Application of Dan'e Fukang preparation in preparation of medicine for treating premature ovarian failure

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to application of a Dan'e Fukang preparation in preparation of a medicine for treating premature ovarian failure. The invention provides application of a Dan'e Fukang preparation in preparation of a medicine for treating premature ovarian failure. Experiments prove that the Dan'e Fukang preparation can improve uterus and ovarian organ indexes of premature ovarian failure mice; the contents of estradiol and progesterone in the serum of the premature ovarian failure mouse are improved. The Dan'e Fukang preparation can reduce the FSH level and the LH level in the serum of a premature ovarian failure rat, and can significantly increase the E2 level of the rat; the Dan'e Fukang preparation disclosed by the invention can be used for inhibiting premature ovarian failure and promoting ovarian function recovery.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to the application of Dan'e Fukang preparation in the preparation of drugs for treating premature ovarian failure. Background Technology

[0002] Premature ovarian failure (POF) is a disease characterized by amenorrhea, infertility, estrogen deficiency, and elevated gonadotropin levels in women before the age of 40. Clinical symptoms include menstrual irregularities, hot flashes, night sweats, irritability, and insomnia, causing premature aging and significant distress to the patient's physical and mental health and marital life. It not only impacts the patient's normal life and health but also has a significant psychological impact. As the disease progresses, it may lead to various serious complications such as heart disease and osteoporosis. POF not only causes amenorrhea, secondary infertility, and low estrogen levels, but it can also affect numerous organs throughout the body, including the nervous, metabolic, cardiovascular, and skeletal systems, severely impacting a woman's normal work.

[0003] Premature ovarian failure is difficult to treat. Western medicine clinically employs hormone replacement therapy, requiring the use of estrogen and progesterone under the guidance of a physician. Studies have shown that estrogen and progesterone therapy has some effect and can improve symptoms, but it is not ideal for restoring ovarian function. Furthermore, symptoms recur after discontinuation of medication, leading to ovarian dependence and potentially further deterioration of ovarian function. Hormone replacement therapy only treats the symptoms, and this method has significant side effects. Long-term use of exogenous hormones carries the risk of endometrial cancer and increases the incidence of coronary heart disease and gallbladder disease.

[0004] Therefore, there is a need to develop a drug for treating premature ovarian failure with fewer complications and significant therapeutic effects. Summary of the Invention

[0005] The purpose of this invention is to provide a new pharmaceutical use for Dan'e Fuke preparation.

[0006] This invention provides the application of Dan'e Fukang preparation in the preparation of drugs for treating premature ovarian failure.

[0007] This invention also provides the application of Dan'e Fuke preparation in the preparation of drugs that improve uterine index and ovarian index.

[0008] This invention also provides the use of Dan'e Fuke preparations in the preparation of drugs that increase the number of follicles at all stages of the ovary.

[0009] This invention also provides the use of Dan'e Fuke preparation in the preparation of a drug for treating elevated serum estradiol and progesterone levels.

[0010] Furthermore, the Dan'e Fuke preparation is a Dan'e Fuke decoction.

[0011] The ingredients of Dan'e Fuke Decoction are: purple salvia miltiorrhiza, turmeric, bupleurum chinense, notoginseng, red peony root, angelica sinensis, sparganium rhizome, cyperus rotundus, corydalis rhizome, and licorice.

[0012] Purple Salvia is the dried root of *Salvia przewalskii* Maxim. or *Salvia przewalskii* Maxim. var. *mandarinorum* (Diels) Stib., belonging to the Lamiaceae family. It has a bitter taste and is slightly cold in nature. It enters the Heart and Liver meridians. It invigorates blood circulation, removes blood stasis, regulates menstruation, relieves pain, clears the heart, and eliminates irritability. It is used for irregular menstruation, amenorrhea, dysmenorrhea, abdominal masses, chest and abdominal pain, hot arthralgia, insomnia, carbuncles, and boils.

[0013] Curcuma zedoaria is the dried rhizome of *Curcuma phaeocaulis* Va L., *Curcum awangsiensis* SGLeeet C.F. Liang, or *Curcuma wenyujin* Y.H.Chenet C.Ling (all belonging to the ginger family). It has the effects of promoting qi circulation, breaking up blood stasis, eliminating stagnation, and relieving pain. It is pungent, bitter, and warm in nature. It enters the liver and spleen meridians. It is used for abdominal masses, amenorrhea due to blood stasis, chest pain, and abdominal distension due to food stagnation.

