performed according to step 1.3. The supernatant was discarded, and 100 μL of 10% CCK-8 reagent was added. After the reaction was complete, the absorbance was measured at 450 nm.

[0074] 1.4.2 Detection of Apoptosis Status

[0075] KGN cells were loaded at 1×10 5 Cells were cultured at the density of cells / wells in 24-well plates. Cell modeling and drug administration were performed according to step 1.3. Flow cytometry was used to detect the cells using Annexin V-FITC / PI dual fluorescent dyes.

[0076] 1.4.3 Apoptosis markers: expression levels of Bcl-2, BaX, and Cleaved-caspase-3 / caspase-3 proteins

[0077] KGN cells were used at 5 × 10 5 Cells were cultured at a density of 6-well plates. Cell modeling and drug treatment were performed according to step 1.3. The original culture medium was discarded, and 500 μL of PBS was added to each well. Cells were scraped with a cell scraper, and the cell suspension was collected. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell pellet was retained for subsequent Western blot analysis.

[0078] 2 Results

[0079] 2.1 Cell viability assay

[0080] The results are shown below. Figure 1 Compared with the blank group, the KGN cell viability in the model group was significantly reduced after acrolein induction (P<0.01); after drug intervention in each group, cell viability was improved, among which the combination group showed the most significant improvement in cell viability (P<0.01).

[0081] 2.2 Apoptosis

[0082] Follicular atresia is a key characteristic of premature ovarian failure (POF) and can be caused by apoptosis of granulosa cells and oocytes. Therefore, ovarian granulosa cell apoptosis is considered an initiating factor in POF. This study investigated the apoptosis rate in each group. The results are shown below. Figure 2Compared with the blank group, the apoptosis rate of the model group was significantly increased (P<0.01); compared with the model group, only the apoptosis rate of the combination group and the control group showed a significant decreasing trend, and the apoptosis rate reduction effect of the combination group was more significant than that of the control group (P<0.01).

[0083] 2.3 Apoptosis-related indicators: expression levels of Bcl-2, BaX, and Cleaved-caspase-3 / caspase-3 proteins

[0084] The Bcl-2 / Bax / Caspase-3 signaling pathway is a core mechanism regulating apoptosis in granulosa cells. The pro-apoptotic protein Bax forms pores in the mitochondrial membrane, increasing membrane permeability and promoting the release of cytochrome C into the cytoplasm; while the anti-apoptotic protein Bcl-2 inhibits this process by binding to Bax, maintaining mitochondrial stability. Results showed… Figure 3 Compared with the control group, the expression levels of pro-apoptotic proteins Bax and Cleaved caspase-3 / caspase-3 were increased, while the expression level of anti-apoptotic protein Bcl-2 was decreased in the model group cells. Among the drug intervention groups, the combination drug showed the most significant effect in inhibiting the expression of pro-apoptotic proteins and increasing the content of anti-apoptotic proteins compared with the model group.

[0085] 3 Discussion

[0086] The above results show that this composition can improve the viability of ovarian granulosa cells after modeling, inhibit acrolein-induced apoptosis of ovarian granulosa cells, downregulate the expression of pro-apoptotic proteins Caspase-3 and Bax, and upregulate the expression of anti-apoptotic protein Bcl-2, effectively protecting ovarian granulosa cells and maintaining normal ovarian function. Among them, the composition of the present invention showed the best improvement effect in all the above indicators. The comparative groups 1-3 showed varying degrees of weakened effect due to the lack of some medicinal ingredients in the formula. This phenomenon verifies at the molecular level the multi-component, multi-target, and synergistic effect of the traditional Chinese medicine preparation of the present invention, which jointly improves premature ovarian failure.

[0087] Experimental Example 2

[0088] 1.1 Experimental reagents

[0089] Composition: 10g tortoise shell glue, 12g Dendrobium officinale, 12g Myrtle, 8g Lycopus lucidus, 4g Nardostachys jatamansi, 4g Prunus mume, 12g Panax quinquefolius, 10g Panax notoginseng, 12g dried scallop, 12g Euryale ferox, 14g Paeonia lactiflora, 5g Amomum villosum, 8g Perilla frutescens stem, 4g fresh ginger.

