A multifunctional traditional Chinese medicine composition, oral preparation, its preparation method and application

Abstract

This invention discloses a multi-functional traditional Chinese medicine composition, an oral preparation, its preparation method, and its applications. The traditional Chinese medicine composition comprises the following components in parts by weight: Epimedium 2-8 parts, dried ginger 2-8 parts, licorice 2-8 parts, ginseng 1-4 parts, Poria cocos 1-4 parts, and Salvia miltiorrhiza 1-4 parts. This traditional Chinese medicine composition has multiple effects, simultaneously preventing and treating impotence, myocardial infarction, estrogen deficiency (typical estrogen deficiency such as postmenopause) related diseases (including osteoporosis, obesity, and uterine atrophy), and anti-fatigue effects, with significant efficacy and a very high degree of applicability. Moreover, the composition is simple, with all components being medicinal and edible herbs, ensuring high safety. It can be extracted using conventional water decoction methods to produce extracts, which can then be formulated into various oral preparations, including drugs, health foods (functional foods), or other food products, possessing significant market value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a multifunctional traditional Chinese medicine composition, oral preparation, preparation method and application thereof. Specifically, it is a traditional Chinese medicine composition for the prevention and treatment of impotence, myocardial infarction, postmenopausal osteoporosis and obesity, as well as anti-fatigue, and its preparation method and application thereof. Background Technology

[0002] As people age and enter middle and old age, their health gradually declines, and the incidence of various diseases increases significantly. One of the most significant physiological changes in the human body during this period is the substantial shift in hormone levels, which leads to a series of problems.

[0003] Erectile dysfunction (ED) is a common condition in men over 40 years of age, and its prevalence increases with age and other comorbidities (such as diabetes and cardiovascular disease). Oral phosphodiesterase-5 inhibitors (PDE5Is) are commonly used to treat ED; for patients who do not respond well to conventional PDE5Is, combination therapy with androgens, anti-oxidative stress agents, and / or drugs that improve microcirculation can be used. my country has approved four selective PDE5Is: sildenafil, tadalafil, vardenafil, and avanafil. However, the use of PDE5 inhibitors carries cardiovascular safety risks, therefore, these drugs have usage restrictions. These restrictions include: contraindications for patients who have had myocardial infarction, stroke, or life-threatening arrhythmia within the past 6 months; patients with hypotension (< 90 / 50 mmHg) or hypertension (> 170 / 100 mmHg); patients with unstable angina or congestive heart failure; and concomitant use with nitrates or alpha-blockers. Adverse reactions to PDE5 inhibitors also include visual disturbances, myalgia, and back pain (Chinese Medical Association Andrology Branch, Guidelines for the Diagnosis and Treatment of Erectile Dysfunction Editorial Group. Guidelines for the Diagnosis and Treatment of Erectile Dysfunction. Chinese Journal of Andrology. 2022; 28:722-755.). In addition, regarding traditional Chinese medicine, ED is called "impotence" in TCM, and available TCM formulas include: Jin Kui Shen Qi Wan, You Gui Wan, Tian Wang Bu Xin Dan, and Si Miao Wan, etc. (China Association of Integrative Medicine and Andrology Professional Committee. Guidelines for the Diagnosis and Treatment of Erectile Dysfunction with Integrative Traditional Chinese and Western Medicine (Trial Version). Chinese Journal of Andrology. 2016; 22:751–757.). However, the variety of available formulas is still limited.

[0004] Myocardial infarction is generally caused by coronary artery blockage, leading to insufficient blood supply to the myocardium and irreversible damage and necrosis. It is the most fatal cardiovascular disease. Metoprolol tartrate is a beta-blocker. Although metoprolol tartrate can cause adverse reactions such as abdominal pain, nausea, vomiting, diarrhea, and constipation, it is still widely used clinically for the prevention and treatment of ischemic myocardial infarction due to the lack of safe and effective causal treatment drugs. In terms of traditional Chinese medicine, Compound Danshen Dripping Pills are a traditional Chinese medicine preparation mainly used clinically to treat acute angina pectoris and prevent acute myocardial infarction, but similar or better traditional Chinese medicine varieties still cannot meet social needs. Developing safe and effective traditional Chinese medicines for the prevention and treatment of myocardial infarction has important medical and health value.

[0005] After menopause, with the decline of ovarian function, estrogen levels drop significantly, and estrogen deficiency can cause a series of health problems. Estrogen deficiency is a major factor contributing to osteoporosis, obesity, and uterine atrophy in older women. Moreover, estrogen deficiency and the resulting overweight and obesity are also major risk factors for serious diseases such as hypertension, diabetes, and cardiovascular disease, significantly increasing the risk of death in the global population and becoming a challenging problem in the global public health field.

[0006] Osteoporosis is a common and frequently occurring disease among middle-aged and elderly people, especially postmenopausal women. It is a systemic metabolic skeletal disease characterized by decreased bone mass and deterioration of bone microstructure, leading to increased bone fragility and susceptibility to fractures. Postmenopausal osteoporosis generally occurs 5 to 10 years after menopause in women. The prevalence of osteoporosis in women aged 50-59 is approximately 20.38%. The prevalence of osteoporosis in women over 65 years of age is as high as 51.6% (Chinese Society of Osteoporosis and Bone Mineral Diseases. Results of Epidemiological Survey of Osteoporosis in China and the "Healthy Bone" Special Action. Chinese Journal of Osteoporosis and Bone Mineral Diseases. 2019; 12:317-318). Bone remodeling is the main process for maintaining normal bone metabolism in the body. Osteoclasts are terminally differentiated cells that perform bone resorption in the body and are also key targets of first-line anti-osteoporosis drugs in clinical practice. Overactivation of osteoclasts disrupts the balance between bone resorption and bone formation, leading to degenerative changes such as decreased bone mass, thinning of cortical bone, and reduced trabecular bone. Anti-osteoporosis drugs can be classified by mechanism of action into bone resorption inhibitors, bone formation promoters, drugs with other mechanisms of action, and traditional Chinese medicine. Drugs with a broad spectrum of fracture resistance are usually the first choice (such as alendronate sodium, zoledronic acid, risedronate sodium, and dinosema). Oral medications are preferred for patients with low to moderate fracture risk (such as young postmenopausal women with low bone mineral density but no history of fracture). Injectable formulations (such as zoledronic acid, teriparatide, or dinosema) may be considered for patients with intolerance, contraindications, poor adherence, or high fracture risk (such as elderly patients with multiple vertebral fractures or hip fractures, or patients with extremely low bone mineral density). For patients with only high risk of vertebral fractures, estrogen or selective hormone receptor modulators may be considered. Short-term use of calcitonin may be considered for patients with new-onset fracture pain. Clinically, drugs used to reduce or inhibit bone resorption mainly include bisphosphonates and nuclear factor κB receptor activator ligand inhibitors. However, long-term use of chemically synthesized bisphosphonates can cause varying degrees of adverse reactions such as osteonecrosis of the mandible, bone and joint pain, muscle pain, and cellulitis. Traditional Chinese medicine considers osteoporosis to fall under the categories of "bone weakness" and "bone pain," with kidney deficiency as the main pathogenesis and spleen deficiency as a contributing factor. Developing safe and effective drugs or health foods derived from traditional Chinese medicine to maintain bone remodeling balance is of great significance. To date, the China Food and Drug Administration (CFDA) has approved only three main traditional Chinese medicine preparations for treating osteoporosis: total flavonoids from *Drynaria fortunei*, icariin preparations, and artificial tiger bone powder preparations (Guidelines for the Diagnosis and Treatment of Primary Osteoporosis (2022). Chinese Journal of Osteoporosis and Bone Mineral Diseases, 2022, 15:573-611).

[0007] Currently, research on obesity and overweight in postmenopausal women is extremely limited. Existing medications for improving or treating obesity and overweight in postmenopausal women often suffer from unsatisfactory long-term efficacy or various side effects. For example, sibutramine and rimonaban carry cardiovascular and psychological risks, respectively; orlistat is prone to causing gastrointestinal discomfort such as oily stools and fecal incontinence; the newly marketed semaglutide has been associated with frequent nausea and vomiting; and hormone replacement therapy with estradiol valerate, in addition to gastrointestinal discomfort and endometrial hyperplasia, may increase the risk of breast cancer, endometrial cancer, and ovarian cancer with long-term use. There is a significant market demand for the development of drugs and functional foods beneficial for weight management in postmenopausal women. Summary of the Invention

[0008] The purpose of this invention is to address the sub-health and various diseases of middle-aged and elderly people by providing a traditional Chinese medicine composition and pharmaceutical preparation that has multiple effects such as preventing and treating impotence, myocardial infarction, estrogen deficiency (typical estrogen deficiency such as postmenopause) related diseases (including osteoporosis, obesity and uterine atrophy), and anti-fatigue.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] Through extensive and long-term in-depth systematic research, our team has developed a traditional Chinese medicine composition consisting of Epimedium, dried ginger, licorice, ginseng, Poria cocos, and Salvia miltiorrhiza. This composition has multiple effects, including the prevention and treatment of impotence, myocardial infarction, estrogen deficiency (typical estrogen deficiency such as postmenopause) related diseases (including osteoporosis, obesity, and uterine atrophy), as well as anti-fatigue effects.

