A pharmaceutical composition for treating perimenopausal overweight / obesity, and a preparation method and use thereof

Abstract

The present application belongs to the technical field of traditional Chinese medicine preparation, and particularly relates to a kind of drug composition for treating perimenopausal overweight / obesity, and further discloses its preparation method and use.The drug composition for treating perimenopausal overweight / obesity disclosed by the present application comprises the following raw materials: Anemarrhena asphodeloides, Phellodendri chinensis, Rehmanniae, Astragali, Angelica sinensis, Herba Epimedii, calcined Ossis Muli, calcined Ostraeae and fried Zizyphi Fructus Preparata.The treatment of the composition disclosed by the present application is based on the principle of benefiting kidney essence, regulating yin and yang and tonifying qi and blood, and the composition is applied to treating perimenopausal overweight / obesity by exerting phytoestrogen-like effect.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation technology, specifically relating to a pharmaceutical composition for treating perimenopausal overweight / obesity, and further disclosing its preparation method and uses. Background Technology

[0002] Currently, with the aging society already taking shape and gradually intensifying in my country, statistics show that there are approximately 130 million women in perimenopause in my country. It is projected that by 2030, the number of perimenopausal women globally will reach 1.2 billion, with my country accounting for 280 million. Among them, over 90% will experience perimenopausal symptoms. Simultaneously, as life expectancy among Chinese women continues to increase, the irreversible state of low estrogen levels will persist. This means that women will spend one-third or even longer in a low-stimulation-hormone state, significantly increasing the likelihood of related complications.

[0003] Due to the decline in estrogen levels accompanying the decline in ovarian function during perimenopause, fat redistribution is common, leading to abdominal obesity caused by visceral fat accumulation. Typically, as women enter perimenopause, ovarian function gradually declines, manifesting as menstrual cycle irregularities accompanied by decreased E2 and increased FSH. At this time, the hypothalamus and pituitary gland experience regulatory dysregulation, leading to a series of central nervous system dysfunctions, resulting in a range of neurological and vasomotor symptoms such as hot flashes, insomnia, sweating, and anxiety. Perimenopausal lipid metabolism disorders are a manifestation of the disruption of the balance between the hypothalamus-pituitary-ovarian axis, the hypothalamus-pituitary-adrenal axis, and the hypothalamus-pituitary-peripheral tissue (including adipose tissue) axis. With the decline in ovarian function, the body's compensatory pathways for stimulating endogenous estrogen production become active. At this time, adipose tissue becomes the main tissue for estrogen production, manifesting as increased visceral fat and exacerbated central obesity. However, the increased visceral fat and the resulting lipid metabolism disorders are precisely the high-risk factors for the occurrence and development of atherosclerotic plaques, cardiovascular and cerebrovascular diseases, diabetes, cognitive impairment, and osteoporosis. This also makes women entering perimenopause more susceptible to related diseases than men of the same age, seriously affecting their physical and mental health.

[0004] Currently, Western medicine primarily uses menopausal hormone therapy (MHT) to intervene in the perimenopausal period in women. This mainly involves the use of estrogen alone or in combination with progesterone, offering rapid onset of action and proven efficacy in treating perimenopausal vasomotor symptoms, urinary tract infections, and osteoporosis. However, strict adherence to indications and contraindications, along with close monitoring for side effects, leads to generally low acceptance and usage rates. Traditional Chinese medicine (TCM) has accumulated considerable experience in treating perimenopausal syndrome using compound formulas. These formulas not only alleviate patients' distressing symptoms but also, as many of these herbs possess phytoestrogens-like effects, can improve estrogen deficiency, achieving a "targeted therapy" effect. Through long-term clinical treatment, many reliable TCM preparations have been developed. However, evidence regarding interventions for perimenopausal overweight / obesity is limited. There is a growing interest in developing a safe and effective TCM formula. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a pharmaceutical composition for treating perimenopausal overweight / obesity, and further disclose its preparation method and use.

[0006] To solve the above-mentioned technical problems, the present invention provides a pharmaceutical composition for treating perimenopausal overweight / obesity, wherein the raw materials of the pharmaceutical composition are: Anemarrhena asphodeloides 5-30 parts by weight, Phellodendron chinense 5-30 parts by weight, Rehmannia glutinosa 5-30 parts by weight, Astragalus membranaceus 5-30 parts by weight, Angelica sinensis 5-30 parts by weight, Epimedium brevicornu 5-30 parts by weight, calcined dragon bone 5-30 parts by weight, calcined oyster shell 5-30 parts by weight, and stir-fried Ziziphus jujuba var. spinosa 5-30 parts by weight.

[0007] Preferably, the pharmaceutical composition for treating perimenopausal overweight / obesity comprises the following raw materials: 8 parts by weight of Anemarrhena asphodeloides, 8 parts by weight of Phellodendron chinense, 14 parts by weight of Rehmannia glutinosa, 14 parts by weight of Astragalus membranaceus, 8 parts by weight of Angelica sinensis, 8 parts by weight of Epimedium brevicornu, 14 parts by weight of calcined dragon bone, 14 parts by weight of calcined oyster shell, and 14 parts by weight of stir-fried Ziziphus jujuba var. spinosa.

