Application of folium artemisiae argyi extract in treatment of cold-dampness stagnation type dysmenorrhea

Abstract

The invention provides application of a folium artemisiae argyi extract for treating cold-damp stagnation type dysmenorrhea and an experimental method, and belongs to the technical field of dysmenorrhea treatment. It is found that 3.6-14.4 g / kg (equal to 2-8 times of human equivalent dose) of the folium artemisiae argyi extract has the effect of treating cold-dampness stagnation type dysmenorrhea, apparent symptoms of cold-dampness stagnation type dysmenorrhea model rats can be relieved, uterine ovarian tissue lesions can be effectively improved, the content of related factors can be adjusted, inflammation can be effectively inhibited, and the folium artemisiae argyi extract can be used for treating cold-dampness stagnation type dysmenorrhea. And a new choice is provided for clinical treatment of primary dysmenorrhea.

Description

Technical Field

[0001] The invention belongs to the technical field of dysmenorrhea treatment, and particularly relates to application of an argyi leaf extract in preparing a medicine for treating cold-dampness stagnation type dysmenorrhea. Background Art

[0002] The classic symptom of dysmenorrhea is cyclical lower abdominal pain, sometimes accompanied by lumbar pain, occurring during or before and after menstruation, significantly impacting women's physical and mental health. Dysmenorrhea can be categorized into two types based on etiology: primary and secondary. The former is primarily caused by increased release of prostaglandins in the endometrium during menstruation. The latter is caused by organic pelvic lesions, including adenomyosis and endometriosis.

[0003] Primary dysmenorrhea (PD), also known as functional dysmenorrhea, is characterized by periodic lower abdominal pain, often cramping, accompanied by nausea, fatigue, chills, and even fainting. It is a common condition among adolescent women. Traditional Chinese Medicine (TCM) classifies dysmenorrhea as "menstrual abdominal pain" and categorizes it into multiple types based on etiology and pathogenesis. The cold-damp stagnation type primarily presents with lower abdominal pain, chills, and internal accumulation of yin and cold. Treatment focuses on regulating Qi and blood circulation to relieve pain.

[0004] Currently, nonsteroidal anti-inflammatory drugs (NSAIDs), primarily ibuprofen, are commonly used to treat dysmenorrhea. However, these drugs only relieve pain during the menstrual cycle and can cause recurrence after their effectiveness wears off, even causing gastrointestinal irritation. Therefore, there is an urgent need for medications that can improve dysmenorrhea symptoms.

[0005] Artemisia argyi (Artemisia argyi Levl. et Vant.), a plant of the Asteraceae family, is known for its warming, hemostatic, cold-dispelling, and pain-relieving properties. It is primarily used for lower abdominal pain, menstrual irregularities caused by cold, infertility due to uterine cold, hematemesis, epistaxis, and bleeding during pregnancy, making it a commonly used gynecological medicine. Artemisia argyi contains volatile oils, flavonoids, linalool, and polysaccharides, exhibiting multiple biological activities, including anti-inflammatory, antibacterial, antiviral, and immunomodulatory. Currently, for the treatment of dysmenorrhea caused by cold-dampness stagnation, artemisia argyi is primarily used in combination formulas or for topical application. There are no reports of its use alone for dysmenorrhea. Summary of the Invention

[0006] The technical solution of the present invention provides a new use of mugwort extract, specifically, its use in the preparation of a drug for treating dysmenorrhea caused by cold-dampness and stagnation. Furthermore, the present invention provides a method for preparing the mugwort extract. The mugwort extract provided by the present invention can alleviate symptoms in rats with a dysmenorrhea model caused by cold-dampness and stagnation, effectively improve uterine and ovarian tissue lesions, effectively reduce serum levels of estradiol (E2) and thromboxane B2 (TXB2), increase progesterone (Prog, P), and effectively inhibit the expression of the inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α).

[0007] To achieve the above objectives, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing an Artemisia argyi extract.

[0009] Specifically, the mugwort leaf extract is a water extract obtained by heating and refluxing mugwort slices or decocting them in a medicine pot.

