Application of frankincense essential oil in preparation of medicine for treating ovarian insufficiency

Abstract

The invention discloses application of frankincense essential oil in preparation of a medicine for treating ovarian insufficiency, and belongs to the technical field of biology. The invention provides the application of the frankincense essential oil in preparing the medicine for treating the ovarian insufficiency, experiments prove that the frankincense essential oil effectively relieves the conditions of mouse oestrus cycle disorder, ovarian granular cell looseness, follicular mother cell quality reduction and the like caused by cyclophosphamide, the expression level of ki67 protein is remarkably improved after the frankincense essential oil is used for treating, and the curative effect of the frankincense essential oil on the ovarian insufficiency is improved. The activity of follicles at all levels of the ovary of the mouse is increased, and apoptosis of granular cells is reduced, so that the cyclophosphamide-induced mouse ovarian insufficiency is improved; boswellia carteri essential oil is wide in raw material source and low in cost, and has wide application value and limit value as a development raw material of the ovarian protection medicine.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of frankincense essential oil in preparing a medicine for treating ovarian insufficiency. Background Art

[0002] Premature Ovarian Insufficiency (POI) is one of the early phenomena of premature ovarian failure (POF) caused by various reasons. It is mainly manifested by a decrease in the number and / or quality of oocytes in the ovaries, which in turn causes a series of symptoms such as decreased female fertility and shortened reproductive cycles. It is characterized by increased levels of follicle-stimulating hormone (FSH), decreased levels of estrogen (Estrogen, E2) and decreased levels of anti-Müllerian hormone (AMH). POI caused by the chemotherapy drug cyclophosphamide mainly leads to excessive consumption of follicles by accelerating the activation of primordial follicles and increasing apoptosis and atresia in the development of primordial follicles, ultimately reducing their ovarian reserve function.

[0003] At present, the treatments for ovarian insufficiency mainly include estrogen and progesterone hormone replacement therapy and ovulation induction therapy. However, these treatments have certain side effects and risks, such as increasing the risk of breast cancer, cardiovascular diseases and other diseases. Therefore, finding safe and effective alternative treatments has become an important research direction. For example, the TrkB agonist antibody Ab4B19 improves cyclophosphamide-induced premature ovarian failure in mice by improving the estrous cycle disorder of POF mice, increasing the number and quality of follicles at all levels, increasing the levels of AMH and E2 hormones and the in vitro fertilization rate; human umbilical cord mesenchymal stem cells act on the TGF-β1 / Smad3, AMPK / mTOR and PI3K / Ak signaling pathways to improve the indicators of ovarian function, affect the growth and development process of follicles, and thus achieve the therapeutic effect on POF. Traditional Chinese medicine has certain potential in the treatment of ovarian insufficiency. Some Chinese herbal medicine ingredients can relieve the symptoms of ovarian insufficiency by reducing ovarian oxidative stress, reducing the apoptosis of ovarian granulosa cells and oocytes, and reducing ovarian tissue fibrosis. Clinically, Qilin Pills, which are made up of traditional Chinese medicine ingredients such as Polygonum multiflorum, have a certain effect in the adjuvant treatment of ovarian insufficiency. Animal experiments have also proved that it can effectively improve the reserve function impairment of mice caused by cyclophosphamide. However, the effectiveness, durability, convenience and economy of the above treatment methods are poor, and a new treatment plan is urgently needed to meet the needs of patients. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide an application of frankincense essential oil in the preparation of a drug for treating ovarian insufficiency, so as to solve the problems of poor effectiveness, durability, convenience and economy of existing methods for treating ovarian insufficiency.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] Application of frankincense essential oil in preparing medicine for treating ovarian insufficiency.