[0014] Yunnan Bupleurum is a processed product of the dried whole herb of *Bupleurum chinense* Wall. ex DC., *Bupleurum microcephalum* Diels., and *Bupleurum tenue* Buch.-Ham. ex D. Don, all belonging to the Apiaceae family. It has a bitter taste and is slightly cold in nature. It enters the Liver and Gallbladder meridians. It harmonizes the exterior and interior, soothes the Liver, and raises Yang. It is used for colds with fever, alternating chills and fever, chest and rib pain, irregular menstruation, uterine prolapse, and rectal prolapse.

[0015] Panax notoginseng is the dried root and rhizome of Panax notoginseng (Burk.) FHChen, a plant belonging to the Araliaceae family. It has a sweet and slightly bitter taste, and is warm in nature. It enters the liver and stomach meridians. It disperses blood stasis, stops bleeding, reduces swelling, and relieves pain. It is used for hemoptysis, hematemesis, epistaxis, hematochezia, metrorrhagia, traumatic bleeding, chest and abdominal pain, and swelling and pain from falls.

[0016] Red peony root is the dried root of *Paeonia lactiflora* Pall. or *Paeonia veitchii* Lynch, both belonging to the Ranunculaceae family. It is bitter and slightly cold in nature. It enters the liver meridian. Its functions include clearing heat and cooling the blood, dispersing blood stasis and relieving pain. It is used for heat entering the blood level, febrile diseases with rashes, hematemesis and epistaxis, red and swollen eyes, liver stagnation causing hypochondriac pain, amenorrhea and dysmenorrhea, abdominal masses and pain, traumatic injuries, carbuncles and sores.

[0017] Angelica sinensis is the dried root of Angelica sinensis (Oliv.) Diels, a plant in the Apiaceae family. It has a sweet and pungent taste, and is warm in nature. It enters the liver, heart, and spleen meridians. It nourishes and invigorates blood, regulates menstruation and relieves pain, and moistens the intestines to relieve constipation. It is used for blood deficiency and chlorosis, dizziness and palpitations, irregular menstruation, amenorrhea and dysmenorrhea, abdominal pain due to deficiency and cold, rheumatic arthralgia, traumatic injuries, carbuncles and sores, and constipation due to intestinal dryness. Angelica sinensis brewed with wine invigorates blood and regulates menstruation. It is used for amenorrhea and dysmenorrhea, rheumatic arthralgia, and traumatic injuries.

[0018] Sparganium stoloniferum Buch.-Ham., a plant in the family Sparganaceae, is a dried tuber. It has a pungent, bitter, and neutral flavor. It enters the liver and spleen meridians. It promotes blood circulation, relieves stagnation, and alleviates pain. It is used for abdominal masses, dysmenorrhea, amenorrhea due to blood stasis, chest pain, and abdominal distension due to food stagnation.

[0019] Cyperus rotundus L., a plant in the Cyperaceae family, is a dried rhizome. [Properties and Channels Entered] Pungent, slightly bitter, slightly sweet; neutral in nature. It enters the Liver, Spleen, and Triple Energizer channels. It soothes the Liver and relieves stagnation, regulates Qi and relieves chest tightness, regulates menstruation and relieves pain. It is used for Liver Qi stagnation, chest and rib pain, hernia pain, breast pain, Spleen and stomach Qi stagnation, abdominal distension and pain, irregular menstruation, amenorrhea, and dysmenorrhea.

[0020] Corydalis rhizome is the dried tuber of Corydalis rhizome W.T. Wang, a plant in the Papaveraceae family. It has a pungent, bitter, and warm nature. It enters the liver and spleen meridians. It invigorates blood circulation, regulates qi, and relieves pain. It is used for chest and rib pain, abdominal pain, angina pectoris, amenorrhea, dysmenorrhea, postpartum blood stasis, and traumatic swelling and pain.

[0021] Licorice is the dried root and rhizome of *Glycyrrhiza uralensis* Fisch., *Glycyrrhiza inflata* Bat., or *Glycyrrhiza glabra* L., all belonging to the Fabaceae family. It has a sweet taste and neutral properties. It enters the heart, lung, spleen, and stomach meridians. Its functions include tonifying the spleen and replenishing qi, clearing heat and detoxifying, resolving phlegm and relieving cough, alleviating spasms and pain, and harmonizing other herbs. It is used for spleen and stomach weakness, fatigue, palpitations, shortness of breath, cough with excessive phlegm, abdominal and limb spasms and pain, carbuncles and boils, and to alleviate the toxicity and harshness of other medications.

