Application of dried panax notoginseng in preparation of medicine for treating polycystic ovary syndrome

Abstract

The application is application of cooked panax pseudoginseng in preparation of medicine for treating polycystic ovary syndrome, discloses a new use of cooked panax pseudoginseng, i.e. application of cooked panax pseudoginseng in preparation of medicine for treating polycystic ovary syndrome, and proves that cooked panax pseudoginseng can relieve estrous cycle disorder state of mice, improve polycystic-like change of ovary, increase number of corpus luteum, reduce number of atretic follicle and cystic follicle, and restore normal function of ovary; the application provides a new idea for research and development of medicine for treating polycystic ovary syndrome, and cooked panax pseudoginseng can become a potential candidate medicine for treating polycystic ovary syndrome, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of processed Panax notoginseng in the preparation of drugs for treating polycystic ovary syndrome. Background Technology

[0002] Polycystic ovary syndrome (PCOS) was first reported in 1935; it was officially named PCOS in 1990. Due to its relatively recent discovery, there are no clear records or treatment experiences related to PCOS in traditional Chinese medicine. Currently, the diagnosis and treatment of PCOS are mainly based on Western medical theories and medications. The main clinical manifestations of PCOS include hirsutism or hyperandrogenemia caused by excess androgens, ovulation dysfunction, and polycystic ovarian lesions. The internationally recognized diagnostic criteria for PCOS are the Rotterdam criteria proposed in 2003, which stipulate that a diagnosis of PCOS must meet two of the following three conditions: hyperandrogenemia, ovulation dysfunction, and polycystic ovarian lesions. PCOS is a common polygenic endocrine disorder with a prevalence of 6-20% in women of reproductive age and is one of the important causes of female infertility.

[0003] Because the etiology and pathogenesis of PCOS are not yet fully understood, there are few clinically available treatments, and existing drugs have limited efficacy and significant side effects. For example, clomiphene citrate can induce ovulation in women with PCOS and is considered a first-line treatment. However, clomiphene citrate is expensive and has significant adverse reactions, such as a high rate of ovulation failure, anti-estrogenic effects on the endometrium and cervical mucus, a long half-life, and multiple follicle ovulation. Metformin can also be used to treat PCOS, but it increases the risk of ectopic pregnancy and congenital malformations. Therefore, screening for effective PCOS treatments with fewer side effects is urgently needed.

[0004] In recent years, herbal medicines have gained increasing popularity in clinical use and drug development due to their rich pharmacological activities and low toxicity. Panax notoginseng (Sanqi) Panax notoginsengAs a traditional and precious Chinese medicinal herb, Panax notoginseng is included in all editions of the Chinese Pharmacopoeia, primarily used for dispersing blood stasis, stopping bleeding, reducing swelling, and relieving pain. The 2020 edition of the Chinese Pharmacopoeia mainly includes raw Panax notoginseng products, which are the original Panax notoginseng raw material washed, dried, ground into a fine powder, or made into saponin extracts. However, in folk medicine, Panax notoginseng is traditionally used for both traumatic injuries and tonifying effects; raw Panax notoginseng is used for traumatic injuries and promoting blood circulation, while cooked Panax notoginseng is used to tonify blood, regulate blood, and improve immunity. Although cooked Panax notoginseng is believed to have various pharmacological activities, its understanding is still insufficient, and it is currently only used as a local Chinese medicinal decoction piece and has not yet been included in the Chinese Pharmacopoeia. This invention is the first to apply cooked Panax notoginseng to the treatment of PCOS. Pharmacological experiments have demonstrated that cooked Panax notoginseng has a good therapeutic effect on PCOS and is a potential candidate drug for treating polycystic ovary syndrome, demonstrating originality. Summary of the Invention

[0005] This invention provides a new use for processed Panax notoginseng, namely, its application in the preparation of drugs for treating polycystic ovary syndrome.

[0006] The drug of the present invention contains processed Panax notoginseng as its main ingredient (or active ingredient). One or more drug-acceptable excipients may also be added to improve the absorption effect of the drug or facilitate its use, such as making it into capsules or pills, powders, tablets, granules, oral liquids and injections, etc., that is, to make it into a suitable dosage form.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] 1. Prepare cooked Panax notoginseng according to the standard operating procedure of Yunnan Provincial Food and Drug Administration: Steamed Panax notoginseng powder (Yun YPBZ-0193-2013);

[0009] 2. Pharmacological experiments have demonstrated the therapeutic effect of processed Panax notoginseng on polycystic ovary syndrome. Pharmacological activity studies have shown that processed Panax notoginseng has a significant therapeutic effect on PCOS mice.