[0007] The invention has the following beneficial effects: the main components of the frankincense essential oil are frankincense acid and volatile oil, and the frankincense essential oil has the effects of anti-inflammatory, antibacterial, anti-tumor, neuroprotection, liver and kidney protection, cell repair, etc. The invention is verified by experiments that when the frankincense essential oil is used to treat mice with ovarian insufficiency induced by cyclophosphamide, the loosening of ovarian granulosa cells in mice, the abnormal morphology and quality decline of follicular cells caused by cyclophosphamide, the increase in the number of apoptotic follicles in mice caused by cyclophosphamide can be alleviated, and the expression level of ki67 protein can be enhanced to increase the vitality of follicles at all levels in the mouse ovaries and reduce the apoptosis of granulosa cells, thereby effectively treating the ovarian insufficiency in mice caused by cyclophosphamide.

[0008] A medicine for treating ovarian insufficiency is a preparation prepared from an active ingredient and auxiliary ingredients, and the active ingredient is frankincense essential oil.

[0009] Furthermore, the auxiliary component is a pharmaceutically acceptable excipient.

[0010] Furthermore, the dosage form of the drug includes oral preparations or injection preparations.

[0011] The present invention has the following beneficial effects:

[0012] The invention provides application of frankincense essential oil in preparing a medicine for treating ovarian insufficiency. Experiments show that treatment with frankincense essential oil effectively alleviates cyclophosphamide-induced estrous cycle disorder, loose ovarian granulosa cells, decreased quality of follicle mother cells and the like in mice. After treatment with frankincense essential oil, the expression level of Ki67 protein is significantly increased, and the cyclophosphamide-induced ovarian insufficiency in mice is improved by increasing the vitality of ovarian follicles at all levels in the mice and reducing the apoptosis of granulosa cells. The frankincense essential oil has a wide source of raw materials and low cost, and has broad application value and realm value as a raw material for developing ovarian protection drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The weight changes of mice before and after treatment, among which a is the general state of mice after treatment, from left to right are frankincense essential oil high-dose group, frankincense essential oil medium-dose group, frankincense essential oil low-dose group, Qilin pill group, natural recovery group and natural growth group. b is the statistical chart of weight changes of mice after 21 days of treatment;

[0014] Figure 2 is the estrous cycle detection result of mice during the treatment period, where ad is the Papanicolaou staining diagram of the estrous cycle of mice, which is proestrus, estrus, metestrus and diestrus in sequence;

[0015] Figure 3 The results of ovarian tissue observation after treatment of mice, where a is a general picture of mouse ovarian tissue, and b is a statistical chart of mouse ovarian weight;

[0016] Figure 4 This is the HE staining image of the ovarian cortex area of ​​mice after treatment, where the length of each image scale from top to bottom is 200μm, 100μm and 20μm;

[0017] Figure 5 This is the distribution diagram of Ki67 protein in the ovaries of mice after treatment. DETAILED DESCRIPTION

[0018] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0019] Example 1: Experiment on the treatment of ovarian insufficiency with frankincense essential oil

[0020] 1. Experimental Methods

[0021] 1. Experimental Animals

[0022] Four-week-old SPF-grade C57BL / 6J female mice, animal license number: SCXK(Ning)2020-0001.

[0023] 2. Experimental Methods

[0024] (1) Construction of ovarian insufficiency mouse model and treatment of experimental groups

[0025] The mice were randomly divided into two groups, one group of 40 mice was a modeling group (POI mice), and the other group of 5 mice was a blank control group. The animals in the modeling group were intraperitoneally injected with 70 mg / kg / mouse cyclophosphamide solution on the first day, and 10 mg / kg / mouse cyclophosphamide solution on the 2nd to 15th days. The blank control group was injected with 10 mL / kg / mouse normal saline solution.

[0026] The 40 mice were divided into a high-dose frankincense essential oil (Dorit (Shanghai)) group (2 mg / kg / mouse by oral administration of frankincense essential oil), a medium-dose frankincense essential oil group (1 mg / kg / mouse by oral administration of frankincense essential oil), a low-dose frankincense essential oil group (0.5 mg / kg / mouse by oral administration of frankincense essential oil), and a Qilin pill group as a positive control (5.46 g / kg / mouse by oral administration of Qilin pill). The natural recovery group was given the same dose of normal saline for 21 consecutive days.