[0008] The present invention also discloses the use of the pharmaceutical composition in the preparation of a medicament for treating perimenopausal overweight / obesity.

[0009] The present invention also discloses a pharmaceutical preparation for treating perimenopausal overweight / obesity, comprising the aforementioned pharmaceutical composition.

[0010] Specifically, the pharmaceutical preparation for treating perimenopausal overweight / obesity includes at least one of the following: tablets, capsules, powders, mixtures, pills, granules, solutions, syrups, decoctions, plasters, suppositories, aerosols, ointments, or injections.

[0011] Specifically, the pharmaceutical preparation for treating perimenopausal overweight / obesity further includes clinically acceptable excipients;

[0012] Preferably, the excipients include at least one of fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, or matrices;

[0013] Preferably, the filler includes at least one of starch, pregelatinized starch, lactose, mannitol, chitosan, or microcrystalline cellulose;

[0014] Preferably, the disintegrant includes at least one of starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, or croscarmellose sodium.

[0015] Preferably, the lubricant comprises at least one of magnesium stearate, sodium dodecyl sulfate, talc, or silica;

[0016] Preferably, the suspending agent includes at least one of polyvinylpyrrolidone, microcrystalline cellulose, agar, or hydroxypropyl methylcellulose;

[0017] Preferably, the adhesive comprises at least one of starch paste, polyvinylpyrrolidone, or hydroxypropyl methylcellulose;

[0018] Preferably, the sweetener includes at least one of sodium saccharin, aspartame, sucrose, cyclamate, or glycyrrhetinic acid;

[0019] Preferably, the flavoring agent includes flavoring;

[0020] Preferably, the preservative includes at least one of parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorethidium acetate, or eucalyptus oil;

[0021] Preferably, the matrix includes at least one of PEG6000, PEG4000, or insect wax.

[0022] The present invention also discloses a method for preparing the pharmaceutical preparation for treating perimenopausal overweight / obesity, comprising the step of mixing selected amounts of Anemarrhena asphodeloides, Phellodendron chinense, Rehmannia glutinosa, Astragalus membranaceus, Angelica sinensis, Epimedium brevicornu, calcined dragon bone, calcined oyster shell and stir-fried Ziziphus jujuba var. spinosa.

[0023] Specifically, the preparation method of the drug formulation for treating perimenopausal overweight / obesity includes: taking a selected amount of Anemarrhena asphodeloides, Phellodendron chinense, Rehmannia glutinosa, Astragalus membranaceus, Angelica sinensis, Epimedium brevicornu, calcined dragon bone, calcined oyster shell and stir-fried Ziziphus jujuba seeds, mixing them together, adding water to decoct them, collecting the decoction and concentrating it into an extract, and adding conventional excipients to process it into a selected dosage form.

[0024] Specifically, the method for preparing the pharmaceutical formulation for treating perimenopausal overweight / obesity:

[0025] The amount of water added is 5-15 times the total weight of the raw material; and / or,

[0026] The decoction process involves decocting 1-3 times; and / or,

[0027] The decocting step takes 1-3 hours; and / or,

[0028] The concentration step involves concentrating the decoction to a thick paste with a relative density of 1.25 at 60°C.

[0029] As an feasible solution, the preparation method of the pharmaceutical preparation of the present invention includes the following steps: weighing Anemarrhena asphodeloides, Phellodendron chinense, Rehmannia glutinosa, Astragalus membranaceus, Angelica sinensis, Epimedium brevicornu, calcined dragon bone, calcined oyster shell and stir-fried Ziziphus jujuba var. spinosa according to the selected weight proportions, mixing them evenly, and decocting them 1-3 times. Based on the total weight of the mixed raw materials, add 5-15 times the weight of water each time and decoct for 1-3 hours. Combine the decoctions, filter, and concentrate the filtrate to a thick paste with a relative density of 1.25 at 60°C.

[0030] As a preferred embodiment, the preparation method of the pharmaceutical preparation of the present invention includes the following steps: weighing Anemarrhena asphodeloides, Phellodendron chinense, Rehmannia glutinosa, Astragalus membranaceus, Angelica sinensis, Epimedium brevicornu, calcined dragon bone, calcined oyster shell, and stir-fried Ziziphus jujuba var. spinosa according to the selected weight proportions, mixing them evenly, and decocting twice. Based on the total weight of the mixed raw materials, add 10 times the weight of water each time and decoct for 1.5 hours. Combine the decoctions, filter, and concentrate the filtrate to a thick paste with a relative density of 1.25 at 60°C, thus obtaining the final product.

[0031] The present invention also discloses the use of the pharmaceutical composition for preparing a medicament having at least one of the following effects (1)-(3):

[0032] (1) Improve perimenopausal overweight / obesity;

[0033] (2) Improve TC and / or TG levels;

[0034] (3) Promotes the production of endogenous estrogen.