[0011] Specifically, the traditional Chinese medicine composition provided by the present invention is composed of the following components in parts by weight: 2-8 parts of Epimedium, 2-8 parts of dried ginger, 2-8 parts of licorice, 1-4 parts of ginseng, 1-4 parts of Poria cocos, and 1-4 parts of Salvia miltiorrhiza.

[0012] Preferably, the traditional Chinese medicine composition comprises the following components in parts by weight: 6 parts Epimedium, 3 parts dried ginger, 3 parts licorice, 2 parts ginseng, 2 parts Poria cocos, and 2 parts Salvia miltiorrhiza.

[0013] The above-mentioned traditional Chinese medicine composition can be prepared into traditional Chinese medicine extracts by conventional water decoction extraction methods, and then into various pharmaceutical preparations, such as oral preparations.

[0014] As an alternative implementation, the conventional water decoction extraction method includes the following steps:

[0015] S1. Water extraction: Take the raw material and decoct it with water 3-4 times; each time add 8-20 times the weight of the raw material in water, decoct for 2-3 hours, cool, filter, and combine the filtrates;

[0016] S2. Concentration: Centrifuge the filtrate, take the supernatant and concentrate it under reduced pressure to obtain a concentrated aqueous extract, which is the Chinese herbal extract.

[0017] Based on this, the Chinese herbal extracts (such as the concentrated liquid prepared above) can be added to excipients to make liquid drug preparations such as oral liquids; or the concentrated liquid can be vacuum dried and excipients added to make solid dosage forms such as granules, solid beverages, and tablets.

[0018] Specifically, as an implementation example, based on the above results, the present invention provides a traditional Chinese medicine granule preparation (i.e., Zhenyuan granules), which is obtained by drying, pulverizing and granulating the above-mentioned traditional Chinese medicine extract.

[0019] Preferably, the method for preparing the vibration element particles is as follows:

[0020] Take the raw materials of the Chinese herbal medicine composition, decoct them three times with water, adding 15 times the amount of water each time, decoct for 2 hours, cool, filter, and combine the filtrates; centrifuge, take the supernatant, concentrate under reduced pressure to a thick paste, dry the thick paste under vacuum at 80℃ to a water content of 5% or less, take it out, cool to room temperature, pulverize, make fine powder, granulate, package, and obtain Zhenyuan granules.

[0021] Furthermore, the present invention also provides a method for establishing the fingerprint spectrum of the oral Chinese medicine preparation (Zhenyuan Granules) and a quality testing method, providing methods and standards for the quality control of pharmaceutical preparations.

[0022] The method for establishing the fingerprint spectrum of the oral Chinese medicine preparation (Zhenyuan Granules) includes the following steps:

[0023] S1. Preparation of mixed reference solution: methanol solution of icariin, icariin A, icariin B, icariin C, glycyrrhizin, ammonium glycyrrhizate, quercetin, and salvianolic acid C;

[0024] S2. Preparation of test solution: Add the oral Chinese medicine preparation to 50%–80% ethanol at a ratio of (0.1–2) g: 100 mL, sonicate (480 W, 40 kHz, 1 h), centrifuge (11000 r / min, 10 min), and take the supernatant, which is the test solution.

[0025] S3. Perform HPLC chromatographic analysis: Analyze the test solution using HPLC, with a mixed reference solution as a control. Both the test solution and the mixed reference solution were filtered through a 0.22 µm microporous membrane before use. The injection volume was 10 µL. The HPLC chromatographic conditions are as follows:

[0026] The chromatographic column was a Kromasil 100-5-C18 (250 mm × 4.6 mm, 5 µm) column.

[0027] Mobile phase A is acetonitrile, and mobile phase B is a 0.1% formic acid aqueous solution.

[0028] The detection wavelength was 254 nm, the column temperature was 30℃, and the flow rate was 1 mL / min.

[0029] The mobile phase elution gradient is as follows:

[0030]

[0031] The elution process continues until completion, thus obtaining the fingerprint chromatogram of the oral Chinese medicine preparation. Specifically, the chromatogram of the test sample is compared with the chromatogram of the mixed reference standard, and specific peaks are identified to obtain the fingerprint chromatogram of the oral Chinese medicine preparation.

[0032] In the S1 mixed reference solution, the concentrations of each reference standard can be set as follows: 0.10 mg / mL for icariin, cuscutaidine A, cuscutaidine B, and cuscutaidine C; 0.75 mg / mL for ammonium glycyrrhizate; and 0.04 mg / mL for glycyrrhizin, quercetin, and salvianolic acid C.

[0033] In the preparation of the S2 test solution, the oral Chinese medicine preparation can be added to 50%–80% ethanol (e.g., 50% ethanol) at a ratio of 1 g: 100 mL.

[0034] The HPLC fingerprint spectrum for identifying components of traditional Chinese medicine established using the above method can identify at least eight components, including icariin, using the corresponding reference standards.

[0035] Based on this, a quality testing method for the oral Chinese medicine preparation is provided, characterized by the following steps: comparing the HPLC chromatogram of the sample to be tested with the fingerprint chromatogram established above, and determining whether the sample is qualified based on the retention time, peak area ratio, and peak order of the HPLC chromatogram of the sample to be tested and the Chinese medicine reference standard; wherein, the method for obtaining the HPLC chromatogram of the sample to be tested is the same as the method for establishing the fingerprint chromatogram of the oral Chinese medicine preparation (Zhenyuan granules) mentioned above.

[0036] Furthermore, the application of the above-mentioned traditional Chinese medicine composition and its extracts in the preparation of products for preventing and treating impotence and fatigue, or in the preparation of products for preventing and treating estrogen deficiency-related diseases, or in the preparation of products for preventing and treating myocardial infarction, or in the preparation of products that simultaneously have multiple effects such as preventing and treating impotence, fatigue, myocardial infarction, and estrogen deficiency-related diseases, should all be within the scope of protection of this invention.

[0037] Among them, the estrogen deficiency-related diseases include osteoporosis, obesity, and uterine atrophy caused by estrogen deficiency (typical estrogen deficiency such as postmenopause).

[0038] The components of the traditional Chinese medicine composition provided by this invention are all medicinal and edible herbs, which are highly safe. Therefore, in practice, the products prepared can be either medicines or health foods (functional foods).

[0039] Product dosage forms can include granules, solid beverages, powders, capsules, tablets, lozenges, suspensions, emulsions, solutions, tinctures, and other dosage forms.

[0040] In the above-mentioned traditional Chinese medicine composition, Epimedium warms and tonifies kidney yang; red ginseng, Poria, and licorice, in accordance with the principles of Sijunzi Decoction, invigorate qi and strengthen the spleen; and Salvia miltiorrhiza invigorates blood and unblocks the meridians, promoting the smooth flow of blood and qi. The above-mentioned traditional Chinese medicine composition has the functions of tonifying the kidney and strengthening the spleen, and invigorating vital energy. The traditional Chinese medicine granule preparation made from the extract of the above composition is called Zhenyuan Granules (ZYKL).

[0041] This invention investigated the effects of castration (removal of the testes) in male rats, which significantly decreased serum testosterone levels, serum NOS activity, NO, and cGMP levels, prolonged penile erection latency and capture latency in female rats, and reduced capture frequency. This study found that ZYKL granules significantly increased serum testosterone levels in castrated rats; increased serum NOS activity; increased serum NO and cGMP levels; shortened penile erection latency and capture latency in female rats; and increased capture frequency. NO is a major mediator of penile erection. After entering smooth muscle cells, it binds to and activates soluble guanylate cyclase, which catalyzes the conversion of GTP to cGMP. cGMP induces vasodilation of the corpus cavernosum smooth muscle and blood vessels, initiating penile erection (Gao Ke, et al. Research progress on the role and regulatory mechanism of nitric oxide synthase in penile erection. Chinese Journal of Andrology. 2018; 32:70-72). Therefore, ZYKL can enhance male virility by activating the NO-cGMP signaling pathway.

[0042] The weight-bearing swimming time of rats can directly reflect exercise endurance; lactic acid and urea nitrogen accumulate after exercise, and their levels can reflect the degree of fatigue. This study found that, compared with the sham-operated control group, castrated rats did not show a significant difference in weight-bearing swimming time, but the whole blood lactate surface area and serum urea nitrogen level were significantly increased after exercise, indicating that castrated rats were more prone to fatigue; while ZYKL can reduce the levels of lactic acid and urea nitrogen in castrated rats after exercise, indicating that it has a fatigue-relieving effect.

