Application of camellia oil in prevention and treatment of ovarian function decline

Abstract

The invention is suitable for the technical field of camellia oil application, and provides application of camellia oil in prevention and treatment of ovarian function decline. The invention reveals that the camellia oil has good prevention and improvement effects on the ovarian function decline for the first time. A series of rigorous scientific experiments prove that the camellia oil can effectively improve weight loss and reproductive hormone disorder caused by mouse ovarian function decline induced by cyclophosphamide, repair ovarian tissue damage and inhibit ovarian granular cell apoptosis, so that normal development of ovaries is promoted, the ovarian index is increased, and the effect of preventing and treating ovarian diseases is achieved. The number of atresia follicles is reduced, the pregnancy rate and the litter size are increased, and the occurrence of ovarian function decline is effectively prevented and improved. The discovery provides a scientific basis for preventing and improving ovarian function decline by using the camellia oil, and avoids tissue hyperplasia and canceration risks caused by strong stimulation of an endocrine system, reproductive organs and mammary gland by exogenous hormones in a hormone therapy.

Description

Technical Field

[0001] The invention belongs to the technical field of camellia oil application, and in particular relates to the application of camellia oil in preventing and treating ovarian function decline. Background Art

[0002] Ovarian dysfunction refers to the decrease in the number of follicles and the decline in the quality of oocytes caused by non-biological factors before the age of 40. It is one of the main causes of female infertility. In recent years, the incidence of ovarian dysfunction has increased year by year. According to statistics, the prevalence is about 10%-35%, and it is showing a trend of younger age, which seriously affects women's reproductive health and quality of life. Ovarian dysfunction is a pathological process, which mainly includes three stages: Diminished Ovarian Reserve (DOR), Premature Ovarian Insufficiency (POI) and Premature Ovarian Failure (POF). DOR is the early stage, POI develops from DOR, and POF is the final stage of the pathological process. Although DOR, POI and POF are different stages of the pathological process, they are clinically characterized by oligomenorrhea or amenorrhea, increased basal gonadotropin, and decreased estrogen and anti-mullerian hormone. Modern research shows that ovarian dysfunction is related to many factors such as age, genetics, immunity, environment, infection, iatrogenic injury, and multiple pregnancy. Its etiology is complex and its mechanism is still unclear, and there is no specific treatment. At present, the treatment of this disease mostly uses estrogen replacement therapy (HRT), which aims to relieve the related symptoms caused by hormone deficiency. However, the treatment effect is not ideal, and exogenous estrogen can easily disrupt the "hypothalamus-pituitary-ovary" endocrine system, and over-stimulate target organs such as the uterus and breasts, increasing the risk of cancer. Therefore, in the current social context of declining fertility rates year by year, it is particularly important and urgent to develop a safe and effective drug that can effectively prevent and improve ovarian dysfunction.

[0003] Camellia oil (CO), also known as tea oil and tea seed oil, is a natural woody edible oil obtained from the seeds of Camellia oleifera Abel. of the Theaceae family by pressing or leaching. Camellia oil is rich in nutrients, and its unsaturated fatty acid content is as high as 80% or more, so it is recommended as a healthy food by the Food and Agriculture Organization of the United Nations. "Compendium of Materia Medica" records: "Camellia oil is cool in nature, and has the effects of cooling blood, stopping bleeding, clearing heat, and detoxifying." Modern research has found that camellia oil is rich in tocopherol, tea polyphenols, phytosterols, tea saponins, squalene and other bioactive substances, and has anti-inflammatory, antibacterial, anti-aging, anti-cancer and other effects. It has certain applications in the fields of medicine and cosmetics, but its research and application in reproductive health has not been reported. For this reason, the present invention proposes the application of camellia oil in preventing and treating ovarian function decline. Summary of the invention

[0004] The purpose of the present invention is to provide an application of camellia oil in preventing and treating ovarian dysfunction, aiming to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions: Application of camellia oil in preparing medicine for preventing or treating ovarian dysfunction.

[0006] Application of camellia oil in preparing health products for preventing or improving ovarian function decline.

[0007] Furthermore, the decreased ovarian function includes diminished ovarian reserve function, premature ovarian insufficiency and premature ovarian failure.

[0008] Furthermore, the dosage forms used include soft capsules, pills, emulsions, pills, tablets, lotions, liniments, ointments or suppositories.