[0035] The pharmaceutical composition for treating perimenopausal overweight / obesity described in this invention comprises the following raw materials: Anemarrhena asphodeloides, Phellodendron chinense, Rehmannia glutinosa, Astragalus membranaceus, Angelica sinensis, Epimedium brevicornu, calcined dragon bone, calcined oyster shell, and stir-fried Ziziphus jujuba var. spinosa. This composition targets the core pathogenesis of perimenopausal women, namely, deficiency of kidney essence, imbalance of yin and yang, and insufficiency of qi and blood. When the kidney nourishes primordial yin and yang, abundant kidney essence leads to a flourishing of Tian Gui (menstrual blood), resulting in regular menstruation. When kidney essence is deficient, Tian Gui is exhausted, yin and yang are imbalanced, and qi and blood decline, leading to amenorrhea and physical decline, manifesting as menstrual disorders, hot flashes and night sweats, anxiety and depression, insomnia and dreaminess, fatigue, palpitations, dizziness, aversion to cold and wind, spontaneous sweating and night sweats, and many other discomforts. The therapeutic principle of the composition described in this invention is to nourish kidney essence, regulate yin and yang, and replenish qi and blood. Based on the theory that "essence and qi are divided into yin and yang, and yin and yang cannot be separated", this formula uses Rehmannia glutinosa and Epimedium as the chief herbs. Rehmannia glutinosa nourishes yin and benefits the kidney, while Epimedium warms the kidney and strengthens yang. The two herbs are combined to balance yin and yang, and together they target the pathological basis of insufficient kidney essence and yin-yang imbalance in perimenopausal women. Anemarrhena asphodeloides and Phellodendron chinense are cold in nature and enter the kidney. Combined with Rehmannia glutinosa, they nourish yin and generate fluids, and clear the deficiency heat in the kidney. Astragalus membranaceus invigorates qi and consolidates the exterior, while Angelica sinensis nourishes blood and regulates menstruation. The above four herbs are used as assistant herbs to work together to restore the physiological state of perimenopausal women. Calcined dragon bone and calcined oyster shell astringe and consolidate, calm the mind and subdue yang. Fried jujube seed is sweet and sour and moist, nourishes blood and liver, and calms the mind. The three herbs are used as adjuvant herbs.

[0036] The pharmaceutical composition for treating perimenopausal overweight / obesity described in this invention specifically exerts a phytoestrogen-like effect through aromatase activity, which aligns with the modern medical understanding of decreased estrogen levels in perimenopausal women. By exerting a phytoestrogen-like effect, it is applied to the treatment of perimenopausal overweight / obesity. Attached Figure Description

[0037] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0038] Figure 1 This is a graph showing the effect of the drug composition described in Experimental Example 1 on the weight gain of OVX rats. * P<0.05, ** P<0.01, compared with the Sham group; # P<0.05, ## P<0.01, compared with the OVX group;

[0039] Figure 2 The effect of the drug composition described in Experimental Example 1 on aromatase and E2 levels; where A: changes in aromatase levels; B: changes in E2 levels; GNS represents the Medium group; * P<0.05, *** P<0.001, compared with the Sham group; # P<0.05, ## P<0.01, compared with the OVX group;&&& P<0.001, compared with the EV group;

[0040] Figure 3 The images and analysis diagrams show the effects of the drug composition described in Experiment 1 on fat CT in OVX rats; where A: fat CT image, vWAT is visceral fat (green), sWAT is subcutaneous fat (yellow), Merged is a combination of visceral fat and subcutaneous fat; B: fat volume analysis diagram; GNS is the Medium group; * P<0.05, *** P<0.001, compared with the Sham group; # P<0.05, ## P<0.01, compared with the OVX group;

[0041] Figure 4 This is a bar chart showing the regulation of blood lipid levels in OVX rats by the drug composition described in Experimental Example 1; where A: comparison of TC levels in serum, vWAT, and iWAT; B: comparison of TG levels in serum, vWAT, and iWAT; iWAT represents inguinal fat; GNS represents the Medium group; * P<0.05, ** P<0.01, *** P<0.001, compared with the Sham group; # P<0.05, ## P<0.01, ### P<0.001, compared with the OVX group; & P<0.05, compared with the EV group;

[0042] Figure 5 The images show the H&E sections and analysis diagrams of visceral fat in OVX rats produced by the drug composition described in Experimental Example 1; where A: H&E section; B: statistical diagram of fat cell area within the field of view; C: statistical diagram of fat cell number within the field of view; scale bar is 100 μm. * P<0.05, ** P<0.01, *** P<0.001, compared with the Sham group; # P<0.05, ## P<0.01, ### P<0.001, compared with the OVX group; & P<0.05, compared with the EV group;

[0043] Figure 6 The graph shows the effect of the drug composition described in Experiment 2 on the weight gain of OVX-LET rats; GNS represents the Medium group. * P<0.05, **P<0.01, compared with the Sham group; # P<0.05, compared with the OVX group;

[0044] Figure 7 The effect of the drug composition described in Experiment 2 on serum aromatase and E2 levels in OVX-LET rats; where A: changes in aromatase level; B: changes in E2 level; GNS is the Medium group; * P<0.05, *** P<0.001, compared with the Sham group; # P<0.05, ### P<0.001, compared with the OVX group; & P<0.05, compared with the LET group;