[0022] Dan'e Fuke Decoction invigorates blood circulation, removes blood stasis, soothes the liver, regulates qi, and relieves menstrual pain. It is used for menstrual disorders and dysmenorrhea caused by blood stasis in women. However, the inventors have discovered through research that Dan'e Fuke Decoction also has a therapeutic effect on premature ovarian failure. Dan'e Fuke Decoction can effectively improve uterine and ovarian function in animals with premature ovarian failure, effectively improve estrous cycle disorders, and restore normal follicle development. Compared with Western estrogen, it has better safety. It can provide more medication options for the clinical treatment of premature ovarian failure. Detailed Implementation

[0023] The present invention will be further elaborated in detail through specific embodiments below. The raw materials and reagents involved in the specific implementation part are all obtained commercially.

[0024] Example 1

[0025] Prepare Dan'e Fukang Decoction Extract. It is prepared according to the method of the national drug standard WS-10227(ZD-0227)2002-2012Z-2016 as follows: Weigh 450 g of Salvia miltiorrhiza, 250 g of Rhizoma zedoariae, 250 g of Bupleurum chinense var. sutchuenense, 150 g of Panax notoginseng, 250 g of Radix paeoniae rubra, 250 g of Angelica sinensis, 175 g of Rhizoma sparganii, 150 g of Cyperus rotundus, 175 g of Corydalis yanhusuo, and 100 g of Glycyrrhiza uralensis;

[0026] For the above ten herbs, Salvia miltiorrhiza, Rhizoma zedoariae, Panax notoginseng, Angelica sinensis, and Corydalis yanhusuo are used as solvents with 70% ethanol. After impregnating for 24 hours, percolation is carried out at a speed of 2-4 mL per minute, and about 5800 mL of percolate is collected. Ethanol is recovered under reduced pressure and concentrated to a thick paste with a relative density of 1.30-1.35 (50 °C). The medicinal residues and the other five herbs including Bupleurum chinense var. sutchuenense are decocted twice with water for 2 hours each time. The decoction liquids are combined, filtered, and the filtrate is concentrated to a thick paste with a relative density of 1.30-1.35 (50 °C). The above two thick pastes are combined, and an appropriate amount of refined honey-refined sugar (100:165) and 3 g of potassium sorbate are added, mixed evenly, and 1000 g is prepared to obtain the product.

[0027] Example 2 Application of Dan'e Fukang Preparation in an animal model of premature ovarian failure in mice

[0028] 1. Experimental materials

[0029] 1.1 Experimental animals: SPF-grade normal female mice (purchased from Guangdong Provincial Center for Medical Laboratory Animals, production license number: SCXK(Yue)2022-0002), 5-6 weeks old, with a body weight of 19-22 g.

[0030] 1.2 Experimental drugs and reagents: Dan'e Fukang Decoction Extract (Yunnan Shengke Pharmaceutical Co., Ltd., national drug approval number: Z20025253, specification: 150 g / bottle), Cyclophosphamide for Injection (Jiangsu Hengrui Medicine Co., Ltd., national drug approval number: H32020857, specification: 0.2 g, batch number: 20190417), Estradiol Valerate Tablets (Guangzhou Branch of Bayer Healthcare Co., Ltd., batch number: 20190812), Progesterone Radioimmunoassay Kit, Estradiol Radioimmunoassay Kit.

[0031] 2. Experimental method:

[0032] 2.1 Establishment of a mouse model of premature ovarian failure

[0033] After purchasing SPF-grade female mice, they were acclimatized for one week. One hundred and twenty mice with normal estrous cycles as determined by transvaginal exfoliative cytology were randomly divided into six groups of 20 mice each: a normal control group, a model control group, a positive control group, a low-dose Dan'e Fuke group, a medium-dose Dan'e Fuke group, and a high-dose Dan'e Fuke group. Except for the normal control group, which received daily intraperitoneal injections of saline, the other groups received daily intraperitoneal injections of cyclophosphamide 30 mg / kg for five consecutive days to establish a premature ovarian failure model.

[0034] After modeling, mice in the low-dose group (2.275 g / kg), medium-dose group (4.55 g / kg), and high-dose group (9.1 g / kg) of Dan'e Fuke were administered via gavage. Mice in the normal control group and model control group were given an equal volume of purified water. The positive control group was given estradiol valerate (0.18 mg / kg) via gavage. All mice were administered the drug once daily for 4 consecutive weeks.