[0010] Compared to normal mice, PCOS model mice exhibited disordered estrous cycles, polycystic ovarian changes, reduced corpus luteum numbers, increased atretic and cystic follicles, and enlarged ovarian volume. After treatment with processed Panax notoginseng, the estrous cycles of the mice significantly recovered, the polycystic ovarian changes reversed, the number of corpus luteum increased, the number of atretic and cystic follicles decreased, and the ovarian volume gradually returned to normal. Furthermore, treatment with Panax notoginseng (commonly known as "raw Panax notoginseng") listed in the Chinese Pharmacopoeia did not show significant improvement in PCOS pathology, indicating that the commonly used Panax notoginseng (raw Panax notoginseng) is not suitable for the treatment of PCOS.

[0011] Based on the above research results, processed Panax notoginseng has a significant effect on improving PCOS in mice and can be a potential candidate drug for the treatment of polycystic ovary syndrome, with important application and development value. Attached Figure Description

[0012] Figure 1 This diagram illustrates the estrous cycle results of mice in each group after Wright staining. Red arrows represent nucleated epithelial cells; blue arrows represent anucleated keratinized epithelial cells; and black arrows represent leukocytes.

[0013] Figure 2 Line graphs of the estrous cycle for each group of mice, where P: Proestrus, proestrus; E: Estrus, estrus; M: Metestrus, metestrus; D: Diestrus, diaestrus;

[0014] Figure 3 Representative morphological images of the uterus of each group of mice;

[0015] Figure 4 Schematic diagram of pathological sections of mouse ovaries in each group, H&E stained (200×);

[0016] In the above figure, Control: blank control group; Model: model group; PCOS-PN: raw Panax notoginseng administration group; PCOS-SPN: processed Panax notoginseng administration group; PCOS-Met: positive control group of metformin. Detailed Implementation

[0017] The following are examples of embodiments of the present invention. These embodiments are intended to further describe the present invention, not to limit it. The specific experimental procedures and results described in the embodiments are only for illustrative purposes. Unless otherwise specified, the methods in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared according to conventional methods. The processed Panax notoginseng used in the embodiments was prepared according to the method in the standard operating procedure of Yunnan Provincial Food and Drug Administration standard: Processed Panax notoginseng Powder (Steamed) (Yun YPBZ-0193-2013). ；

[0018] Example 1: Construction of a PCOS mouse model and grouping for drug administration

[0019] Forty 3-week-old female SPF-grade C57BL / 6 mice, weighing 18–20 g, were housed at the Experimental Animal Center of Kunming University of Science and Technology under a temperature of 24±2℃, humidity of 60±5%, and a light duration of 12 h / d. After one week of acclimatization, they were randomly divided into two groups: a control group (n=8) and a model group (n=32). The mouse model was established using letrozole (LE) combined with a high-fat diet (HFD). Mice in the model group were fed a high-fat diet (71.5% normal diet, 12% lard, 1% cholesterol, 10% egg yolk powder, 0.5% bile salts, and 5% sucrose) and were given 100 μL of 1 mg / kg LE by gavage daily. Mice in the control group were fed a normal diet and were given an equal volume of 1% carboxymethyl cellulose (CMC) solution by gavage daily at the same time every morning for 21 consecutive days.

[0020] Mice that successfully developed the model were randomly divided into four groups: Model group, raw Panax notoginseng treatment group (PCOS-PN), processed Panax notoginseng treatment group (PCOS-SPN), and positive control group (PCOS-Met). All mice were continued to be fed a high-fat diet. The dosage for mice was calculated using a human-to-animal drug dosage conversion method. The raw Panax notoginseng group (PCOS-PN) was administered 1600 mg / kg of raw Panax notoginseng by gavage; the processed Panax notoginseng group (PCOS-SPN) was administered 1600 mg / kg of processed Panax notoginseng by gavage; the positive control group was administered 300 mg / kg of metformin (Met) by gavage; and the Model group and the control group were administered an equal volume of physiological saline by gavage. Gavage was performed for 28 consecutive days. Starting on day 19 of treatment, vaginal cytology smears were collected daily at 8:00 AM for 10 consecutive days to observe the estrous cycle. After treatment, the mice were sacrificed, and bilateral ovaries and livers were collected for subsequent experiments.

[0021] Example 2: Cytological morphological assessment of mouse vaginal exfoliated cells

[0022] Mouse vaginal exfoliated cell smears were stained with Wright's stain (methylene blue-eosin). The specific steps were as follows: Every morning after exposing the mouse vagina, 20 μL of 0.9% physiological saline was pipetted into the mouse vagina using a 100 μL pipette, and a drop of mucus was aspirated back and spread evenly on a glass slide. The slide was allowed to stand at room temperature to dry naturally. Wright's staining solution was added and stained for 3 minutes to fix the specimen with methanol. An equal volume of pH 6.4 phosphate buffer was added and the slide was gently shaken to mix with Wright's staining solution. The slide was allowed to stand for 5 minutes. The slide was washed with water and dried. The specimen was examined under a microscope.