[0045] Figure 8 The images and analysis diagrams show the effects of the drug composition described in Experimental Example 2 on fat CT in OVX-LET rats; where A: fat CT image, vWAT is visceral fat (green), sWAT is subcutaneous fat (yellow), Merged is a combination of visceral fat and subcutaneous fat; B: fat volume analysis diagram; GNS is the Medium group; * P<0.05, ** P<0.01, *** P<0.001, compared with the Sham group; # P<0.05, compared with the OVX group;

[0046] Figure 9 This is a bar chart showing the effect of the drug composition described in Experimental Example 2 on the regulation of serum lipid levels in OVX-LET rats; where A: comparison of TC levels in serum; B: comparison of TG levels in serum; GNS represents the Medium group; * P<0.05, ** P<0.01, *** P<0.001, compared with the Sham group; # P<0.05, compared with the OVX group;

[0047] Figure 10 The image shows the analysis of H&E sections of visceral fat in OVX-LET rats using the drug composition described in Experimental Example 2; where A: H&E section; B: statistical graph of adipocyte area within the field of view; C: statistical graph of adipocyte number within the field of view; GNS represents the Medium group; the scale bar is 100 μm. * P<0.05, ** P<0.01, compared with the Sham group. Detailed Implementation

[0048] Example 1

[0049] The pharmaceutical composition for treating perimenopausal overweight / obesity described in this embodiment comprises the following raw materials: 8 parts by weight of Anemarrhena asphodeloides, 8 parts by weight of Phellodendron chinense, 14 parts by weight of Rehmannia glutinosa, 14 parts by weight of Astragalus membranaceus, 8 parts by weight of Angelica sinensis, 8 parts by weight of Epimedium brevicornu, 14 parts by weight of calcined dragon bone, 14 parts by weight of calcined oyster shell, and 14 parts by weight of stir-fried Ziziphus jujuba var. spinosa.

[0050] Weigh out the following ingredients according to the selected weight proportions: Anemarrhena asphodeloides, Phellodendron chinense, Rehmannia glutinosa, Astragalus membranaceus, Angelica sinensis, Epimedium brevicornu, calcined dragon bone, calcined oyster shell, and stir-fried Ziziphus jujuba var. spinosa. Mix them well and decoct twice. Using the total weight of the mixed raw materials as a base, add 10 times the weight of water each time and decoct for 1.5 hours. Combine the decoctions, filter, and concentrate the filtrate to a thick paste with a relative density of 1.25 at 60℃.

[0051] Example 2

[0052] The pharmaceutical composition for treating perimenopausal overweight / obesity described in this embodiment comprises the following raw materials: 5 parts by weight of Anemarrhena asphodeloides, 30 parts by weight of Phellodendron chinense, 5 parts by weight of Rehmannia glutinosa, 30 parts by weight of Astragalus membranaceus, 5 parts by weight of Angelica sinensis, 30 parts by weight of Epimedium brevicornu, 5 parts by weight of calcined dragon bone, 30 parts by weight of calcined oyster shell, and 5 parts by weight of stir-fried Ziziphus jujuba var. spinosa.

[0053] Weigh out the following ingredients according to the selected weight proportions: Anemarrhena asphodeloides, Phellodendron chinense, Rehmannia glutinosa, Astragalus membranaceus, Angelica sinensis, Epimedium brevicornu, calcined dragon bone, calcined oyster shell, and stir-fried Ziziphus jujuba var. spinosa. Mix them well and decoct them three times. Using the total weight of the mixed raw materials as a reference, add 5 times the weight of water each time and decoct for 1 hour. Combine the decoctions, filter them, and concentrate the filtrate to a thick paste with a relative density of 1.25 at 60°C.

[0054] Example 3

[0055] The pharmaceutical composition for treating perimenopausal overweight / obesity described in this embodiment comprises the following raw materials: 30 parts by weight of Anemarrhena asphodeloides, 5 parts by weight of Phellodendron chinense, 30 parts by weight of Rehmannia glutinosa, 5 parts by weight of Astragalus membranaceus, 30 parts by weight of Angelica sinensis, 5 parts by weight of Epimedium brevicornu, 30 parts by weight of calcined dragon bone, 5 parts by weight of calcined oyster shell, and 30 parts by weight of stir-fried Ziziphus jujuba var. spinosa.

[0056] Weigh out the following ingredients according to the selected weight proportions: Anemarrhena asphodeloides, Phellodendron chinense, Rehmannia glutinosa, Astragalus membranaceus, Angelica sinensis, Epimedium brevicornu, calcined dragon bone, calcined oyster shell, and stir-fried Ziziphus jujuba var. spinosa. Mix them well and decoct twice. Based on the total weight of the mixed raw materials, add 15 times the weight of water each time and decoct for 3 hours. Combine the decoctions, filter, and concentrate the filtrate to a thick paste with a relative density of 1.25 at 60℃.

[0057] Experimental Example

[0058] In the following experimental example of this invention, 40 healthy female SD rats, 3 months old, were purchased from Changchun Yisi Experimental Animal Technology Co., Ltd. and housed at the Animal Center of Changchun University of Traditional Chinese Medicine. The rats were housed under specific pathogen-free (SPF) conditions and fed a standard diet. The ambient temperature was controlled at 22-25℃, the humidity at 55±5%, and a 12 / 12-hour light / dark cycle was maintained (light time 7:00-19:00). They had free access to food and water.