[0027] (2) Detection of the estrous cycle in mice

[0028] Fix the mouse tail, draw 20μm saline with a pipette and insert it into the mouse vagina, blow slowly 2-3 times, then apply the mixture on the slide and let it air dry naturally. Before staining, immerse the slide in 95% alcohol for 2-3 minutes, then stain it in nuclear dye for 2 minutes, wash it with water, then stain it in pulp dye for 2 minutes, and finally add the dye enhancer for 10 seconds and then dry it with filter paper. When sealing, use anhydrous ethanol to dehydrate 1-2 times, and add gum to seal the slide.

[0029] (3) Sampling and fixation of experimental animal tissues

[0030] The mice were weighed, and blood samples were collected from the apex of the heart after anesthesia, left to stand at room temperature for 2 hours, centrifuged at 4°C and 3000 rpm for 20 minutes, and the supernatant was stored at -80°C. The mice were placed on the operating table with their abdomen facing upward, the abdominal cavity was opened, the ovaries were removed, the surrounding fat tissue was removed, and the weight was recorded on an analytical balance and placed in a PBS solution for later use.

[0031] (4) Preparation of paraffin sections of ovarian tissue

[0032] Place the ovarian tissue in 4% paraformaldehyde at 4°C overnight. Place the ovary in an embedding box and immerse it in gradient alcohol concentrations for dehydration. After xylene is transparent, place it in a 60°C oven for wax treatment. After completion, embed it in paraffin at room temperature overnight. After trimming the wax block, slice it continuously at a thickness of 5 microns. Expand the slices at about 40°C in a slice bleaching and drying temperature controller, adhere them to a polylysine-coated slide, and mark them. Place the slide in a 62°C baking machine for 1-2 hours, then put it in a slide box and store it in a 4°C refrigerator for long-term storage.

[0033] (5) HE staining of ovarian tissue

[0034] The baked slices were immersed in xylene dewaxing solution I twice, anhydrous ethanol twice, 95% alcohol, and 85% alcohol in order to remove the paraffin of the slice tissue. After washing the slices for 2 minutes, they were immersed in hematoxylin dye solution for 1 minute, washed with water to return to blue for 5-8 minutes, and the coloring was observed under a microscope. The slices were placed in eosin dye solution for 1 minute and rinsed with running water. After dyeing, dehydration was performed to make them transparent. Finally, 50 microliters of sealing glue were added to the slides and the slides were dried in a fume hood for standby use.

[0035] (6) Immunohistochemistry

[0036] After dewaxing, the slices were placed in a slide box, and freshly prepared sodium citrate was added to repair the microwave twice. After cooling at room temperature, they were washed with PBS. The tissue was circled with a brush, and a 3% hydrogen peroxide solution was added and placed in a wet box for incubation at room temperature in the dark for 10 minutes. After washing with PBS, 10% goat serum was added and blocked at room temperature for 30 minutes. Ki67 antibody diluted with PBS was added and incubated in a wet box at 4°C overnight. The next day, after washing with PBS, the secondary antibody was added and incubated at 37°C for 30 minutes. After washing with PBS, DAB solution was added, and the positive area was observed under a microscope and then stopped by water. Finally, the slices were immersed in hematoxylin staining solution to counterstain the nuclei, dehydrated with alcohol, transparent, sealed, observed under a microscope, and counted.

[0037] 2. Experimental Results

[0038] (1) Effects of frankincense essential oil on body weight and physiological status of POI mice

[0039] The experimental results are as follows Figure 1 As shown in Figure 2, during the treatment, it was found that the weight of mice in each group varied greatly. Figure 1 As shown in Figure b, compared with naturally growing mice, the mice in the natural recovery group had a slightly lower body weight. There was no significant increase in the body weight of the mice after treatment with various doses of frankincense essential oil, and only after treatment with high doses of frankincense and Qilin Pills were significant fluctuations in the body weight of the mice found.