[0010] Preferably, the wormwood water extract is prepared by heating and refluxing the wormwood slices in a round-bottom flask connected to a condenser.

[0011] In a second aspect, the present invention provides therapeutic uses of the Artemisia argyi extract.

[0012] Specifically, the use is based on the cold-dispelling and analgesic effects of mugwort to treat clinical diseases caused by traditional Chinese medicine symptoms.

[0013] Preferably, the use is to treat primary dysmenorrhea caused by cold environmental stimulation, i.e., cold-dampness stagnation type dysmenorrhea.

[0014] In a third aspect, the present invention provides a pharmaceutical preparation for treating dysmenorrhea of cold-dampness stagnation type.

[0015] Specifically, the pharmaceutical preparation is an oral preparation.

[0016] Preferably, the oral preparation is a liquid preparation or a solid preparation containing Artemisia argyi extract.

[0017] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0018] Preferably, the excipients include but are not limited to solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, thickeners, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, release agents, coating agents, flavoring agents or antioxidants.

[0019] The beneficial effects of the present invention are:

[0020] The present invention successfully established a rat model of dysmenorrhea caused by cold-dampness stagnation. The mugwort extract provided by the present invention can alleviate the pain symptoms of rats in this model, improve tissue and organ lesions, reduce the occurrence of blood stasis, and inhibit the production of inflammatory factors, providing a new option for the clinical treatment of primary dysmenorrhea. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Weight, diet, and dietary changes.

[0022] Figure 2 Number of twists.

[0023] Figure 3 The appearance of the uterus and ovaries and the ovarian index after administration.

[0024] Figure 4 The contents of various biochemical indicators in different samples.

[0025] Figure 5 HE-stained sections of uterine tissue.

[0026] Figure 6 HE-stained sections of ovarian tissue. DETAILED DESCRIPTION

[0027] The present invention is described below with reference to specific examples. The following examples are not intended to limit the present invention, but are merely intended to illustrate the present invention so that the technical solutions of the present invention are easier to understand and grasp. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0028] 1. Experimental equipment: 1 / 10,000 analytical balance; electric heating mantle; round-bottom flask; condenser; powder mixer; beaker; measuring cylinder; volumetric flask; filter bag; syringe; gavage needle.

[0029] 2. Experimental reagents: Artemisia argyi, purchased from Chongqing Zhongjing Chinese Medicine Pieces Co., Ltd., production batch number 230606, identified by Professor Liu Xiaofen of Chengdu University of Traditional Chinese Medicine as the dried leaves of Artemisia argyi of the Asteraceae family; estradiol benzoate injection (Hefei Xinkexin Animal Pharmaceutical Co., Ltd., production batch number 20240102); oxytocin injection (Jiangxi Bolai Pharmaceutical Factory Co., Ltd., production batch number 20240227); Tongjingbao granules (Zhongjing Wanxi Pharmaceutical Co., Ltd., production batch number 231016); purified water.

[0030] 3. Animals: SPF-grade sexually mature female SD rats, naive, 7–8 weeks old, weighing 200 ± 20 g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., license number SCXK (Xiang) 2021-0002.

[0031] 4. Data processing and statistical analysis

[0032] SPSS 21.0 and Origin 2022 software were used to perform statistical analysis on the research results. The differences between the two groups were analyzed by t test, ### or Indicates p<0.001, there is a very significant difference; ## or Indicates p<0.01, there is a significant difference; # or Indicates p<0.05, the difference is significant (# represents the difference compared with the blank group, represents the comparison with the model group).

[0033] Experimental Example Preparation of Artemisia argyi extract of the present invention

[0034] Weigh 160 g of Artemisia argyi leaves, place them in a 1000 mL round-bottom flask, add 10 times the amount of pure water, soak for 30 min, heat under reflux for 1 h, filter, concentrate, and add pure water to make the volume to 100 mL to obtain an Artemisia argyi leaf extract with a crude drug concentration of 1.6 g / mL.