[0023] The estrous cycle of mice was observed by examining vaginal exfoliated cytology smears. The estrous cycle of mice is generally 4-5 days, which can be divided into four stages: proestrus, estrus, metestrus, and mitrus. The results of vaginal exfoliated cytology smears in each stage have their own characteristics, but the main cell types observed are nucleated epithelial cells, anucleated keratinized epithelial cells, and leukocytes, which appear in different proportions in each stage of the estrous cycle. ① Proestrus: Nucleated epithelial cells constitute a large proportion, appearing as round or oval cells larger than leukocytes. Small, round leukocytes and irregularly keratinized epithelial cells are occasionally seen. This phase can be identified as proestrus. ② Estrus: If irregularly shaped anucleated keratinized epithelial cells constitute a high proportion, with a very small number of nucleated epithelial cells and almost no leukocytes, this phase can be identified as estrus. ③ Metestrus: If the proportions of irregularly shaped anucleated keratinized epithelial cells, nucleated epithelial cells, and leukocytes are similar, this phase is metestrus. ④ Diestrus: If leukocytes constitute the vast majority, with occasional nucleated epithelial cells, this phase is diestrus. After treatment with prepared Panax notoginseng (SPN), the vaginal exfoliated cell smears and estrous cycle curves of mice are shown below. Figure 1 , 2 As shown, the estrous cycle of mice in the blank control group (Control) was regular and could last for 4-5 days. The estrous cycle of mice in the model group (Model) was disordered and had not fully recovered, with most mice in the interestrous phase. The estrous cycle of the raw Panax notoginseng group (PCOS-PN) was slightly improved, but the recovery effect was not good. The processed Panax notoginseng group (PCOS-SPN) and the positive drug metformin group (PCOS-Met) basically recovered their estrous cycles.

[0024] Example 3: Morphological and pathological changes in the mouse reproductive system

[0025] After mice were anesthetized and euthanized, the uterus and ovaries were completely removed. The adipose tissue surrounding the ovaries was carefully removed, and the mice were washed with physiological saline. Changes in the morphology of the uterus and ovaries were compared among different groups. Representative morphologies of the mouse uterus are shown in the figure below. Figure 3 As shown, the mice had a bilateral "Y"-shaped distribution of the uterus and coupled ovaries. In the model group, the bilateral uterus was shrunken and the ovaries were enlarged. Compared with the blank control group, cystic protrusions could be observed with the naked eye. After treatment with raw Panax notoginseng, the uterus of the mice did not improve significantly, and the cystic protrusions of the ovaries were still obvious. After treatment with processed Panax notoginseng (SPN), the shrinkage of the uterus was reversed and the volume of the ovaries was reduced, and the normal function of the ovaries was basically restored.

[0026] Example 4: Pathological section of mouse ovarian tissue

[0027] The mouse ovary was completely removed, and the surrounding adipose tissue was carefully removed and washed with physiological saline. Mouse ovarian tissue sections were prepared according to standard procedures, specifically as follows: The ovarian tissue was soaked in PBS buffer for 24 hours, followed by gradient dehydration with ethanol: 60% ethanol for 10 hours, 70% ethanol for 8 hours, 80% ethanol for 6 hours, 95% ethanol for 4 hours, and finally anhydrous ethanol for 1 hour. After dehydration, the tissue was soaked in xylene for 2 hours until it became clear, and then embedded in paraffin. The paraffin embedding procedure was as follows: Paraffin was placed in a 65°C oven, and then the ovarian tissue was placed inside. The tissue was immersed in paraffin for 2 hours, with the paraffin changed every hour, repeated three times. After paraffin embedding, the tissue was allowed to cool and solidify at room temperature, completing the tissue embedding. The embedded tissue was sectioned: the tissue block was serially sectioned using a microtome to a thickness of 4 μm. Suitable sections were then selected and spread using 30% ethanol for the first spreading. After this, the sections were transferred to a 40°C water bath for a second spreading until the tissue sections were completely attached to the slide. The final step was drying: the slides were placed on a support and dried in a 37°C oven before being used for tissue staining. After drying, the prepared sections were stained with hematoxylin and eosin (H&E). Following the kit instructions, the tissue sections were stained and then observed under a light microscope to examine ovarian morphology and the development of follicles at each stage.

[0028] After 28 days of drug administration, HE staining of mouse ovarian tissue revealed pathological sections of the ovaries as shown in the image. Figure 4 Compared with the blank control group, the model group had more cystic dilated follicles and an increase in atretic follicles in the ovaries. The number of granulosa cells did not recover significantly after the initial decrease, the number of granulosa cell layers decreased, the number of corpora lutea decreased, and the morphological structure of the ovaries remained disordered. After treatment with raw Panax notoginseng, there were still many cystic and atretic follicles in the ovaries, and the number of corpora lutea and granulosa cells did not increase significantly. The morphological structure of the ovaries had not yet recovered. After treatment with processed Panax notoginseng (SPN), the number of cystic and atretic follicles decreased, the number of follicles at different developmental stages increased, the number of corpora lutea increased, the number of granulosa cells increased, and the morphological structure of the ovaries basically recovered.

Claims

1. Use of dried panax notoginseng as the only active ingredient for the preparation of a medicament for the treatment of polycystic ovary syndrome.

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