[0043] The experimental results of this invention provide a pharmacodynamic basis for developing Zhenyuan granules into a drug or health food (functional food) for tonifying the kidney and strengthening yang and relieving fatigue. It also provides pharmacological experimental evidence for using Zhenyuan granules to manufacture new traditional Chinese medicines for tonifying the kidney and strengthening yang and relieving fatigue, or to manufacture drugs or health foods (functional foods) that are beneficial to adults with kidney yang deficiency and easy fatigue.

[0044] The following is a description of the relevant pharmacodynamic functions of this invention: The traditional Chinese medicine granule preparation made from the traditional Chinese medicine composition of this invention has the effect of preventing and treating erectile dysfunction in adult men caused by kidney yang deficiency, and improving the fatigue of adult men caused by kidney yang deficiency; the medicine and health food (functional food) made from the traditional Chinese medicine composition of this invention are beneficial for the prevention or adjunctive treatment of symptoms of erectile dysfunction in adult men caused by kidney yang deficiency, and beneficial for the prevention or adjunctive treatment of fatigue in adult men or patients caused by kidney yang deficiency.

[0045] Furthermore, the term "impotence" refers to the phenomenon or symptom of erectile dysfunction in men as described in modern medicine, characterized by difficulty or weakness in achieving or maintaining an erection.

[0046] Furthermore, the impotence described is male erectile dysfunction in modern medicine, characterized by prolonged penile erection latency and capture latency of female mice in impotence model experimental animals, and a reduced number of captures.

[0047] Furthermore, the therapeutic effect on erectile dysfunction is characterized by improving the serum NO and cGMP levels in the serum of experimental animals with erectile dysfunction models.

[0048] Furthermore, the anti-fatigue effect is to improve the abnormally elevated lactic acid and serum urea nitrogen levels in the impotence model experimental animals and alleviate physical fatigue.

[0049] In an experiment investigating the preventive effect (pre-intervention) of ZYKL granules on isoproterenol-induced myocardial infarction in rats, the inventors observed a significant decrease in ejection fraction and shortening fraction on echocardiography, indicating that ZYKL can alleviate the damage to cardiac function caused by isoproterenol. This invention also examined cardiomyocytes in rats with isoproterenol-induced myocardial infarction using HE staining, showing that ZYKL can alleviate isoproterenol-induced ischemic myocardial injury. Furthermore, this invention detected serum levels of cardiac enzymes CK and CK-MB, finding that ZYKL can downregulate these levels, indicating that ZYKL has a protective effect against isoproterenol-induced myocardial damage. Finally, this invention's electrocardiographic observation revealed that ZYKL can reduce ST segment elevation, alleviating ischemic injury. This study, by detecting the inflammatory factors TNF-α, IL-1β, and IL-6 in rat myocardium, found that isoproterenol significantly increased the levels of TNF-α, IL-1β, and IL-6 in the myocardium of rats with myocardial infarction; while ZYKL downregulated the levels of TNF-α, IL-1β, and IL-6 in the myocardium, indicating that ZYKL can alleviate isoproterenol-induced myocardial inflammation. Since myocardial damage in ischemic myocardial infarction is related to oxidative stress, by detecting the levels of myocardial antioxidants SOD and GSH, and lipid oxidation products TBARS and LOOH, it was found that: isoproterenol significantly decreased the levels of SOD and GSH in the myocardium of rats with myocardial infarction, while ZYKL upregulated the levels of SOD and GSH in the myocardium; isoproterenol significantly increased the levels of TBARS and LOOH in the myocardium of rats with myocardial infarction, while ZYKL downregulated the levels of TBARS and LOOH in the myocardium, indicating that ZYKL can alleviate isoproterenol-induced ischemic myocardial infarction by relieving oxidative stress. This invention also conducted a post-intervention experiment on the therapeutic effect of ZYKL on isoproterenol-induced myocardial infarction in rats. The experimental results showed that the treatment (post-intervention) with ZYKL can also reduce the damage to cardiac function caused by isoproterenol and has a protective effect against myocardial damage caused by isoproterenol. It can also reduce isoproterenol-induced ischemic myocardial infarction by alleviating oxidative stress.

[0050] The relevant pharmacodynamic data of this invention are as follows: The traditional Chinese medicine granule preparation made from the traditional Chinese medicine composition of this invention has the effect of preventing and treating ischemic myocardial infarction; the drugs and health foods (functional foods) made from the traditional Chinese medicine composition of this invention are beneficial to the prevention or adjuvant treatment of ischemic myocardial infarction patients, and can relieve or improve symptoms.

[0051] Furthermore, the myocardial infarction is characterized by pathological changes of ischemic myocardial injury in model animals or patients.

[0052] Furthermore, the myocardial infarction is manifested as P-wave inversion on the electrocardiogram in the model animal or patient.

[0053] Furthermore, the myocardial infarction is characterized by abnormalities in ejection fraction and shortening fraction in the model animals or patients.

[0054] Furthermore, the myocardial infarction is characterized by a significant and abnormally high level of serum CK and CK-MB in the model animals or patients.

[0055] Furthermore, the myocardial infarction is characterized by an abnormally significant increase in the levels of myocardial inflammatory factors TNF-α, IL-1β, and IL-6 in the model animals or patients.

[0056] Furthermore, the myocardial infarction is characterized by an abnormally significant decrease in the levels of myocardial antioxidant stress protective factors SOD and GSH in model animals or patients.

[0057] Furthermore, the myocardial infarction was characterized by a significant and abnormal increase in the levels of TBARS and LOOH, markers of myocardial oxidative stress injury, in the model animals or patients.

[0058] Furthermore, the effect of the Zhenyuan granules in preventing and treating ischemic myocardial infarction is that they can alleviate oxidative stress and thus reduce ischemic myocardial infarction.

[0059] This invention uses a bilateral ovariectomized C57BL / 6J female mouse model of estrogen deficiency as the research subject to simulate estrogen-induced osteoporosis. It was found that ZYKL particles can improve bone tissue parameters such as bone mineral density and bone volume fraction, improve the trabecular microstructure of bone in estrogen-deficient osteoporotic mice, alleviate bone loss and decreased bone mass, and inhibit osteoclast formation to improve bone reduction and bone loss. This invention also found that ZYKL can inhibit bone resorption-related genes. Car2 , Ctsk , MMP9 and TRAF6 The expression of mRNA and the inhibition of bone resorption-related proteins Trap, CTSK, and c-fos significantly inhibits osteoclast formation and differentiation, thus suppressing bone resorption. Simultaneously, it upregulates the expression levels of serum antioxidant markers SOD and GPX, downregulates the expression levels of oxidative stress markers MDA and ALP, activates the erythrofollicle nuclear factor-2 (Nrf2) / heme oxygenase-1 (HO-1) pathway, promotes the expression of Nrf2, HO-1, and NQO1 proteins, reduces oxidative damage to bone tissue, and decreases bone loss. Zhenyuan granules can significantly inhibit the expression of osteoclast-specific proteins and genes, suppressing bone resorption, providing a new, safe, and highly effective source of pharmacologically active substances for the prevention and treatment of osteoporosis, especially osteoporosis caused by estrogen deficiency.

[0060] The efficacy data of this invention are as follows: The traditional Chinese medicine granules prepared from the herbal composition of this invention have the effect of preventing and treating osteoporosis caused by estrogen deficiency; the drugs and health foods (functional foods) prepared from the herbal composition of this invention are beneficial for improving and adjuvantly treating osteoporosis caused by estrogen deficiency. Moreover, compared with the prior formulation CN113491757B, it has better efficacy and reduces the intake of four herbs: Polygonum cuspidatum, processed Aconitum carmichaelii, Phellodendron chinense, and Scutellaria baicalensis, making the formulation simpler and safer.

[0061] Furthermore, the osteoporosis caused by estrogen deficiency is postmenopausal osteoporosis.

[0062] Furthermore, the prevention and treatment of osteoporosis involves inhibiting the differentiation and formation of osteoclasts within bone tissue.

[0063] Furthermore, the prevention and treatment of osteoporosis involves increasing bone density, bone volume fraction, and the number of trabeculae.

[0064] Furthermore, the prevention and treatment of osteoporosis involves inhibiting bone resorption, specifically by inhibiting genes related to bone resorption. Car2 , Ctsk , MMP9 and TRAF6 The expression of mRNA and bone resorption-related proteins Trap, CTSK and c-fos.