[0059] The following experimental examples of this invention include the following related verification experiments.

[0060] 1. OVX animal experiments

[0061] Experimental methods

[0062] Experimental grouping and administration method

[0063] After one week of acclimatization, all rats underwent either OVX or Sham surgery. Following a two-week recovery period, successful model establishment was confirmed by vaginal smear examination, and rats were randomly assigned to groups of six. The specific groupings are as follows:

[0064] ①Sham group: This group underwent sham surgery, with the same surgical procedure as the other groups, but only the same volume of fat around the ovary was removed. They were fed regular feed and given an equal volume of sterile drinking water via gavage once daily.

[0065] ②OVX group: also known as the model group, the surgical procedure involved a dorsal incision to remove both ovaries, followed by layered suturing. Patients were fed regular feed and administered an equal volume of sterile drinking water via gavage once daily.

[0066] ③OVX+EV positive control group: In addition to OVX, estradiol valerate tablets (trade name: Progynova) were administered by gavage at a dose of 1 mg / day once daily.

[0067] ④ OVX+GNS (Low) low-dose group: In addition to OVX, the drug composition finally prepared in Example 1 was administered in equal volume once daily at 0.5 times the body weight.

[0068] ⑤ OVX+GNS (Mid) medium-dose group: In addition to OVX, the drug composition finally prepared in Example 1 was administered by gavage in equal volume once a day;

[0069] ⑥ OVX+GNS(Hig) high-dose group: In addition to OVX, the drug composition finally prepared in Example 1 was administered at twice the body weight and administered by gavage in equal volume once daily.

[0070] Administration method

[0071] During the drug administration process, soybean feed was removed, and each group was given drug intervention simultaneously (drug administration according to the experimental group information above), administered orally by gavage, with an intervention period of 9 weeks.

[0072] Serum preparation

[0073] Samples were collected 24 hours after the last administration, with fasting but free access to water. Anesthesia was administered via intraperitoneal injection of 0.3% sodium pentobarbital (40 mg / kg). After fixation in a supine position, the abdominal cavity was disinfected and opened to expose the abdominal aorta. 5–10 ml of blood was collected using a vacuum blood collection tube containing a coagulant. The blood was allowed to stand at room temperature for 2 hours, then centrifuged at 3500 rpm for 10 minutes. The supernatant was collected, aliquoted, and stored in cryovials at -80°C.

[0074] Detection indicators and methods

[0075] (1) Body weight: The rats were weighed and recorded using an electronic weighing device once a week at a fixed time.

[0076] (2) The content of TC was detected by CHOD-PAP method and TG was detected by GPO-PAP method. The specific operation method was strictly followed according to the kit instructions and the detection was performed by Toshiba fully automated biochemical analyzer (TBA40FR).

[0077] (3) Radioimmunoassay was used to detect E2 levels. The specific operation method was strictly followed according to the kit instructions. The test was performed using a γ-radioimmunoassay counter (XH-6080) from Xi'an Nuclear Instrument Factory.

[0078] (4) The aromatase level was detected by ELISA. The specific operation method was strictly followed according to the kit instructions. The detection was performed using a multifunctional microplate detection system (BioTek Synergy Neo2).

[0079] miro-CT detection of fat volume

[0080] At the end of the experiment, all rats were anesthetized with isoflurane and scanned using a micro-CT system (PerkinElmer, Inc., Waltham, MA, United States). Scanning parameters: 90 kV tube voltage, 88 uA, 86 mm FOV, 360° rotation, 30 frames per second, and 2 min scan time. The system's built-in image analysis software was used for fat color marking and 3D reconstruction, and the volumes of visceral fat (vWAT) and subcutaneous fat (sWAT) were assessed separately.

[0081] H&E staining

[0082] H&E staining was performed to study the morphology of subcutaneous adipose tissue in different groups and to assess the area and number of lipid droplets (Wang et al., 2021). After staining, images of each section were acquired using an M8 microscope and scanner (Precision Point, Thuringia, Germany), and the fat area and number were analyzed using Image Pro Plus 6.0 software (Media Cybemetics, INC., Rockville, MD, United States).

[0083] Data Analysis

[0084] SPSS 27.0 software was used for statistical analysis, and univariate statistical tests were employed to compare differences between samples from different groups. For normally distributed data, ANOVA (multiple-group test) and Student's t-test (two-group test) were used for analysis; for non-normally distributed data, Kruskal-Wallis H-test (multiple-group test) and Mann-Whitney U-test (two-group test) were used for analysis.

[0085] Experimental results

[0086] Weight comparison

[0087] As attached Figure 1 The results showed that after 9 weeks of GNS intervention, the OVX group had a significantly higher body weight compared to the Sham group (P<0.05). Exogenous EV supplementation (OVX+EV group) significantly reduced body weight, with a statistically significant difference compared to the OVX group (P<0.05). Medium-dose GNS intervention (OVX+GNS Mid group) also significantly reduced body weight after 9 weeks, with a statistically significant difference compared to the OVX group (P<0.05).