[0009] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention reveals for the first time that camellia oil has a good preventive and improving effect on ovarian dysfunction. Through a series of rigorous scientific experiments, the present invention confirms that camellia oil can effectively improve the weight loss and reproductive hormone disorders caused by cyclophosphamide-induced ovarian dysfunction in mice, repair ovarian tissue damage, inhibit ovarian granulosa cell apoptosis, and promote the normal development of the ovaries, improve the ovarian index, reduce the number of atretic follicles, increase the pregnancy rate and the number of litters, and effectively prevent and improve the occurrence of ovarian dysfunction. This discovery provides a scientific basis for the use of camellia oil to prevent and improve ovarian dysfunction, and avoids the risk of tissue hyperplasia and carcinogenesis caused by strong stimulation of the endocrine system, reproductive organs and mammary glands by exogenous hormones in hormone therapy.

[0010] 2. The camellia oil used in the present invention is widely planted in southern my country, with sufficient raw material sources, and has the characteristics of being both a medicine and food. Long-term use will not cause damage to other organs such as the liver and kidneys. It is rich in unsaturated fatty acids and a variety of bioactive ingredients, and can fully nourish and regulate the body through a multi-target and multi-pathway approach, effectively improving the decline of ovarian function and delaying the ovarian aging process. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the research roadmap of the present invention.

[0012] Figure 2 The figure shows the body weight change trend of mice in each group.

[0013] Figure 3 The results of serum reproductive hormone changes in each group of mice.

[0014] Figure 4 The results of ovarian index changes in each group of mice.

[0015] Figure 5 Paraffin sections and HE staining results of ovarian tissues of mice in each group.

[0016] Figure 6 The results of apoptosis in ovarian tissues of mice in each group; a is the result of TUNEL staining, and b is the fluorescence positive rate of each group.

[0017] Figure 7 The figures are for the litter size of mice in each group. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0019] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments. In the embodiments, cyclophosphamide is referred to as CTX; camellia oil is referred to as CO; follicle-stimulating hormone is referred to as FSH; estradiol is referred to as E2; and anti-mullerian hormone is referred to as AMH.

[0020] Example 1: Study on the preventive and improving effects of camellia oil on cyclophosphamide-induced ovarian dysfunction in mice. Figure 1 shown.

[0021] 1 Materials and methods; 1.1 Establishment of an animal model of ovarian dysfunction; Cyclophosphamide is a commonly used alkylating anti-tumor drug, which is mainly used in the clinical treatment of malignant lymphoma, multiple myeloma, leukemia and breast cancer. Studies have found that cyclophosphamide has a strong selective toxicity to the ovaries, and can induce apoptosis of ovarian granulosa cells by organizing DNA replication, thereby causing ovarian function damage, leading to ovarian reserve function decline, early-onset sexual dysfunction, and even premature aging. Due to its high toxicity, long duration of action and stable effect, it has been widely used in the establishment of animal models of ovarian dysfunction.

[0022] Female KM mice with normal estrous cycle at 8 weeks old were selected and intraperitoneally injected with CTX (50 mg / kg.bw) once a day for 7 consecutive days to establish a mouse model of ovarian dysfunction. The judgment criteria are: if the mice have estrous cycle disorders, increased serum FSH levels, and decreased E2 and AMH levels, the animal model of ovarian dysfunction is successfully established; otherwise, the animal model is considered to be unsuccessful.

[0023] 1.2 Experimental grouping and drug administration; Seventy-six 8-week-old female KM mice with normal estrous cycle were randomly divided into Control group, Model group, CTX+CO 1 group (camellia oil high-dose group) and CTX+CO 2 group (camellia oil low-dose group), with 19 mice in each group. They were allowed to drink water and eat freely. After adapting to the feeding, the mice were given medication according to the scheme in Table 1.

[0024]

[0025] Animal status was monitored during the experiment. On the 22nd day, 10 mice were randomly selected from each group for anesthesia, blood sampling, and sacrifice, and samples were collected for further testing. The remaining mice were caged with male mice to observe their reproductive status.

[0026] 1.3 Indicator measurement and data collection; (1) Observe the mental state of each group of mice every day, weigh them, and draw a graph of their weight change trends.

[0027] (2) Ten mice were randomly selected from each group for anesthesia and blood collection. The mice were anticoagulated with sodium heparin and the plasma was separated. The levels of FSH, E2, and AMH in the plasma were detected by ELISA. (3) Remove the ovaries, weigh the wet weight, and calculate the ovarian index. The calculation formula is: Ovarian index = ovarian wet weight / body weight * 100%.

[0028] (4) The left ovary was fixed in 4% paraformaldehyde solution, embedded in paraffin, sectioned, and stained with HE, and its tissue structure changes were observed under a microscope.