[0065] This invention investigates the effects of Zhenyuan granules on adipogenic differentiation of preadipocytes using a 3T3-L1 mouse preadipocyte model. A bilateral ovariectomized estrogen-deficient mouse model was also used to evaluate the effects of Zhenyuan granules on weight gain and fat accumulation induced by estrogen deficiency. This study found that Zhenyuan granules significantly inhibited adipocyte formation and key genes involved in adipogenesis during the induction of 3T3-L1 preadipocyte differentiation into adipocytes. PPARγ , C / EBPα , SREBP-1c and ACC1The expression of proteins PPARγ, SREBP-1c, FAS and ACC1 was significantly upregulated, as were the levels of p-AMPK / AMPK and p-ACC1 / ACC1. Compared with the sham-operated control group, the model control group mice showed significantly increased body weight, gonadal fat index (fat weight / body weight), and inguinal fat index, significantly enlarged adipocytes, significantly elevated serum total cholesterol and leptin levels, significantly atrophied uterus, significantly increased expression levels of SREBP-1c, PPARγ, FAS, and ACC1 proteins, and significantly decreased p-AMPK / AMPK and p-ACC1 / ACC1 levels. In contrast, Zhenyuan granules inhibited weight gain and fat accumulation in estrogen-deficient mice, improved uterine atrophy, reduced serum total cholesterol and leptin levels, improved adipocyte dysfunction and cholesterol metabolism abnormalities caused by estrogen deficiency, significantly upregulated p-AMPK / AMPK and p-ACC1 / ACC1 levels, and downregulated the expression levels of SREBP-1c, PPARγ, FAS, and ACC1. Not only at the cellular level, but also at the animal level, Zhenyuan granules significantly inhibited adipogenesis by activating AMPK / ACC signaling.

[0066] The following is a description of the relevant pharmacodynamic data of this invention: The traditional Chinese medicine granule preparation made from the traditional Chinese medicine composition of this invention has the effect of preventing and treating obesity (including obesity and overweight) caused by estrogen deficiency; the medicine and health food (functional food) made from the traditional Chinese medicine composition of this invention are beneficial for weight control and adjuvant treatment of people with obesity (including obesity and overweight) caused by estrogen deficiency.

[0067] Furthermore, the estrogen deficiency-induced obesity includes overweight and obesity.

[0068] Furthermore, the overweight refers to weight gain caused by estrogen deficiency, reaching the overweight standard (BMI=27) or above.

[0069] Furthermore, the obesity is estrogen deficiency-induced obesity, reaching the obesity standard (BMI=28) or above.

[0070] Furthermore, the stated effect is to reduce the adipogenic differentiation rate of preadipocytes and inhibit the formation of adipocytes.

[0071] Furthermore, the reduction of preadipocyte adipogenic differentiation rate and inhibition of adipocyte formation is characterized by significant inhibition of key genes for adipogenesis (…). PPARγ , C / EBPα , SREBP-1c , ACC1 The expression of ) and proteins (PPARγ, SREBP-1c, FAS, ACC1) was significantly upregulated, as were the levels of p-AMPK / AMPK and p-ACC1 / ACC1.

[0072] Furthermore, the efficacy is to slow down the weight gain trend in overweight / obese animals caused by estrogen deficiency, characterized by a significant reduction in the weight of gonadal fat and groin fat in overweight / obese animals, and a significant reduction in the average diameter and average cross-sectional area of ​​adipocytes, with effects similar to those of the positive control drugs orlistat and estradiol valerate.

[0073] Furthermore, the method for mitigating overweight and obesity in ovariectomized mice is characterized by significantly inhibiting the expression of lipogenesis-related proteins PPARγ, SREBP-1c, FAS, and ACC1 in mouse adipose tissue, and significantly upregulating the levels of p-AMPK / AMPK and p-ACC1 / ACC1.

[0074] Furthermore, the efficacy is to improve and protect the uterus of estrogen-deficient mice and slow its atrophy.

[0075] The present invention has the following beneficial effects:

[0076] This invention provides a multi-effect traditional Chinese medicine composition that simultaneously prevents and treats impotence, myocardial infarction, estrogen deficiency (typical estrogen deficiency such as postmenopause) related diseases (including osteoporosis, obesity and uterine atrophy), and has anti-fatigue effects. It has significant effects and has a very high advantage in wide application.

[0077] Moreover, the formula has a simple composition, and all components are medicinal and edible materials, making it highly safe. It is a new, safe, and effective drug that can be used as a medicine or a health food (functional food). Attached Figure Description

[0078] Figure 1 HPLC chromatograms of the mixed reference standard (top) and Zhenyuan granules test sample (bottom). 1. Glycyrrhizin; 2. Quercetin; 3. Asarumin A; 4. Asarumin B; 5. Asarumin C; 6. Icariin; 7. Tanshinone C; 8. Ammonium glycyrrhizate. Horizontal axis: Time (min).

[0079] Figure 2 Effect of Zhenyuan granules on erectile latency in rats. Compared with the sham-operated control group (Sham), ** P <0.01; compared with the orchiectomy model control group, ## P <0.01. TP, testosterone propionate; ZYKL, Zhenyuan granules.

[0080] Figure 3 Effects of Zhenyuan granules on the capture latency and number of captures in male rats. (A) Capture latency; (B) Number of captures. Compared with the sham-operated control group, * P<0.05; compared with the model control group (Orchiectomy), # P <0.05, ## P <0.01. TP, testosterone propionate; ZYKL, Zhenyuan granules.

[0081] Figure 4 Effects of Zhenyuan granules on testosterone levels in rats. Compared with the sham-operated control group (Sham), ** P <0.01; compared with the model control group (Orchiectomy), # P <0.05, ## P <0.01. TP, testosterone propionate; ZYKL, Zhenyuan granules.

[0082] Figure 5 Effects of Zhenyuan granules on NOS, NO, and cGMP in rats. (A) NOS; (B) NO; (C) cGMP. Compared with the sham-operated control group, ** P <0.01; compared with the model control group (Orchiectomy), # P <0.05, ## P <0.01. TP, testosterone propionate; ZYKL, Zhenyuan granules.

[0083] Figure 6 Effects of Zhenyuan Granules on Weight-Bearing Swimming Time in Rats. Sham, sham-operated control group; Orchiectomy, model control group; TP, testosterone propionate; ZYKL, Zhenyuan Granules.

[0084] Figure 7 Effect of Zhenyuan granules on lactate levels in rats after exercise. (A) Whole blood lactate; (B) Area under the curve (AUC) of whole blood lactate. Compared with the sham-operated control group, * P <0.05, ** P <0.01; compared with the model control group (Orchiectomy), # P <0.05, ## P <0.01. TP, testosterone propionate; ZYKL, Zhenyuan granules.

[0085] Figure 8Effect of Zhenyuan granules on serum blood urea nitrogen (BUN) levels in castrated rats after swimming. Compared with the sham-operated control group, ** P <0.01; compared with the model control group (Orchiectomy), ## P <0.01. TP, testosterone propionate; ZYKL, Zhenyuan granules.

[0086] Figure 9 Effects of Zhenyuan granules on glycogen reserves in rats. Compared with the sham-operated control group (Sham), * P <0.05, ** P <0.01; compared with the model control group (Orchiectomy), ## P <0.01. TP, testosterone propionate; ZYKL, Zhenyuan granules.

[0087] Figure 10 Effects of Zhenyuan Granules on Serum SOD and MDA in Rats. Sham, sham-operated control group; Orchiectomy, model control group; TP, testosterone propionate; ZYKL, Zhenyuan Granules.

[0088] Figure 11 Pre-intervention with Zhenyuan granules can alleviate isoproterenol-induced cardiac dysfunction. Compared with the model control group, *** P <0.001, ** P <0.01. ZYKL, Zhenyuan granules; positive control, metoprolol.

[0089] Figure 12 Pre-intervention with Zhenyuan granules can alleviate isoproterenol-induced myocardial pathological damage. Compared with the model control group, *** P <0.001, ** P <0.01, * P <0.05. ZYKL, Zhenyuan granules; positive control, metoprolol.

[0090] Figure 13 Pre-intervention with Zhenyuan granules can alleviate ischemic electrocardiographic abnormalities in rats with isoproterenol-induced myocardial infarction. ZYKL, Zhenyuan granules; positive control, metoprolol.

[0091] Figure 14 Pre-intervention with Zhenyuan granules reduced myocardial inflammation levels in isoproterenol-induced myocardial infarction rats. Compared with the model control group,*** P <0.001, ** P <0.01, * P <0.05. ZYKL, Zhenyuan granules; positive control, metoprolol.

[0092] Figure 15 Pre-intervention with Zhenyuan granules can reduce the levels of oxidative stress-related indicators in rats with isoproterenol-induced myocardial infarction. Compared with the model control group, *** P <0.001, ** P <0.01, * P <0.05. ZYKL, Zhenyuan granules; positive control, metoprolol.

[0093] Figure 16 Post-treatment with Zhenyuan granules can alleviate isoproterenol-induced cardiac function impairment in rats. Compared with the model control group, *** P <0.001, * P <0.05. ZYKL, Zhenyuan granules; positive control, metoprolol.

[0094] Figure 17 Post-treatment with Zhenyuan granules can alleviate isoproterenol-induced myocardial pathological damage in rats. ZYKL, Zhenyuan granules; positive control, metoprolol.

[0095] Figure 18 Post-intervention (treatment) with Zhenyuan granules can alleviate ischemic electrocardiographic abnormalities in rats with isoproterenol-induced myocardial infarction. ZYKL, Zhenyuan granules; positive control, metoprolol.