[0035] Experimental Example 1 Establishment of a rat model of dysmenorrhea caused by cold-dampness stagnation

[0036] 1. Grouping

[0037] Forty SPF sexually mature female SD rats, 7-8 weeks old, weighing (200±20) g, were evenly divided into six groups according to body weight: control group (CG), model group (MG), low-dose Artemisia argyi Levl. et Vant. group (LAG), middle-dose Artemisia argyi Levl. et Vant. group (MAG), high-dose Artemisia argyi Levl. et Vant. group (HAG), and TongJingBao granules (positive control group, TJBG), with 8 rats in each group.

[0038] 2. Modeling

[0039] A rat model of cold-dampness stagnation-type dysmenorrhea was established using cold stimulation combined with estradiol benzoate and oxytocin. Rats were immersed in a (0±1)°C ice-water mixture for 10 minutes daily (room temperature 23±2°C) for 19 consecutive days (inability to climb and shivering were considered appropriate). Simultaneously, each rat received a subcutaneous injection of 0.4 mg of estradiol benzoate daily, with 0.8 mg administered on days 1 and 19 (double the first and last doses to ensure a constant blood concentration). One hour after the final gavage, each rat received an intraperitoneal injection of 4 units of oxytocin.

[0040] 3. Medication

[0041] All rats were given corresponding drugs by gavage starting from the 6th day after model establishment and 75 minutes after model establishment. Low, medium and high doses of 3.6 g / kg, 7.2 g / kg and 14.4 g / kg of Artemisia argyi extract were given by gavage. The normal group and model group were given 2 mL of pure water by gavage at the same time, once a day for 14 consecutive days.

[0042] Experimental Example 2 Monitoring and Detection of Indicators in the Dysmenorrhea Model of Cold-Damp Stagnation

[0043] 1. Apparent indicators

[0044] 1.1 Manifestations of Deficiency and Coldness. Rats exhibit pronounced chills, prefer to huddle together, have weak breathing, arch their backs with erect hair, curl up and sluggish movements, dark purple claws and tails, dark red ears, with withered fur, dark purple tongues, thickened and enlarged sublingual veins, and unformed feces.

[0045] 1.2 Number of writhings and writhing latency. Immediately after oxytocin injection, timing was started, and the time from oxytocin injection to the onset of the writhing response (i.e., latency) and the number of writhings per rat within 30 min were recorded.

[0046] 1.3 Changes in Body Weight, Diet, and Water Consumption. Each group was weighed daily to monitor weight changes. Daily diet and water consumption of rats in each group was recorded, and increases and decreases were calculated.

[0047] 1.4 Uterine Appearance. The uteri were separated and photographed for comparison.

[0048] 1.5 Organ Index. Separate the uterus, ovaries, and spleen and weigh each organ. Organ index = organ mass / body weight.

[0049] 2. Biochemical indicators

[0050] 2.1 Serum Parameter Assays. Blood was collected from the abdominal aorta, allowed to rest at room temperature for 30 minutes, and then centrifuged at 3500 rpm for 15 minutes. Serum was collected and stored at -80°C until assayed. Serum levels of thromboxane TXB2, estradiol E2, progesterone P, interleukin-6, and tumor necrosis factor α were determined using a microplate reader according to the ELISA kit instructions.

[0051] 2.2 Detection of Indicators in Tissue Homogenates. An appropriate amount of uterine tissue was minced and added to the corresponding volume of PBS buffer (1:9 mass-to-volume ratio). Steel grinding beads were added and the sample was ground and homogenized to obtain a 10% tissue homogenate. After homogenization, the sample was centrifuged at 5000 x g for 10 minutes. The supernatant was collected and stored for testing. The IL-6 and TNF-α levels in the uterine tissue were determined using a microplate reader according to the ELISA kit instructions.

[0052] 3. Pathological indicators

[0053] Uterine and ovarian pathological sections. Uterine and ovarian tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin for histological observation. Vascular distribution and inflammatory cell infiltration in the uterine tissue were primarily observed.