[0035] 2.2 Evaluation Indicators

[0036] 2.2.1 Detection of progesterone and estradiol levels in mice with premature ovarian failure: After the experiment, blood was collected from the eyeballs of mice and immediately incubated in a 37°C water bath for 2 hours. After centrifugation at 3000 r / min for 8 minutes, the supernatant was collected and the serum progesterone and estradiol levels were measured.

[0037] 2.2.2 Determination of uterine and ovarian organ indices in mice with premature ovarian failure: After cervical dislocation and sacrifice, the ovaries and uterus were removed by abdominal dissection and weighed precisely on an analytical balance. The organ indices were calculated separately.

[0038] Organ index = organ wet weight (mg) / mouse body weight (g) × 100%.

[0039] 2.2.3 Statistical methods: Results were analyzed using SPSS 23.0 statistical software. Data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, and t-tests were used for comparisons between two groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered statistically significant.

[0040] 3. Experimental Results

[0041] See Table 1 and Table 2.

[0042] Table 1. Organ index results of mice in each group.

[0043] Group Uterine Index Ovarian index normal control group 139.34±16.62 35.28±7.86 Model control group 72.63±13.27* 20.46±6.31* Positive control group <![CDATA[141.34±14.68 ## ]]> <![CDATA[34.60±8.71 ## ]]> Dangofukang low-dose group <![CDATA[123.69±15.94 # ]]> <![CDATA[32.16±5.28 # ]]> Dan'e Fuke medium dose group <![CDATA[127.37±12.43 # ]]> <![CDATA[33.54±6.55 # ]]> Dango Fucang High-Dose Group <![CDATA[130.85±13.52 ## ]]> <![CDATA[34.85±4.67 ## ]]>

[0044] Note: Compared with the normal control group, *p<0.05; compared with the model control group, ##p<0.01, #p<0.05

[0045] As can be seen from Table 1, compared with the normal control group, the uterine and ovarian indices of the mice in the model control group were significantly decreased (P < 0.05), indicating that the model was successfully established. Compared with the model control group, both the low and medium dose groups of Dan'e Fukang could increase the uterine index and ovarian index of the mice (P < 0.05), while the high dose group of Dan'e Fukang could significantly increase the uterine and ovarian indices of the mice (P < 0.01). The results showed that the Dan'e Fukang preparation could increase the uterine and ovarian organ indices and had a certain effect on slowing down the decline of ovarian function.

[0046] Table 2 Results of the contents of progesterone and estradiol in the sera of mice in each group

[0047]

[0048]

[0049] Note: Compared with the normal control group, *p < 0.05; compared with the model control group, ##p < 0.01, #p < 0.05.

[0050] As can be seen from Table 2, compared with the normal control group, the contents of progesterone and estradiol in the sera of the mice in the model control group were decreased (p < 0.05), indicating that the model was successfully established. Compared with the model control group, both the low and medium dose groups of Dan'e Fukang could increase the contents of progesterone and estradiol in the sera of the mice (p < 0.05). The results showed that the Dan'e Fukang preparation increased the contents of estradiol and progesterone in the sera to a certain extent and had a certain effect on slowing down the decline of ovarian function.

[0051] Example 3 Application of the Dan'e Fukang preparation in an animal model of premature ovarian failure in rats

[0052] 1. Experimental materials

[0053] 1.1 Experimental animals: SPF-grade normal female rats (purchased from Guangdong Provincial Medical Experimental Animal Center, production license number: SCXK(Yue)2020 - 0002), weighing 200 - 250 g.

[0054] 1.2 Experimental drugs and reagents: Dan'e Fukang decoction extract (Yunnan Shengke Pharmaceutical Co., Ltd., national drug approval number: Z20025253, specification: 150 g / bottle), cyclophosphamide for injection (Jiangsu Hengrui Medicine Co., Ltd., national drug approval number: H32020857, specification: 0.2 g, batch number: 20190417), busulfan (B₂₆₃₅; Sigma, USA)

[0055] 2. Experimental method:

[0056] Establishment of the rat model of premature ovarian failure

[0057] Female SD rats aged 8-12 weeks, weighing 200±20g, and with normal estrous cycles were selected and randomly divided into 5 groups of 10 rats each after 1 week of acclimatization: normal control group, model control group, low-dose Dan'e Fuke group, medium-dose Dan'e Fuke group, and high-dose Dan'e Fuke group.