[0090] Take the above-mentioned medicinal materials (except for the tortoise shell glue), add water to completely cover the medicinal materials, soak for 30 minutes, then decoct for 30 minutes. Then add the melted tortoise shell glue to the decoction, mix well, and concentrate to a raw medicinal content of 1g / ml for later use.

[0091] Zuogui Pill: Purchased from Zhongjing Wanxi Pharmaceutical Co., Ltd.

[0092] Estradiol Valerate: Bayer Healthcare Company, Guangzhou Branch.

[0093] 1.2 Laboratory Animals

[0094] SPF-grade 8-week-old female C57 / BL6 mice, weighing (20±0.5) g, were provided by Liaoning Changsheng Biotechnology Co., Ltd.

[0095] 1.3 Modeling Methods and Grouping

[0096] Mice were acclimatized for 7 days, and vaginal smear screening was used to ensure that no estrous cycle disorders were observed. Six mice were randomly selected as the control group using a random number table. Except for the control group, which received an equal volume of saline, the remaining mice underwent modeling. On the first day, mice were intraperitoneally injected with 60 mg / kg cyclophosphamide, followed by 8 mg / kg cyclophosphamide intraperitoneally for 14 consecutive days to establish a mouse model of premature ovarian failure. After modeling, animals exhibiting estrous cycle disorders were randomly divided into a model group, a high-dose combination group, a medium-dose combination group, a low-dose combination group, a positive control group (Zuogui Wan) using traditional Chinese medicine, and a positive control group (estradiol valerate). The high, medium, and low dose combination groups were administered 32 g / kg via gavage. -1 16g·kg -1 8g·kg -1 The positive group of Western medicine was administered 0.15 mg / kg via gavage. -1 Estradiol valerate, 2g / kg was administered orally to the positive control group of the traditional Chinese medicine treatment. -1 Zuogui Pills were administered to both the control group and the model group via gavage with an equal volume of physiological saline for 4 weeks.

[0097] 1.4 Detection Indicators

[0098] 1.4.1 Estrogenic cycle in mice with premature ovarian failure

[0099] Vaginal exfoliative cytology was performed daily during the last two weeks of drug administration to record the estrous cycle of mice. The estrous cycle disorder rate in each group was calculated as: (Number of mice with estrous cycle disorder / Total number of mice in each group) × 100%.

[0100] 1.4.2 Ovarian index in mice with premature ovarian failure

[0101] Record the bilateral ovarian weight and body weight of the mice, and calculate the ovarian index = single ovarian weight (mg) / mouse body weight (g) × 100%.

[0102] 1.4.3 Serum levels of E2, AMH, and FSH in mice with premature ovarian failure

[0103] Mouse serum was collected and the levels of E2, AMH, and FSH in the serum were detected according to the ELISA kit instructions.

[0104] 1.4.4 Birth rate of offspring from mice with premature ovarian failure

[0105] Male and female mice were grouped at a ratio of 2:1 at 18:00 daily. The vaginal plugs of the female mice were examined at 8:00 the following day; the day of plug examination was defined as day 0.5 of gestation, and the cycle continued for 5 days, with the above examination repeated daily. Female mice with plugs were removed and housed individually. The date of parturition was recorded to calculate gestation duration, and offspring indicators were collected. Litter size was recorded within 24 hours of parturition.

[0106] 1.5 Statistical Methods

[0107] Statistical analysis was performed on the data using GraphPad Prism 8.0 software, and one-way ANOVA was used for comparisons among multiple groups.

[0108] 2 Results

[0109] 2.1 Estrogenic cycle of mice in each group

[0110] The resumption of the estrous cycle is a signal of ovarian function recovery. Observation results of the estrous cycle show... Figure 4 The control group mice generally exhibited regular estrous cycles, while the model group mice all showed varying degrees of estrous cycle disorder, arrest, or prolongation. After drug intervention, the number of normal mice in the high-, medium-, and low-dose groups of the composition was higher than that in the other two positive drug groups. This suggests that treatment with all three doses of the composition can restore normal estrous cycles in mice to varying degrees, indicating that the ovaries can ovulate normally, with the high-dose group showing the best recovery effect.