[0040] (2) Frankincense essential oil improves estrous cycle disorders in POI mice

[0041] In order to observe the effect of frankincense essential oil on estrous cycle disorders in POI mice, vaginal smear results and estrous cycle changes were continuously observed during the treatment period. Figure 2 As shown. Under the microscope, it can be seen that the mouse estrous cycle is divided into four periods, namely proestrus, estrus, metestrus and diestrus. Proestrus is characterized by a large number of nucleated epithelial cells and a small number of keratinized cells, estrus is characterized by a large number of keratinized epithelial cells, metestrus shows keratinized epithelial cells and leukocytes, and metestrus is characterized by a large number of leukocytes and a small amount of mucus.

[0042] During the treatment with frankincense essential oil, it was found that the estrous cycle of mice in the natural recovery group was significantly disturbed, indicating that the chemotherapy drug cyclophosphamide can affect the estrous cycle of mice. After treatment, it was found that the estrous cycle of mice in both the frankincense essential oil group and the Qilin Pill group had a significant recovery effect.

[0043] (3) Effects of frankincense essential oil on ovarian weight and follicle quality in POI mice

[0044] The experimental results are as follows Figure 3 and Figure 4 As shown. Figure 3 It can be seen that there was no significant difference in the morphology, volume and weight of the ovaries of POI mice compared with the natural growth group, while the volume and weight of the ovaries decreased after treatment with various doses of frankincense essential oil and Qilin Pills. Figure 4 It can be seen that the granulosa cells in the follicles of POI mice were relatively loose, the oocytes were abnormal in morphology and the quality was reduced, and the number of apoptotic follicles increased significantly. After treatment with frankincense essential oil and Qilin Pills, the above phenomena were significantly alleviated, among which the frankincense essential oil group showed better therapeutic effect.

[0045] (4) Effects of frankincense essential oil on ovarian granulosa cells of POI mice

[0046] In order to explore whether frankincense essential oil can improve the apoptosis of follicles and granulosa cells at all levels in the ovaries of POI mice, Ki67 protein was used for observation. Figure 5 As shown in the figure, the expression of Ki67 protein in POI mice after cyclophosphamide modeling was significantly decreased, which inhibited the proliferation of follicular cells at all levels of mice. After treatment with frankincense essential oil, the expression of ki67 increased significantly, which had a restorative effect on the inhibition of proliferation of follicles at all levels of mice. However, there was no significant change in ki67 expression after Qilin Pills treatment.

[0047] In summary, the present invention proves through experiments that after receiving low-dose frankincense essential oil treatment, the estrous cycle of mice gradually becomes regular, indicating that frankincense essential oil can effectively alleviate the disordered estrous cycle of mice caused by cyclophosphamide; at the same time, low-dose frankincense essential oil treatment effectively alleviates the loosening of mouse ovarian granulosa cells caused by cyclophosphamide, the abnormal morphology of follicle mother cells, the quality decline, etc., and reduces the number of apoptotic follicles; in addition, after frankincense essential oil treatment, the vitality of follicles at all levels of mouse ovaries can be effectively increased, the expression of ki67 protein significantly increases, and the apoptosis of granulosa cells is reduced. Frankincense essential oil can improve cyclophosphamide-induced mouse ovarian insufficiency by increasing the vitality of follicles at all levels of mouse ovaries and reducing the apoptosis of granulosa cells.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of frankincense essential oil in the preparation of medicines for treating ovarian insufficiency.

2. A drug for treating ovarian insufficiency, characterized in that: The medicine is a preparation prepared from active ingredients and auxiliary ingredients, and the active ingredient is the frankincense essential oil according to claim 1.

3. The drug for treating ovarian insufficiency according to claim 2, characterized in that: The auxiliary components are pharmaceutically acceptable auxiliary materials.

4. The drug for treating ovarian insufficiency according to claim 3, characterized in that: The dosage form of the drug includes oral preparations or injection preparations.

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