[0029] (5) The ovaries were sliced ​​and then stained with TUNEL, and the apoptosis of ovarian tissue cells was observed under a fluorescence microscope.

[0030] (6) The remaining 9 mice in each group were housed together in a male:female ratio of 3:1 for 10 days, and their pregnancy rate and litter size were followed up.

[0031] 1.4 Data statistical analysis; SPSS 27.0 statistical software was used for statistical analysis of the data, and one-way ANOVA test was used for variance analysis. P >0.05, the LSD method was used for multiple comparisons between groups. P <0.05), Tamhane's T2 method was used for multiple comparisons between groups. The results are expressed as "mean ± standard error". P <0.01 indicates a very significant difference. P <0.05 indicates significant difference. P >0.05 indicated that the difference was not significant.

[0032] 2 Results; 2.1 Effects of camellia oil on body weight and mental state (physical changes) of mice with cyclophosphamide-induced ovarian dysfunction; Body weight and mental state can directly reflect the effect of camellia oil against cyclophosphamide damage. In terms of mental state, there was no significant difference between the CTX+CO 1 group, CTX+CO 2 group and the Control group. The mice were lively and active, sensitive to external stimuli, and had shiny fur. However, the mice in the Model group were listless, with hunched necks and arched backs, and their fur stood up, and they had hair loss.

[0033] Weight changes such as Figure 2 As shown in the data, the body weight of mice in the CTX+CO 1 and CTX+CO 2 groups remained stable, with no significant difference from the Control group, while the body weight of mice in the Model group continued to decrease after injection of cyclophosphamide, which was significantly lower than that of the Control group and the CTX+CO 1 and CTX+CO 2 groups, indicating that camellia oil can effectively counteract the weight loss caused by cyclophosphamide.

[0034] 2.2 Effect of camellia oil on serum reproductive hormones (FSH, E2 and AMH) in mice with cyclophosphamide-induced ovarian dysfunction; Although ovarian dysfunction has different clinical pathological stages, they are all characterized by infrequent or amenorrhea, elevated basal gonadotropin, and decreased estrogen and anti-Mullerian hormone. Changes in FSH, E2, and AMH hormones are important indicators for clinical diagnosis and evaluation of the effectiveness of treatment of the disease. The results of this test are shown in Table 2 and Figure 3As shown in the figure, compared with the Control group, the FSH level in the Model group was significantly increased, and the E2 and AMH levels were significantly decreased, indicating that the ovarian function decline model was successfully established. Compared with the Model group, the FSH level in the CTX+CO 1 group and the CTX+CO 2 group was significantly decreased, and the E2 and AMH levels were significantly increased. This shows that camellia oil can effectively regulate the hormone disorder caused by ovarian function decline and improve ovarian function decline.

[0035]

[0036] Note: “*” represents a significant difference compared with the Control group, P<0.05; “**” represents an extremely significant difference compared with the Control group, P<0.01; “#” represents a significant difference compared with the Model group, P<0.05; “##” represents an extremely significant difference compared with the Model group, P<0.01.

[0037] 2.3 Effects of camellia oil on the ovaries of mice with cyclophosphamide-induced ovarian dysfunction; 2.3.1 Effect of camellia oil on ovarian index; The ovarian index is the ratio of the ovaries to the total body weight of the animal, and is mainly used to evaluate the development of the ovaries and monitor ovarian function. In reproductive research, the ovarian index is an important indicator for evaluating the effects of different factors on the ovaries. The results of this experiment are shown in Table 3 and Figure 4 As shown in the figure, the ovarian index of the CTX+CO 1 group was significantly higher than that of the Control group and the Model group. The ovarian index of the CTX+CO 2 group was higher than that of the Control group and the Model group, and the ovarian index of the Control group was higher than that of the Model group. This shows that camellia oil can effectively promote ovarian development and improve ovarian function.

[0038]

[0039] Note: “#” indicates significant difference compared with the Model group, P<0.05; “##” indicates extremely significant difference compared with the Model group, P<0.01.