[0054] 4. Experimental Results

[0055] After treatment with mugwort leaf extract, it can effectively relieve pain, improve tissue inflammation and blood stasis. The specific results are as follows:

[0056] 4.1 Apparent indicators

[0057] 4.1.1 Changes in body weight, diet, and drinking water. Six days after modeling, the body weight, diet, and drinking water in the model and drug groups showed a significant downward trend compared to the blank group; two weeks after drug administration, both the model and drug groups showed an upward trend, but the overall increase in the model group was lower than that in the drug group. The results are as follows: Figure 1 shown.

[0058] 4.2.2 Writhing reaction. After administration of mugwort extract, the number of writhing reactions in rats decreased, and the difference between the medium-dose group and the control group was significant, indicating that the pain caused by modeling can be alleviated to a certain extent. Figure 2 shown.

[0059] 4.2.3 Tissue Observation and Organ Indexes. The uterus of the model group showed significant edema and enlargement. After administration of mugwort extract, the uterine tissue showed some improvement, with reduced edema and vascular proliferation ( Figure 3 A). After modeling, the ovarian index of the model group increased. Different concentrations of mugwort decoction can make the ovarian index return to normal ( Figure 3 B).

[0060] 4.2 Biochemical indicators

[0061] 4.2.1 Serum E2 and TXB2 levels. E2 is a primary dysmenorrhea pain factor. Increased E2 levels can directly or indirectly promote uterine vasospasm and smooth muscle contraction. Different concentrations of mugwort extract reduced E2 to a certain extent, with significant differences ( Figure 4 A). When the body's TXB2 content increases, platelets mediate thrombosis, resulting in poor blood flow. The TXB2 concentration in the model group was higher than that in the blank group. Compared with the model group, the concentrations of each drug-treated group decreased to varying degrees. Among them, the medium-dose group (MAG) showed significant differences ( Figure 4 B).

[0062] 4.2.2 Inflammatory factor content. The increase in IL-6 represents the occurrence of inflammation. By measuring the content in uterine tissue homogenate, it was found that mugwort extract can reduce the content of the inflammatory factor IL-6 and reduce the occurrence of inflammation in a dose-concentration dependent manner ( Figure 4 C).

[0063] 4.3 Pathological indicators

[0064] Compared with the model group, administration of Artemisia argyi extract can reduce the degree of endometrial vacuolation and uterine gland expansion ( Figure 5 After administration, the degree of hydropic degeneration of the corpus luteum in the ovarian tissue was alleviated compared with the model group, the necrosis of the oocytes was reduced, and the morphology was more regular ( Figure 6 ).

Claims

1. Use of an extract of Artemisia argyi in preparing a preparation for relieving dysmenorrhea caused by cold-dampness stagnation, characterized in that: The mugwort leaf extract is an active ingredient obtained by extracting mugwort leaves with water, and the use is mainly aimed at its traditional effect of dispelling cold and relieving pain.

2. The wormwood extract according to claim 1, wherein The extraction solvent of the wormwood leaf extract is water, and the extraction method is heating reflux.

3. The cold-dispelling and analgesic effect according to claim 1, characterized in that The efficacy can be used to relieve primary dysmenorrhea of cold-damp stagnation type.

4. The use according to claim 3, characterized in that The use is for clinical conditions caused by TCM symptoms, which is more in line with the therapeutic properties of TCM.

5. A medicine for treating primary dysmenorrhea of cold-dampness stagnation type, characterized in that: The raw material components of the medicine include mugwort leaf extract.

6. The drug according to claim 5, characterized in that The weight percentage of the wormwood leaf extract in the medicine is 5% to 99%.

7. The pharmaceutical preparation according to claim 5, characterized in that The medicine further contains pharmaceutically acceptable excipients.

8. The medicine according to claim 5, characterized in that The medicine is an oral preparation or a non-oral preparation.

9. The drug according to claim 8, characterized in that The oral preparation is one or more of capsules, tablets, oral liquids, mixtures, granules, pills, powders, pills or pastes.

10. The medicine according to claim 8, characterized in that The non-oral preparation is one or more of a cream, a patch, an ointment or a spray.

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