[0088] Aromatase levels and E2 levels

[0089] As attached Figure 2 As shown in Figure A, after 9 weeks of GNS intervention, the aromatase level in the OVX group was significantly higher than that in the Sham group (P<0.05); the aromatase level in the EV group was significantly lower than that in the OVX group (P<0.05); and compared with various treatment factors, the GNS group significantly increased the aromatase level (P<0.05).

[0090] As attached Figure 2As shown in Figure B, after 9 weeks of GNS intervention, compared with the Sham group, the E2 level in the OVX group was significantly lower (P<0.05); compared with the OVX group, the E2 level in the EV group was significantly higher (P<0.05); and compared with the OVX group, the E2 level in the GNS group was significantly higher, but still lower than that in the Sham group, with statistically significant differences (P<0.05).

[0091] Comparison of visceral fat and subcutaneous fat volume

[0092] As attached Figure 3 As shown in Figures A and B, after 9 weeks of GNS intervention, the OVX group exhibited a significant increase in both visceral fat (vWAT) and subcutaneous fat (sWAT), with statistically significant differences (P<0.05). Compared with the OVX group, both the EV group and the GNS group inhibited the increase in vWAT and sWAT volume, with statistically significant differences (P<0.05). However, the vWAT volume in the GNS group was still higher than that in the Sham group, with a statistically significant difference (P<0.05).

[0093] Comparison of blood lipid levels

[0094] As attached Figure 4 As shown in Figures A and B, after 9 weeks of GNS intervention, the OVX group showed a significant increase in serum and inguinal fat (iWAT) TC levels, while TG levels were significantly increased in both vWAT and iWAT (P<0.05). The EV group significantly reduced serum and iWAT TC levels, and significantly reduced vWAT and iWAT TG levels (P<0.05). The GNS group significantly reduced serum and iWAT TC levels, and significantly reduced vWAT and iWAT TG levels (P<0.05).

[0095] H&E section analysis of visceral adipose tissue

[0096] As attached Figure 5As shown in the AC results, after 9 weeks of GNS intervention, compared with the Sham group, the OVX group showed an increase in adipocyte volume and a decrease in adipocyte number within the visual field, with statistically significant differences (P<0.05). Compared with the OVX group, the EV group inhibited the increase in adipocyte volume and increased the adipocyte number within the visual field, with statistically significant differences (P<0.05). Compared with the OVX group, the GNS group inhibited the increase in adipocyte volume and increased the adipocyte number within the visual field, with statistically significant differences (P<0.05). However, compared with the Sham group, the volume and number of adipocytes within the visual field in the GNS group did not recover to the levels of the Sham group (P<0.05). Moreover, the volume of adipocytes within the visual field in the GNS group was consistent with that after EV treatment (P>0.05), but the number of adipocytes within the visual field was still lower than that after EV treatment (P<0.05).

[0097] 2. OVX-LET animal experiments

[0098] Experimental methods

[0099] Experimental grouping and administration method

[0100] After one week of acclimatization, all rats underwent either OVX or Sham surgery. Following a two-week recovery period, successful model establishment was confirmed by vaginal smear examination, and rats were randomly assigned to groups of six. The specific groupings are as follows:

[0101] ①Sham group: This group underwent sham surgery, with the same surgical procedure as the other groups, but only the same volume of fat around the ovary was removed. They were fed regular feed and given an equal volume of sterile drinking water via gavage once daily.

[0102] ②OVX group: also known as the model group, the surgical procedure involved a dorsal incision to remove both ovaries, followed by layered suturing. Patients were fed regular feed and administered an equal volume of sterile drinking water via gavage once daily.

[0103] ③OVX+LET group: In addition to OVX, letrozole (LET, trade name: Frolon, Novartis Pharmaceuticals, Switzerland) was administered by gavage at a dose of 2.5 mg / day once daily.

[0104] ④ OVX+LET+GNS medium dose group (i.e. optimal dose group): Based on OVX, LET and GNS were administered in combination. The drug composition finally prepared in Example 1 was taken and administered by gavage in equal volume once a day.

[0105] Administration method

[0106] During the administration of medication, soybean-free feed was used, and each group was simultaneously given drug intervention (administered according to the experimental grouping information above) via oral gavage. The intervention period was 8 weeks. The gavage interval for LET and GNS was 30 minutes.

[0107] Serum preparation

[0108] Samples were collected 24 hours after the last administration, with fasting but free access to water. Anesthesia was administered via intraperitoneal injection of 0.3% sodium pentobarbital (40 mg / kg). After fixation in a supine position, the abdominal cavity was disinfected and opened to expose the abdominal aorta. 5–10 ml of blood was collected using a vacuum blood collection tube containing a coagulant. The blood was allowed to stand at room temperature for 2 hours, then centrifuged at 3500 rpm for 10 minutes. The supernatant was collected, aliquoted, and stored in cryovials at -80°C.

[0109] Detection indicators and methods

[0110] (1) Body weight: The rats were weighed and recorded using an electronic weighing device once a week at a fixed time.

[0111] (2) The content of TC was detected by CHOD-PAP method and TG was detected by GPO-PAP method. The specific operation method was strictly followed according to the kit instructions and the detection was performed by Toshiba fully automated biochemical analyzer (TBA40FR).