[0096] Figure 19 Post-treatment with Zhenyuan granules reduced myocardial inflammation levels in rats with isoproterenol-induced myocardial infarction. Compared with the model control group, *** P <0.001, ** P <0.01, * P <0.05. ZYKL, Zhenyuan granules; positive control, metoprolol.

[0097] Figure 20 Post-treatment with Zhenyuan granules reduced the levels of oxidative stress-related indicators in rats with isoproterenol-induced myocardial infarction. Compared with the model control group, *** P<0.001, ** P <0.01, * P <0.05. ZYKL, Zhenyuan granules; positive control, metoprolol.

[0098] Figure 21 Femoral CT scan parameters of ovariectomized osteoporotic mice in each group. Compared with the sham-operated control group (S), ** P <0.01, *** P <0.001; compared with the model control group (O), # P <0.05, ## P <0.01, ### P <0.001. BMD, bone mineral density; BV, bone volume; BV / TV, bone volume fraction; BS, bone surface area; iS, cross-sectional area; BS / TV, solid surface area to tissue ratio; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; Tb.N, number of trabeculae. ZL, low-dose group of Zhenyuan granules; ZM, medium-dose group of Zhenyuan granules; ZH, high-dose group of Zhenyuan granules.

[0099] Figure 22 Effects of Zhenyuan granules on bone microstructure in ovariectomized osteoporotic mice. Sham, sham-operated control group; OVX, model control group; ZY-L, ZY-M, and ZY-H, representing low, medium, and high dose groups of Zhenyuan granules, respectively.

[0100] Figure 23 Zhenyuan granules alleviated bone tissue damage in ovariectomized osteoporotic mice (HE staining, 100×). Sham, sham-operated control group; OVX, model control group; ZYKL-L, ZYKL-M, and ZYKL-H were the low, medium, and high dose groups of Zhenyuan granules, respectively.

[0101] Figure 24 Zhenyuan granules alleviated bone tissue damage in ovariectomized osteoporotic mice (TRAP staining, 100×). Sham, sham-operated control group; OVX, model control group; ZYKL-L, ZYKL-M, and ZYKL-H were the low, medium, and high dose groups of Zhenyuan granules, respectively.

[0102] Figure 25 Effects of Zhenyuan granules on serum ALP, SOD, MDA, and GPX levels (n = 6). Compared with the sham-operated control group (Sham), ** P <0.01, ***P <0.001; compared with the model control group (OVX), # P <0.05, ## P <0.01, ### P <0.001. ZYKL-L, ZYKL-M, and ZYKL-H represent the low, medium, and high dose groups of Zhenyuan granules, respectively.

[0103] Figure 26 Effects of Zhenyuan granules on the expression of bone resorption-related gene mRNAs in the femur. Compared with the sham-operated control group (Sham), * P <0.05, ** P <0.01, *** P <0.001; compared with the model control group (OVX), # P <0.05, ## P <0.01, ### P <0.001. ZYKL-L, ZYKL-M, and ZYKL-H represent the low, medium, and high dose groups of Zhenyuan granules, respectively.

[0104] Figure 27 Effects of Zhenyuan granules on the expression of bone resorption-related proteins c-fos, Trap, and CTSK in the femur. Compared with the sham-operated control group (Sham),* P <0.05,** P <0.01, *** P <0.001; compared with the model control group (OVX), # P <0.05, ## P <0.01, ### P <0.001. ZYKL-L, ZYKL-M, and ZYKL-H represent the low, medium, and high dose groups of Zhenyuan granules, respectively.

[0105] Figure 28 Effects of Zhenyuan granules on the expression of oxidative stress-related proteins Nrf2, HO-1, and NQO1 in the femur. Compared with the sham-operated control group (Sham), *** P <0.001; compared with the model control group (OVX), # P <0.05, ## P <0.01,### P <0.001. ZYKL-L, ZYKL-M, and ZYKL-H represent the low, medium, and high dose groups of Zhenyuan granules, respectively.

[0106] Figure 29 Effects of Zhenyuan Granules on the differentiation of mouse 3T3-L1 preadipocytes (Oil Red O staining, 100×). Undifferentiated group, no differentiation inducer added; Differentiation control group, differentiation inducer added, no Zhenyuan Granules or lovastatin; ZYKL, Zhenyuan Granules; Lovastatin.

[0107] Figure 30 Zhenyuan granules affect key genes in adipogenic differentiation of mouse 3T3-L1 preadipocytes ( PPARγ , C / EBPα , SREBP-1c and ACC1 The effect of mRNA expression. DMI, differentiation inducer; ZYKL, Zhenyuan granules; - indicates no differentiation inducer. + indicates addition of differentiation inducer. Compared with the undifferentiated group, * P <0.05; *** P <0.001; compared with the differentiation-inducing control group, # P <0.05, ## P <0.01, ### P <0.001.

[0108] Figure 31 Effects of Zhenyuan Granules on the expression levels of AMPK signaling pathway and downstream adipogenic key proteins in mouse 3T3-L1 preadipocytes. A: Protein bands. B-G: Quantitative analysis of protein expression (n = 3). Compared with the differentiation control group, # P <0.05, ## P <0.01, ### P <0.001. ZYKL, Zhenyuan granules; DMI, differentiation inducer; + indicates the addition of differentiation inducer.

[0109] Figure 32 Effect of Zhenyuan granules on body weight gain in estrogen-deficient mice. A: Trend of mouse body weight gain during treatment. B: Net body weight gain at the end of treatment. Compared with the sham-operated control group (Sham), *** P <0.001; compared with the model control group (OVX), # P <0.05,## P <0.01, ### P <0.001. ZYKL-L, low-dose group of Zhenyuan granules; ZYKL-H, high-dose group of Zhenyuan granules; Orlistat; EV, estradiol valerate.

[0110] Figure 33 Effects of Zhenyuan granules on gonadal and inguinal adipose tissue in estrogen-deficient mice. Microscopic examination of adipocytes stained with HE (400 ×). A: Gonadal adipose tissue (pWAT) index = pWAT weight / body weight (g / g), histopathological images, and adipocyte size (mean diameter, mean area) analysis results; B: Inguinal adipose tissue (ingWAT) index = ingWAT weight / body weight (g / g), histopathological images, and adipocyte size (mean diameter, mean area) analysis results. Compared with the sham-operated control group (Sham), * P <0.05, ** P <0.01, *** P <0.001; compared with the model control group (OVX), # P <0.05, ## P <0.01, ### P <0.001. ZYKL-L, low-dose group of Zhenyuan granules; ZYKL-H, high-dose group of Zhenyuan granules; Orlistat; EV, estradiol valerate.

[0111] Figure 34 Effects of Zhenyuan Granules on Uterine Atrophy in Estrogen-Deficient Mice. Uterus examined under HE staining (40 ×). Sham, sham-operated control group; OVX, model control group; ZYKL-L, low-dose Zhenyuan Granules group; ZYKL-H, high-dose Zhenyuan Granules group; Orlistat; EV, estradiol valerate.

[0112] Figure 35 Effects of Zhenyuan Granules on Serum Total Cholesterol and Leptin Levels in Estrogen-Deficient Mice. A: Serum total cholesterol level (n = 10); B: Serum leptin level (n = 10). Compared with the sham-operated control group (Sham), *** P <0.001; compared with the model control group (OVX), # P <0.05, ## P <0.01,### P <0.001. ZYKL-L, low-dose group of Zhenyuan granules; ZYKL-H, high-dose group of Zhenyuan granules; Orlistat; EV, estradiol valerate.

[0113] Figure 36 Effects of Zhenyuan Granules on the expression levels of AMPK signaling pathway and downstream adipogenic key proteins in adipose tissue of estrogen-deficient mice. A: Protein bands. a: Sham, sham-operated control group; b: OVX, model control group; c: EV, estradiol valerate group; d: Orlistat, orlistat group; e: ZYKL-L, low-dose Zhenyuan Granules group; f: ZYKL-H, high-dose Zhenyuan Granules group. B-G: Quantitative analysis of protein expression (n = 3). Compared with the sham-operated control group, * P <0.05, ** P <0.01, *** P <0.001; compared with the model control group, # P <0.05, ## P <0.01, ### P <0.001. Detailed Implementation

[0114] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention, but do not limit the present invention in any way.

[0115] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0116] Example 1: Preparation and Quality Control of Vibration Element Particles

[0117] 1. Preparation of Zhenyuan Particles

[0118] Take the Chinese herbal combination (6 parts Epimedium, 3 parts dried ginger, 3 parts licorice, 2 parts ginseng, 2 parts Poria cocos, and 2 parts Salvia miltiorrhiza), add water and decoct 3 times, each time adding 15 times the amount of water of the herbal pieces, decoct for 2 hours, cool, filter, and combine the filtrates; centrifuge, take the supernatant, concentrate under reduced pressure to a thick paste, vacuum dry the thick paste at 80℃ to a water content of 5% or less, take it out, cool to room temperature, pulverize, make fine powder, granulate, and package to obtain Zhenyuan granules.