[0111] 2.2 Ovarian index detection in each group of mice

[0112] The results are shown below. Figure 5 Compared with the blank group, the ovarian index of mice in the model group decreased significantly (P<0.01); compared with the model group, the ovarian index of all other dose groups increased to varying degrees, with the high dose group of the composition showing the best effect, indicating that the composition has the effect of repairing and nourishing ovarian tissue.

[0113] 2.3 Serum levels of E2, AMH, and FSH in each group of mice

[0114] Elevated FSH and decreased E2 are the main clinical diagnostic indicators for premature ovarian failure; AMH can accurately and rapidly reflect ovarian reserve function. Results are shown in... Figure 6 Compared with the blank group, the serum levels of E2 and AMH in the model group mice were significantly reduced, while the FSH level was significantly increased (P<0.01). After drug treatment, all drug treatment groups showed a trend of recovery to normal, and the high and medium dose groups of the combination were more effective than the other two positive drug groups.

[0115] 2.4 Birth rate of offspring in mice with premature ovarian failure

[0116] The results are shown below. Figure 7 Compared with the blank group, the model group mice had a lower birth rate and fewer pups (P<0.01); after drug treatment, the reproductive status of mice in all groups improved, with the high and medium dose groups of the combination showing better effects and significant differences (P<0.01).

[0117] 3. Discussion

[0118] This invention establishes a mouse model of premature ovarian failure (POF) using cyclophosphamide. The model mice exhibited disordered, prolonged, or arrested estrous cycles, along with decreased ovarian indices, indicating impaired ovarian function. After drug intervention, ovarian function improved to some extent in all dosage groups of the composition and the two positive control groups, with high and medium doses showing the best results. Following treatment with the composition, the estrous cycles of the mice gradually returned to normal, the ovarian indices increased, endocrine system disorders were regulated, and ovarian function was restored (increasing serum E2 and AMH levels and inhibiting FSH levels). Furthermore, in animal reproductive observations, this composition significantly improved reproductive health. This provides a potential and promising treatment method for patients with PSF and even infertility.

[0119] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for improving premature ovarian failure, characterized in that, The composition comprises the following raw materials in parts by weight: 3-18 parts tortoise shell glue, 5-22 parts Dendrobium officinale, 8-20 parts Myrtle, 2-18 parts Lycopus lucidus, 1-15 parts Nardostachys jatamansi, 1-15 parts Prunus mume, 3-24 parts Panax quinquefolius, 3-25 parts Panax notoginseng, 5-35 parts dried scallop, 2-20 parts Euryale ferox, 6-25 parts Paeonia lactiflora, 1-12 parts Amomum villosum, 2-20 parts Perilla frutescens stem, and 1-10 parts fresh ginger.

2. The composition according to claim 1, characterized in that, The composition is made from the following raw materials in parts by weight: 6-12 parts tortoise shell glue, 10-15 parts Dendrobium officinale, 10-15 parts Myrtle, 6-12 parts Lycopus lucidus, 3-6 parts Nardostachys jatamansi, 3-6 parts Prunus mume, 9-15 parts Panax ginseng, 9-12 parts Panax notoginseng, 10-25 parts dried scallop, 10-15 parts Euryale ferox, 10-15 parts Paeonia lactiflora, 3-6 parts Amomum villosum, 6-12 parts Perilla frutescens stem, and 3-6 parts fresh ginger.

3. The composition according to claim 1, characterized in that, The composition is made from the following raw materials in parts by weight: 10 parts tortoise shell glue, 12 parts Dendrobium officinale, 12 parts Myrtle, 8 parts Lycopus lucidus, 4 parts Nardostachys jatamansi, 4 parts Prunus mume, 12 parts Codonopsis pilosula, 10 parts Panax notoginseng, 12 parts dried scallop, 12 parts Euryale ferox, 14 parts Paeonia lactiflora, 5 parts Amomum villosum, 8 parts Perilla frutescens stem, and 4 parts fresh ginger.

4. The use of the composition according to any one of claims 1 to 3 in the preparation of a product for improving premature ovarian failure, characterized in that, The composition is prepared according to conventional methods into any one of the following oral dosage forms: pills, powders, granules, oral liquids, tablets, capsules, and ointments.

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