[0112] (3) Radioimmunoassay was used to detect E2 levels. The specific operation method was strictly followed according to the kit instructions. The test was performed using a γ-radioimmunoassay counter (XH-6080) from Xi'an Nuclear Instrument Factory.

[0113] (4) The aromatase level was detected by ELISA. The specific operation method was strictly followed according to the kit instructions. The detection was performed using a multifunctional microplate detection system (BioTek Synergy Neo2).

[0114] miro-CT detection of fat volume

[0115] At the end of the experiment, all rats were anesthetized with isoflurane and scanned using a micro-CT system (PerkinElmer, Inc., Waltham, MA, United States). Scanning parameters: 90 kV tube voltage, 88 uA, 86 mm FOV, 360° rotation, 30 frames per second, and 2 min scan time. Fat color labeling and 3D reconstruction were performed using the system's built-in image analysis software, and the volumes of vWAT and sWAT were evaluated separately.

[0116] H&E staining

[0117] H&E staining was performed to study the morphology of subcutaneous adipose tissue in different groups and to assess the area and number of lipid droplets (Wang et al., 2021). After staining, images of each section were acquired using an M8 microscope and scanner (Precision Point, Thuringia, Germany), and the fat area and number were analyzed using Image Pro Plus 6.0 software (Media Cybemetics, INC., Rockville, MD, United States).

[0118] Data Analysis

[0119] SPSS 26.0 software was used for statistical analysis, and univariate statistical tests were employed to compare differences between samples from different groups. For normally distributed data, ANOVA (multiple-group test) and Student's t-test (two-group test) were used for analysis; for non-normally distributed data, Kruskal-Wallis H-test (multiple-group test) and Mann-Whitney U-test (two-group test) were used for analysis.

[0120] Experimental results

[0121] OVX-LET weight comparison

[0122] As attached Figure 6 The results showed that after 8 weeks of GNS intervention, the body weight in the OVX group was significantly higher than that in the Sham group (P<0.05). Compared with the Sham group, the body weight in the LET group and the LET+GNS group was also significantly higher (P<0.05). This suggests that after LET inhibits the increase of endogenous estradiol, GNS cannot play a role in reducing body weight.

[0123] Aromatase levels and E2 levels

[0124] As attached Figure 7 As shown in Figure A, after 8 weeks of GNS intervention, compared with the Sham group, the aromatase level in the OVX group was significantly increased (P<0.05); compared with the OVX group, the aromatase level in the LET group was significantly decreased (P<0.05). The LET+GNS group significantly decreased the aromatase level (P<0.05).

[0125] As attached Figure 7As shown in Figure B, after 8 weeks of GNS intervention, compared with the Sham group, the E2 level in the OVX group was significantly lower (P<0.05); compared with the OVX group, the E2 level in the LET group was further significantly lower (P<0.05); compared with the OVX group, the E2 level in the LET+GNS group was also significantly lower, significantly higher than that in the LET group, but still lower than that in the Sham group, and the differences were all statistically significant (P<0.05).

[0126] OVX-LET Comparison of Visceral Fat and Subcutaneous Fat Volume

[0127] As attached Figure 8 As shown in Figures A and B, after 8 weeks of GNS intervention, the OVX group showed a significant increase in both vWAT and sWAT, with statistically significant differences (P<0.05). Compared with the Sham group, the LET group showed an increase in both vWAT and sWAT volume, with statistically significant differences (P<0.05). Compared with the OVX group, the LET group showed a significant increase in sWAT volume (P<0.05). Compared with the Sham group, the LET+GNS group showed a significant increase in both vWAT and sWAT volume (P<0.05), while there was no significant difference compared with the OVX group (P>0.05).

[0128] OVX-LET blood lipid levels comparison

[0129] As attached Figure 9 As shown in Figures A and B, after 8 weeks of GNS intervention, compared with the Sham group, serum TC levels in the OVX group, LET group, and LET+GNS group were significantly increased (P<0.05). Compared with the Sham group, serum TG levels in the OVX group and LET group were significantly decreased (P<0.05), while no significant change was observed in TG levels in the LET+GNS group (P>0.05). Compared with the OVX group, serum TG levels in the LET group were significantly decreased (P<0.05).

[0130] OVX-LET visceral adipose tissue H&E section analysis

[0131] As attached Figure 10 As shown in the AC results, after 8 weeks of GNS intervention, compared with the Sham group, the OVX group, LET group, and LET+GNS group all showed an increase in adipocyte volume in the visual field, and the differences were statistically significant (P<0.05). Compared with the Sham group, the OVX group, LET group, and LET+GNS group all reduced the number of adipocytes in the visual field, and the differences were statistically significant (P<0.05).

[0132] In summary, the pharmaceutical composition of this invention can improve body weight in a perimenopausal overweight / obesity model, increase aromatase and E2 levels, inhibit the increase of visceral and subcutaneous fat, and simultaneously inhibit intrafacial TC and TG levels. However, after the inhibition of aromatase activity leads to a further decrease in estrogen levels, the intervention effect of the pharmaceutical composition of this invention on perimenopausal overweight / obesity disappears. This suggests that the pharmaceutical composition of this invention specifically inhibits perimenopausal overweight / obesity by exerting a phytoestrogen-like effect through aromatase activity, providing a treatment option for perimenopausal overweight / obesity.