[0058] Establishing a rat model of premature ovarian failure: In addition to the normal control group, the model control group, the low-dose group of Dan'e Fuke, the medium-dose group of Dan'e Fuke, and the high-dose group of Dan'e Fuke were each given a single intraperitoneal injection of 0.1 mL of cyclophosphamide solution (120 mg / kg) and busulfan solution (12 mg / kg). Vaginal secretions were collected daily with cotton swabs, smeared, and Papanicolaou stained to observe changes in exfoliated vaginal cells. When a continuous interestrus period was observed for 10 consecutive days, the premature ovarian failure model was considered to be successfully established. After successful modeling, the low-dose group of Dan'e Fuke (1.575 g / kg), the medium-dose group of Dan'e Fuke (3.15 g / kg), and the high-dose group of Dan'e Fuke (6.3 g / kg) were administered via gavage. Mice in the normal control group and the model control group were given an equal volume of purified water once a day for 14 consecutive days.

[0059] Twenty-four hours after the last administration, blood was collected from the abdominal aorta, centrifuged at 3000 rpm for 10 minutes, and serum was separated. The levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) in the serum were measured. After sacrificing the rats, the ovaries were dissected, weighed, fixed in 10% formaldehyde, dehydrated with graded ethanol, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE) as usual, and then observed under an optical microscope.

[0060] 3. Experimental Results:

[0061] See Tables 3 and 4.

[0062] Table 3. Effects of Dan'e Fuke preparation on serum sex hormones in rats.

[0063] Group FSH (mIU / mL) LH (mIU / mL) <![CDATA[E2(pg / mL)]]> normal control group 10.56±0.35** 3.92±0.16* 142.19±6.32** Model control group 24.84±0.27 5.23±0.28 93±5.53 Dangofukang low-dose group 18.71±0.56* 4.42±0.64* 119±4.71** Dan'e Fuke medium dose group 15.63±0.76* 3.45±0.73* 126±7.66** Dango Fucang High-Dose Group 12.58±0.69** 3.31±0.26* 132±5.63**

[0064] Note: Compared with the model control group, *P<0.05, **P<0.01

[0065] Table 4. Effects of Danshen Fuke preparation on rat follicular development.

[0066] Group Number of primordial follicles / Number of primary follicles / Number of secondary follicles / normal control group 8.6±0.6* 7.5±0.4* 6.6±0.7* Model control group 4.2±0.2 3.2±0.5 3.1±0.2 Dangofukang low-dose group 5.4±0.3* 5.2±0.7* 4.2±0.3* Dan'e Fuke medium dose group 6.3±0.8* 6.8±0.5* 4.8±0.5* Dango Fucang High-Dose Group 7.9±0.4* 7.1±0.3* 5.7±0.6*

[0067] Note: Compared with the model control group, *P<0.05, **P<0.01

[0068] Table 3 shows that, compared with the normal control group, the serum FSH and LH levels of rats in the model control group were increased, and the E2 level was decreased, indicating successful surface modeling. The low, medium, and high doses of Dan'e Fukang all reduced serum FSH and LH levels in rats and significantly increased E2 levels.

[0069] As shown in Table 4, the number of follicles at each stage in the low, medium, and high dose groups of Dan'e Fukang increased to varying degrees compared with the model control group. The follicles grew actively, the number of mature follicles increased, and the follicles developed well. This indicates that Dan'e Fukang preparation has a promoting effect on follicle development and ovarian repair in rats with premature ovarian failure, and restores the endocrine function of the ovary, thus effectively treating premature ovarian failure.

[0070] In summary, the Dan'e Fuke preparation of this invention can increase the uterine and ovarian organ indices in mice with premature ovarian failure (POF); and increase the levels of estradiol and progesterone in the serum of POF mice. The Dan'e Fuke preparation of this invention can reduce the serum FSH and LH levels in rats with POF, and significantly increase the E2 level in rats; it also increases the number of primordial, primary, and secondary follicles, indicating that the Dan'e Fuke preparation of this invention can inhibit POF and promote the recovery of ovarian function.

Claims

1. Application of Dan'e Fuke preparation in the preparation of drugs for treating premature ovarian failure.

2. Application of Dan'e Fuke preparations in the preparation of drugs that improve uterine index and ovarian index.

3. Application of Dan'e Fuke preparations in the preparation of drugs that increase the number of follicles at all stages of ovarian development.

4. Application of Dan'e Fuke preparations in the preparation of drugs for treating elevated serum estradiol and progesterone levels.

5. The application according to claim 1, characterized in that: The Dan'e Fuke preparation is a Dan'e Fuke decoction.

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