[0040] 2.3.2 Effect of camellia oil on ovarian pathological changes; The ovary is the core organ in the female reproductive system. Its main function is to produce oocytes and promote their development, maturation and release. In addition, the ovary is an important endocrine organ that secretes estrogen, progesterone and a small amount of androgen, which are essential for maintaining normal menstrual cycles, sexual development and reproductive ability. Ovarian pathological changes directly affect ovarian function. Figure 5As shown in Table 4, there was no obvious case change in the ovarian tissue sections of the Control group. The granulosa cells surrounding the oocytes were closely arranged and relatively sparse around the fluid cavity. The granulosa cell nuclei were round, the chromatin was relatively dense, the cytoplasm was basophilic and blue-stained, and a small amount of apoptosis was occasionally seen. The granulosa cells in the primary and secondary follicles were normal in morphology. The ovaries of the Model group had obvious pathological changes. There were varying numbers of apoptotic cells in the granulosa cell layer of the primary and secondary follicles. The nuclear chromatin was condensed and darkly stained, the nucleus was fragmented, the cytoplasm was eosinophilic, or the cells were dispersed into several vesicle-like structures. The central oocyte was necrotic, the volume was reduced, and the nuclear chromatin was condensed. Only a small amount of mild granulosa cell apoptosis was seen in the ovarian tissue sections of the CTX+CO 1 group and the CTX+CO 2 group, which was close to the Control group and significantly better than the Model group. By counting the number of atretic follicles in the anterior, middle and posterior sections of the ovarian tissue, the results are shown in Table 4. The number of atretic follicles in the CTX+CO 1 group was significantly less than that in the Model group and the Control group. This indicates that camellia oil can effectively improve ovarian damage caused by cyclophosphamide and reduce follicular atresia.

[0041]

[0042] Note: “*” represents a significant difference compared with the Control group, P<0.05; “**” represents an extremely significant difference compared with the Control group, P<0.01; “#” represents a significant difference compared with the Model group, P<0.05; “##” represents an extremely significant difference compared with the Model group, P<0.01.

[0043] 2.3.3 Effect of camellia oil on apoptosis of ovarian granulosa cells; Granulosa cells are cells that surround the egg during its growth. Mature granulosa cells secrete a large amount of estrogen, regulatory factors and growth factors, and form gap junctions between cells to promote the growth, maturation and ovulation of the egg. Therefore, the development and growth of granulosa cells to a certain extent reflects the developmental capacity of the egg. Granulosa cell apoptosis will directly lead to follicular atresia. The results of this experiment are as follows Figure 6 As shown in a and b, there was a large amount of apoptosis in the ovarian granulosa cells of the mice in the Model group, with a wide range and a large number in the ovarian tissue, while there was only a small amount of apoptotic granulosa cells in the CTX+CO 1 group and the CTX+CO 2 group, which was similar to the Control group. This indicates that camellia oil can effectively inhibit the apoptosis of ovarian granulosa cells.

[0044] 2.4 Effects of camellia oil on pregnancy rate and litter size (reproduction status) in mice with cyclophosphamide-induced ovarian dysfunction; The pregnancy rate and litter size of female mice are the most direct indicators of reproductive capacity. The results of this experiment are shown in Table 5 and Figure 7As shown in the figure, the female mice in the Model group were extremely emaciated and in poor mental state, and no female mice were pregnant after 10 days of cohabitation. Six mice in the CTX+CO 1 group were pregnant and delivered, while 100% of the mice in the CTX+CO 2 group and the Control group were pregnant and delivered; the number of litters in the CTX+CO 1 group and the CTX+CO 2 group was less than that in the Control group, but the difference was not significant, P>0.05. This shows that camellia oil can significantly improve the reproductive performance of mice with ovarian dysfunction induced by cyclophosphamide.

[0045]

[0046] Note: “*” represents a significant difference compared with the Control group, P<0.05; “**” represents an extremely significant difference compared with the Control group, P<0.01; “#” represents a significant difference compared with the Model group, P<0.05; “##” represents an extremely significant difference compared with the Model group, P<0.01.

[0047] 3 Conclusion: (1) Camellia oil can effectively counteract the damage caused by cyclophosphamide and maintain stable body weight.

[0048] (2) Camellia oil can effectively correct the reproductive hormone disorder caused by cyclophosphamide and restore it to normal levels.

[0049] (3) Camellia oil can effectively improve ovarian tissue damage, inhibit granulosa cell apoptosis, and reduce the number of atretic follicles.

[0050] (4) Camellia oil can effectively promote ovarian development, increase pregnancy rate and birth quantity.

[0051] In summary, camellia oil can effectively prevent and improve ovarian function decline and promote reproductive health.

[0052] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. Application of camellia oil in the preparation of a drug for preventing or treating ovarian dysfunction.

2. Application of camellia oil in the preparation of health products for preventing or improving ovarian function decline.

3. The use according to claim 1 or 2, characterized in that: The decreased ovarian function includes diminished ovarian reserve, premature ovarian insufficiency and premature ovarian failure.

4. The use according to claim 1 or 2, characterized in that: The dosage forms used include soft capsules, drop pills, emulsions, pills, tablets, lotions, liniments, ointments or suppositories.

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