[0119] 2. Quality control of Zhenyuan particles

[0120] HPLC fingerprint of Zhenyuan particles:

[0121] Mixed reference solution: Accurately weigh the reference standards of icariin, citrogine A, citrogine B, citrogine C, glycyrrhizin, ammonium glycyrrhizate, quercetin, and salvianolic acid C, dissolve them in methanol, filter through a 0.22 µm microporous membrane, and dilute to prepare a mixed reference solution (the concentrations of the reference standards are 0.10 mg / mL for icariin, citrogine A, citrogine B, and citrogine C, 0.75 mg / mL for ammonium glycyrrhizate, and 0.04 mg / mL for glycyrrhizin, quercetin, and salvianolic acid C).

[0122] Zhenyuan granule test solution: Accurately weigh 0.2 g of Zhenyuan granules, place them in a stoppered conical flask, add 20 mL of 50% ethanol, weigh, sonicate (480 W, 40 kHz) for 1 h, cool, add weight, shake well, centrifuge at 11000 r / min for 10 min, take the supernatant, filter through a 0.22 µm microporous membrane, and the solution is obtained.

[0123] Chromatographic conditions: A Kromasil 100-5-C18 column (250 mm × 4.6 mm, 5 µm) was used with acetonitrile as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B. The detection wavelength was 254 nm, the column temperature was 30 °C, the flow rate was 1 mL / min, and the injection volume was 10 µL. The elution gradient is shown in Table 1.

[0124] Table 1. Elution gradient of mobile phase

[0125]

[0126] Recording the HPLC fingerprint of Zhenyuan granules: The mixed reference solution and the Zhenyuan granules test solution were injected separately for analysis. 10 μL of the mixed reference solution and 10 μL of the Zhenyuan granules test solution were injected, and the chromatograms were recorded. Based on the retention times of each traditional Chinese medicine reference standard, the corresponding chromatographic peaks in the test sample chromatogram were designated. The established HPLC fingerprint of Zhenyuan granules is shown below. Figure 1 As shown, when compared with the chromatograms of the corresponding Chinese herbal reference standards, the Chinese herbal components identified from the fingerprint chromatograms include glycyrrhizin, quercetin, chomopodophyllin A, chomopodophyllin B, chomopodophyllin C, icariin, salvianolic acid C, and ammonium glycyrrhizate.

[0127] Example 2: Preparation and Quality Control of Zhenyuan Particles

[0128] Take the following traditional Chinese medicine composition (8 parts Epimedium, 8 parts dried ginger, 8 parts licorice, 3 parts ginseng, 3 parts Poria cocos, and 3 parts Salvia miltiorrhiza), decoct it three times with water, adding 10 times the amount of water each time, decoct for 2 hours, cool, filter, and combine the filtrates; centrifuge, collect the supernatant, concentrate under reduced pressure to a thick paste, vacuum dry the paste at 80℃ until the water content is 5% or less, remove, cool to room temperature, pulverize, make fine powder, granulate, and package to obtain Zhenyuan granules. The quality control of Zhenyuan granules is the same as in Example 1.

[0129] Example 3: Preparation and Quality Control of Zhenyuan Particles

[0130] Take the following traditional Chinese medicine composition (2 parts Epimedium, 2 parts dried ginger, 2 parts licorice, 3 parts ginseng, 3 parts Poria cocos, and 3 parts Salvia miltiorrhiza), add water and decoct 4 times, adding 20 times the amount of water each time, decoct for 3 hours, cool, filter, and combine the filtrates; centrifuge, take the supernatant, concentrate under reduced pressure to a thick paste, vacuum dry the paste at 80℃ to a moisture content of 5% or less, remove, cool to room temperature, pulverize, make fine powder, granulate, and package to obtain Zhenyuan granules. The quality control of Zhenyuan granules is the same as in Example 1.

[0131] Example 4: Preparation and Quality Control of Zhenyuan Particles

[0132] Take the following traditional Chinese medicine composition (5 parts Epimedium, 4 parts dried ginger, 4 parts licorice, 3 parts ginseng, 3 parts Poria cocos, and 3 parts Salvia miltiorrhiza), add water and decoct three times, adding 8 times the amount of water as the medicinal slices each time, decoct for 2 hours, cool, filter, and combine the filtrates; centrifuge, take the supernatant, concentrate under reduced pressure to a thick paste, vacuum dry the paste at 80℃ to a moisture content of 5% or less, remove, cool to room temperature, pulverize, make fine powder, granulate, and package to obtain Zhenyuan granules. The quality control of Zhenyuan granules is the same as in Example 1.

[0133] The following examples use the Zhenyuan particles prepared in Example 1 as an example to test the efficacy of the Zhenyuan particles.

[0134] Example 5: The kidney-tonifying and aphrodisiac (prevention and treatment of impotence) and anti-fatigue effects of Zhenyuan granules

[0135] 1. Experimental Materials and Methods

[0136] 1.1 Experimental Reagents

[0137] Testosterone propionate, estradiol benzoate (Shanghai Aladdin Biochemical Technology Co., Ltd.); progesterone (Sigma); penicillin (Shanghai Macklin Biochemical Technology Co., Ltd.); rat testosterone ELISA kit, rat cGMP ELISA kit (Shanghai Enzyme-linked Biotechnology Co., Ltd.); total NOS assay kit, NO assay kit, whole blood lactate assay kit, urea nitrogen test kit, total superoxide dismutase (SOD) assay kit, malondialdehyde (MDA) test kit, muscle / liver glycogen assay kit (Nanjing Jiancheng Bioengineering Institute).

[0138] 1.2 Animal grouping and treatment

[0139] Sprague-Dawley (SD) male rats (SPF grade, 4 weeks old) were purchased from the Guangdong Provincial Medical Experimental Animal Center (Experimental Animal Production License No.: SCXK (Guangdong) 2022-0002; Animal Quality Certificate No.: No. 44007200116302, No. 44007200116410, No. 44007200116599).

[0140] This experiment was approved by the Animal Experiment Ethics Committee of Guangzhou University of Chinese Medicine (No. 20220907002) and was completed in the Animal Experiment Center of Guangzhou University of Chinese Medicine (Experimental Animal Use License No.: SYXK (Guangdong) 2018-0001) in accordance with the animal care guidelines of Guangzhou University of Chinese Medicine.

[0141] After one week of adaptive feeding, all animals were healthy and divided into the following 6 groups, with 10 animals in each group:

[0142] (1) Sham operation control group (equal volume of pure water);

[0143] (2) Model control group (equal volume of pure water);

[0144] (3) Testosterone propionate group (2 mg / kg);

[0145] (4) Low-dose Zhenyuan Granule group (0.94 g / kg);

[0146] (5) Medium-dose Zhenyuan Granule group (3.76 g / kg);

[0147] (6) High-dose Zhenyuan Granule group (7.52 g / kg).

[0148] Except for the sham surgery control group, all other groups underwent bilateral orchiectomy (castration) under anesthesia. In the sham surgery control group, the skin was opened and sutured, but the testes were not removed. Postoperatively, penicillin 20,000 units / kg was injected subcutaneously for 3 consecutive days. After 3 days, except for the testosterone propionate group which received intramuscular injection, all other groups received oral administration (20 mL / kg) once daily for 21 consecutive days at the same dosage.

[0149] 1.3 Efficacy Experiments and Index Determination

[0150] 1.3.1 Penile erection latency test

[0151] After the last administration, the rats were anesthetized, and one electrode of the electroacupuncture needle was placed at the external urethral orifice, and the other electrode was placed on the penile skin. The current intensity was 4 mA, and the rat's penis was locally stimulated. The time from the start of electrical stimulation to penile erection was recorded, which is the erection latency.

[0152] 1.3.2 Capture Experiment

[0153] One day after the erection latency test, male rats were placed in a (50 × 50 × 40) cm cage, one rat per cage. 3 In the rat cages, the rats were allowed to acclimatize for 2 minutes. One female rat in estrus was added to each cage (administered subcutaneously with estradiol benzoate 0.1 mg / kg 24 h before the experiment and progesterone 2 mg / kg subcutaneously 4 h before the experiment). The latency period and number of attacks by male rats on female rats were observed and recorded within 20 minutes. If no attacking behavior occurred within 20 minutes, the attack latency period was calculated to be 1200 s.

[0154] 1.3.3 Weighted Swimming Experiment

[0155] Add a load (lead wire) to the tail of the rat at 5% of its body weight, place the rat in a water container (the height of which exceeds the sum of the head and tail of the rat), and set the water temperature to 25°C. Record the swimming time of the rat (the end of swimming is judged when the rat's nose is submerged in water for 10 seconds and its head cannot be exposed above the water surface).