[0133] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A pharmaceutical composition for treating perimenopausal overweight / obesity, characterized in that, The active pharmaceutical ingredients of the pharmaceutical composition are: Anemarrhena asphodeloides 5-30 parts by weight, Phellodendron chinense 5-30 parts by weight, Rehmannia glutinosa 5-30 parts by weight, Astragalus membranaceus 5-30 parts by weight, Angelica sinensis 5-30 parts by weight, Epimedium brevicornu 5-30 parts by weight, calcined dragon bone 5-30 parts by weight, calcined oyster shell 5-30 parts by weight, and stir-fried Ziziphus jujuba var. spinosa 5-30 parts by weight.

2. The pharmaceutical composition for treating perimenopausal overweight / obesity according to claim 1, characterized in that, The active pharmaceutical ingredients of the pharmaceutical composition are: 8 parts by weight of Anemarrhena asphodeloides, 8 parts by weight of Phellodendron chinense, 14 parts by weight of Rehmannia glutinosa, 14 parts by weight of Astragalus membranaceus, 8 parts by weight of Angelica sinensis, 8 parts by weight of Epimedium brevicornu, 14 parts by weight of calcined dragon bone, 14 parts by weight of calcined oyster shell, and 14 parts by weight of stir-fried Ziziphus jujuba var. spinosa.

3. Use of the pharmaceutical composition of claim 1 or 2 in the preparation of a medicament for treating perimenopausal overweight / obesity.

4. A pharmaceutical preparation for treating perimenopausal overweight / obesity, characterized in that, Prepared from the pharmaceutical composition according to claim 1 or 2.

5. The pharmaceutical preparation for treating perimenopausal overweight / obesity according to claim 4, characterized in that, The pharmaceutical preparation is selected from at least one of the following: tablets, capsules, powders, mixtures, pills, granules, solutions, syrups, decoctions, plasters, suppositories, aerosols, and ointments.

6. The pharmaceutical preparation for treating perimenopausal overweight / obesity according to claim 4 or 5, characterized in that, The pharmaceutical formulation also includes clinically acceptable excipients selected from at least one of fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, or matrices.

7. The pharmaceutical preparation for treating perimenopausal overweight / obesity according to claim 6, characterized in that, The filler is selected from at least one of starch, pregelatinized starch, lactose, mannitol, chitin, or microcrystalline cellulose; The disintegrant is selected from at least one of starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, or croscarmellose sodium. The lubricant is selected from at least one of magnesium stearate, sodium dodecyl sulfate, talc, or silica. The suspending agent is selected from at least one of polyvinylpyrrolidone, microcrystalline cellulose, agar or hydroxypropyl methylcellulose; The adhesive is selected from at least one of starch paste, polyvinylpyrrolidone, or hydroxypropyl methylcellulose; The sweetener is selected from at least one of sodium saccharin, aspartame, sucrose, cyclamate, or glycyrrhetinic acid; The flavoring agent is selected from flavorings; The preservative is selected from at least one of parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorethidium acetate, or eucalyptus oil; The matrix is ​​selected from at least one of PEG6000, PEG4000 or insect wax.

8. A method for preparing a pharmaceutical formulation for treating perimenopausal overweight / obesity as described in any one of claims 1-7, characterized in that, The process includes the step of mixing selected amounts of the following herbs: Anemarrhena asphodeloides, Phellodendron chinense, Rehmannia glutinosa, Astragalus membranaceus, Angelica sinensis, Epimedium brevicornu, calcined dragon bone, calcined oyster shell, and stir-fried Ziziphus jujuba var. spinosa.

9. The method for preparing the pharmaceutical formulation for treating perimenopausal overweight / obesity according to claim 8, characterized in that, include: Take a selected amount of Anemarrhena asphodeloides, Phellodendron chinense, Rehmannia glutinosa, Astragalus membranaceus, Angelica sinensis, Epimedium brevicornu, calcined dragon bone, calcined oyster shell and stir-fried Ziziphus jujuba seeds, mix them together, add water and decoct, collect the decoction and concentrate it into an extract, and add conventional excipients to process the selected dosage form.

10. The method for preparing the pharmaceutical preparation for treating perimenopausal overweight / obesity according to claim 9, characterized in that: The amount of water added is 5-15 times the total weight of the raw material; and / or, The decoction process involves decocting 1-3 times; and / or, The decocting step takes 1-3 hours; and / or, The concentration step involves concentrating the decoction to a thick paste with a relative density of 1.25 at 60°C.

11. Use of the pharmaceutical composition of claim 1 or 2, the pharmaceutical preparation of claims 4-7, or the pharmaceutical preparation prepared by any one of the preparation methods of claims 8-10 for the preparation of a medicament for improving perimenopausal overweight / obesity.

12. The use according to claim 11, characterized in that, The use of the medication for improving perimenopausal overweight / obesity includes improving TC and / or TG levels.

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