[0156] 1.3.4 Determination of lactic acid and blood urea nitrogen

[0157] One day after the weighted swimming experiment, a non-weighted swimming experiment was conducted to determine lactate and urea nitrogen concentrations. Blood was collected from rats before swimming, and they were immediately immersed in water for 10 minutes without weight (water temperature 25℃). Blood was collected immediately after swimming, and again after a 20-minute rest period. Whole blood lactate was measured based on the blood samples collected before swimming, 10 minutes after swimming, and 20 minutes after rest. The area under the lactate curve (AUC) was calculated as: (AUC) = 1 / 2 × (lactate value before swimming + lactate value after 10 minutes of swimming) × 10 + (lactate value after 10 minutes of swimming + lactate value after 20 minutes of rest) × 20. Serum was separated 20 minutes after swimming for urea nitrogen concentration measurement. The detection method followed the kit instructions.

[0158] 1.3.5 Detection of testosterone, NOS, NO, cGMP, SOD, and MDA

[0159] Rats were fasted overnight, anesthetized, and blood was collected. Serum was separated, and the levels of testosterone, NO, cGMP, and MDA, as well as the activity levels of NOS and SOD, were measured. The detection methods followed the kit instructions.

[0160] 1.3.6 Muscle / Liver Glycogen Detection

[0161] Muscle glycogen and liver glycogen were measured from foreleg muscle and liver tissue. The detection method was as described in the kit instructions.

[0162] 1.4 Statistical Analysis

[0163] Data were statistically analyzed using SPSS 26.0. Results are expressed as mean ± standard deviation. The Kolmogorov-Smirnov test was used to test the normality of the data. If the data conformed to a normal distribution, one-way ANOVA and the post-hoc Tukey test (assuming homogeneity of variance) or Dunnett's T3 test (assuming homogeneity of variance) were used for comparisons between groups. If the data did not conform to a normal distribution, the rank-sum test was used to compare differences between groups. P A value <0.05 indicates a statistically significant difference.

[0164] 2. Experimental Results

[0165] 2.1 The kidney-tonifying and aphrodisiac effects of Zhenyuan granules

[0166] 2.1.1 Effect of Zhenyuan Granules on Penile Erection Latency in Castrated Rats

[0167] Compared with the sham-operated control group, the penile erection latency of rats in the model control group was significantly prolonged by electrical stimulation. Compared with the model control group, the penile erection latency of rats in the testosterone propionate group was significantly shortened, approaching that of the sham-operated control group; the penile erection latency of rats in all dose groups of Zhenyuan granules was significantly shortened, and a certain dose-response relationship was observed. Among them, the penile erection latency of rats in the medium and high dose groups of Zhenyuan granules was significantly shorter than that of the model control group. Figure 2 ).

[0168] 2.1.2 Effects of Zhenyuan Granules on the Latency Period and Frequency of Castrated Rats Capturing Female Rats

[0169] Compared with the sham-operated control group, the latency period for male rats to capture female rats was significantly prolonged and the number of captures within 20 minutes was significantly reduced in the model control group. Compared with the model control group, the latency period for male rats to capture female rats was significantly shortened and the number of captures was significantly increased in the testosterone propionate group; all doses of Zhenyuan granules showed a significant shortening of the latency period for male rats to capture female rats and a significant increase in the number of captures; among them, the high dose of Zhenyuan granules significantly shortened the latency period for male rats to capture female rats and increased the number of captures. Figure 3 ).

[0170] 2.1.3 Effect of Zhenyuan Granules on Testosterone Levels in Castrated Rats

[0171] Compared with the sham-operated control group, the serum testosterone level in the model control group was significantly decreased. Compared with the model control group, the testosterone propionate group showed a significant increase in testosterone level; all dose groups of Zhenyuan granules showed a significant increase in testosterone level, and a dose-response relationship was observed; among them, the testosterone levels in the medium and high dose groups of Zhenyuan granules were significantly higher than those in the model control group. Figure 4 ).

[0172] 2.1.4 Effects of Zhenyuan Granules on NOS, NO, and cGMP in Castrated Rats

[0173] Compared with the sham-operated control group, the serum NOS activity, NO, and cGMP levels in the model control group were significantly decreased. Compared with the model control group, the NOS activity, NO, and cGMP levels in the testosterone propionate group were significantly increased; the serum NOS activity, NO, and cGMP levels in rats at all doses of Zhenyuan granules were increased to varying degrees, showing a dose-response relationship, and were significantly higher than those in the model control group. Figure 5 ).

[0174] 2.2 Anti-fatigue effect of Zhenyuan particles

[0175] 2.2.1 Effect of Zhenyuan Granules on Weight-Bearing Swimming Time of Castrated Rats

[0176] Compared to the sham-operated control group, there was no significant difference in the weight-bearing swimming time of rats in the model control group. Compared to the model control group, there were no significant differences in the weight-bearing swimming time of rats in the testosterone propionate group and each dose group of Zhenyuan granules. Figure 6).

[0177] 2.2.2 Effects of Zhenyuan Granules on Lactate Levels in Castrated Rats After Exercise

[0178] Compared with the sham-operated control group, the area under the curve (AUC) of whole blood lactate in rats in the model control group was significantly increased after swimming. Compared with the model control group, the AUC of whole blood lactate in rats in the testosterone propionate group was increased to some extent after swimming, but the difference was not significant; the AUC of whole blood lactate in rats in all dose groups of Zhenyuan granules was significantly decreased after swimming. Figure 7 Zhenyuan particles are superior to the testosterone propionate group.

[0179] 2.2.3 Effect of Zhenyuan Granules on Blood Urea Nitrogen Levels in Castrated Rats After Exercise

[0180] Compared with the sham-operated control group, the serum urea nitrogen level of rats in the model control group was significantly increased after swimming. Compared with the model control group, there was no significant difference in serum urea nitrogen level in the testosterone propionate group after swimming; the serum urea nitrogen level of rats in each dose group of Zhenyuan granules was significantly decreased after swimming. Figure 8 (), which is superior to the testosterone propionate group.

[0181] 2.2.4 Effect of Zhenyuan Granules on Glycogen Levels in Castrated Rats

[0182] Compared with the sham-operated control group, the levels of muscle and liver glycogen in the model control group were significantly decreased. Compared with the model control group, there was no significant difference in muscle glycogen content and a significant increase in liver glycogen content in the testosterone propionate group; there were no significant differences in muscle and liver glycogen content among the different dosage groups of Zhenyuan granules. Figure 9 ).

[0183] 2.2.5 Effects of Zhenyuan Granules on SOD and MDA in Castrated Rats

[0184] Compared with the sham-operated control group, there were no significant differences in serum SOD activity and MDA levels in the model control group rats. Compared with the model control group, there were no significant differences in serum SOD activity and MDA levels in the testosterone propionate group and each dose group of Zhenyuan granules rats. Figure 10 ).

[0185] Example 6: The effect of Zhenyuan granules in alleviating myocardial infarction

[0186] This experiment used an isopropanol-induced rat model of myocardial infarction to study the effect of Zhenyuan granules in alleviating myocardial infarction.

[0187] 1. Experimental Materials and Methods

[0188] 1.1 Animal grouping and treatment

[0189] SD female rats (SPF grade, 8 weeks old, 10 weeks old) were purchased from the Guangdong Provincial Center for Medical Laboratory Animals (Production License for Laboratory Animals: SCXK (Guangdong) 2022-0002; Animal Quality Certificate: No. 44007200118067).

[0190] This experiment was approved by the Animal Experiment Ethics Committee of Guangzhou University of Chinese Medicine (No. 20240109005) and was completed in the Animal Experiment Center of Guangzhou University of Chinese Medicine (License for the Use of Laboratory Animals: SYXK (Guangdong) 2023-0347) in accordance with the animal care guidelines of Guangzhou University of Chinese Medicine.

[0191] Animal experiments were conducted in a SPF environment. SD rats were placed in a 26°C light / dark cycle of 12 h.

[0192] 1.1.1 Grouping and treatment of the pre-intervention effect experiment

[0193] Thirty 8-week-old SD female rats were used in the experiment of Zhenyuan Granules pre-intervening on isoproterenol-induced myocardial infarction. The animals were randomly divided into:

[0194] (1) Pre-intervention normal control group (equal volume of normal saline), 6 rats;

[0195] (2) Pre-intervention model control group (equal volume of normal saline), 9 rats;

[0196] (3) Pre-intervention Zhenyuan Granules group (9.25 g / kg), 7 rats;

[0197] (4) Pre-intervention metoprolol group (positive control drug, metoprolol tartrate 50 mg / tablet, prepared with normal saline into a suspension with a concentration of 10 mg / mL, dose of 10 mg / kg), 8 rats.

[0198] Gavage administration was performed at the above doses (administration volume ratio 1 mL / kg), and continuous administration was carried out for pre-intervention for 14 days, once a day. After gavage on the 13th and 14th days, except for the pre-intervention normal control group, other groups were given subcutaneous injection of isoproterenol 85 mg / kg at the posterior neck.

[0199] 1.1.2 Grouping and treatment of the post-intervention (treatment) effect experiment

[0200] Thirty-five 10-week-old SD female rats were used in the experiment of Zhenyuan Granules post-intervening, that is, treating isoproterenol-induced myocardial infarction. The animals were randomly divided into: post-intervention normal control group (equal volume of normal saline), 4 rats, and post-intervention model group, 31 rats. The post-intervention model group was injected with isoproterenol once on the 1st and 2nd days of the experiment, with a dose of 85 mg / kg. Results: 15 rats died and 16 rats had successful modeling.

[0201] The 16 rats that successfully developed the model were randomly divided into the following groups:

[0202] (1) Six animals were in the post-intervention model control group (equal volume of physiological saline);

[0203] (2) Five animals in the post-intervention Zhenyuan granule group (9.25 g / kg);

[0204] (3) Five animals were in the post-intervention metoprolol group (positive control drug metoprolol tartrate 50 mg / tablet, prepared with physiological saline to a concentration of 10 mg / mL suspension, dose of 10 mg / kg).

[0205] On the third day of the experiment, the normal control group and the modeling group were administered the above-mentioned dose by gavage (dose volume ratio 1 mL / kg) for 14 consecutive days after the intervention (treatment), once a day.

[0206] 1.2 Experiment and index determination

[0207] The relevant indicators of rats in the pre-intervention experiment and the post-intervention (treatment) experiment were tested according to the following methods.

[0208] 1.2.1 Cardiac function testing

[0209] Rats were anesthetized by inhalation of 1% isoflurane. Hair was removed from the rats' chests using Pure Hair Removal Cream (Veet, ReckittBenckiser, LSE:RB), and Medical Ultrasonic Coupling (Qingdao HYNAUT Medical Supplies Co., LTD, China) was applied to the area. Cardiac function was assessed using a high-resolution small animal ultrasound machine (Vevo, Visual Sonics Co., LTD, Canada), and left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were measured. Relevant indicators were calculated.

[0210] (1) Shorten the fraction FS

[0211] FS = (LVEDD LVESD / LVEDD) ×100%

[0212] (2) Left ventricular end-diastolic volume (EDV)

[0213] EDV = 7 × LVEDD 3 / (2.4 + LVEDD)

[0214] (3) End-systolic volume of the left ventricle (ESV)

[0215] ESV = 7 × LVESD 3 / (2.4 + LVESD)

[0216] (4) Ejection fraction EF

[0217] EF = (EDV ESV) / EDV×100%

[0218] (5) Left ventricular stroke volume (SV)

[0219] SV = EDV ESV

[0220] 1.2.2 Electrocardiogram (ECG) testing

[0221] After completing the cardiac function test, the five leads of the small animal electrocardiograph (model: ECG-3306B, Guangzhou 3ray electronic Technology Co., LTD) were fixed to the limbs and chest of the rats according to the instructions, and the rat electrocardiogram data were collected.

[0222] 1.2.3 Cardiac tissue collection and related indicator testing

[0223] After anesthetizing rats with phenobarbital (50 mg / kg), the 1st to 7th ribs were cut along the left side of the costochondral joint using surgical scissors to expose the heart. The heart was then separated from the ascending aorta, pulmonary artery, pulmonary vein, pericardium, and other tissues using forceps and scissors. The ventricles were then divided into upper and lower parts in a coronal plane at the midpoint between the left and right ventricles. The upper part was placed in 4% paraformaldehyde for pathological examination. The lower part was placed in cryovials.

[0224] (1) Cardiac pathological examination: Rat heart tissue was fixed in 4% paraformaldehyde (BIOSHARP, Labgic Technology Co., Ltd, China) solution and then subjected to HE staining.

[0225] (2) Serum collection: After obtaining rat heart samples, the abdominal skin of the rat was cut open with scissors, and the tissue covering the abdominal aorta was removed with hemostatic gauze to fully expose the abdominal aorta. Blood was drawn from the rat through the abdominal aorta. The blood was centrifuged at 3000g for 15 min, and the serum was collected.

[0226] (3) Serum marker detection: The concentrations of TNF-α, IL-6, IL-1β, SOD, GSH, CK, and CK-mb in serum were detected using commercially available kits (Nanjing Jiancheng Bioengineering Institute, China) according to the instructions, using a microplate reader (Multiskan Mk3, Thermo Fisher Scientific Shanghai Instruments Co., Ltd.). The concentrations of thiobarbituric acid reactants and lipid hydroperoxides in tissues were detected using a human thiobarbituric acid reactant (TBARS) assay kit (Cayman Chemical, China) and a lipid peroxide (LPO) assay kit (Cayman Chemical, China).

[0227] 1.3 Statistical Analysis

[0228] SPSS 23 software was used to analyze the results. Data are expressed as mean ± standard error (sem). When the dataset is normally distributed and has homogeneous variance, one-way ANOVA and Fisher's minimum significance analysis were used to determine the variance. P Value. Otherwise, use the Kruskal-Wallis test to determine. P value. P A value <0.05 is considered statistically significant. For count data, if the minimum frequency is not less than 5, use the chi-square test; otherwise, use Fisher's exact test.

[0229] 2. Experimental Results

[0230] 2.1 Pre-intervention can reduce cardiac function damage in rats with myocardial infarction.

[0231] like Figure 11 As shown, pre-intervention with Zhenyuan granules has a mitigating effect on isoproterenol-induced cardiac function impairment: compared with the pre-intervention model control group, Zhenyuan granules significantly improved ejection fraction (EF). P <0.001, with an effect similar to the positive control drug metoprolol ( P = 0.005); compared with the pre-intervention model control group, Zhenyuan particles significantly improved the reduction of the fractional FS (FS). P <0.001, with an effect similar to the positive control drug metoprolol ( P = 0.009) consistent.

[0232] 2.2 Pre-intervention can reduce myocardial pathological damage in rats with myocardial infarction.

[0233] By HE staining ( Figure 12It was observed that the cardiomyocytes of the rats in the pre-intervention model control group underwent significant necrosis, with extensive inflammatory cell infiltration in the necrotic areas. Necrotic myocardial fibers were replaced by collagen fibers, resulting in significant atrophy of the myocardial fibers and a marked widening of the interstitial spaces. In contrast, the cardiomyocytes of the rats in the pre-intervention Zhenyuan granule group showed small-pattern necrosis, with the necrotic cardiomyocytes replaced by newly formed collagen fibers. Small capillaries were visible, surrounded by fibroblast and inflammatory cell infiltration. Some myocardial atrophy and widening of the interstitial spaces were observed. Significant atrophy of myocardial cells and widening of the interstitial spaces were also observed, along with a small amount of inflammatory cell infiltration and dissolution of a small number of myocardial fibers. The improvement effect on the lesions was most pronounced in the pre-intervention Zhenyuan granule group. Simultaneously, the protective effect of Zhenyuan granules on cardiomyocyte damage was observed: compared with the pre-intervention normal control, the myocardial injury indicators creatine kinase (CK) and creatine kinase isoenzyme (CK-MB) in the pre-intervention model control group were significantly elevated. P <0.001 or P =0.001), compared with the pre-intervention model control group, the CK and CK-MB levels in the pre-intervention Zhenyuan particle group were significantly lower ( P <0.05), similar to the effect of positive control drugs; the results indicate that Zhenyuan granules have a protective effect against myocardial injury in animals with infarcted myocardium.

[0234] 2.3 Pre-intervention can alleviate isoproterenol-induced ischemic electrocardiographic abnormalities.

[0235] like Figure 13 As shown:

[0236] Pre-intervention normal control group: normal electrocardiogram, no significant changes in the ST segment of any lead;

[0237] Pre-intervention model control group: mild ST segment elevation in lead I; no obvious abnormalities in lead II; no obvious abnormalities in lead III; downsloping ST segment depression in lead aVR; Q wave and ST segment depression in lead aVL; no obvious abnormalities in lead aVF.

[0238] Pre-intervention positive control group: mild ST segment elevation in lead I; no obvious abnormalities in lead II; mild ST segment elevation in lead III; T wave inversion in lead aVR; T wave inversion in lead aVL; no obvious abnormalities in lead aVF;

[0239] Pre-intervention Zhenyuan Particle Group: Q wave in lead I, no significant ST segment changes; biphasic T wave in lead II; no significant ST segment changes in lead III; no significant ST segment changes in lead aVR; Q wave in lead aVL, no significant ST segment changes; biphasic T wave in lead aVF; Compared with the pre-intervention model control group, the pre-intervention Zhenyuan Particle Group had fewer abnormal leads and a lower degree of abnormal ST segment elevation.

[0240] The results showed that pre-intervention with Zhenyuan granules could alleviate ischemic electrocardiogram abnormalities induced by isoproterenol.

[0241] 2.4 Pre-intervention can reduce myocardial inflammation and oxidative stress in rats with myocardial infarction.

[0242] like Figure 14 As shown, isoproterenol can significantly increase the levels of myocardial TNF-α, IL-1β, and IL-6 in rats with myocardial infarction; while pre-intervention with Zhenyuan granules can downregulate the levels of myocardial TNF-α, IL-1β, and IL-6, indicating that Zhenyuan granules can alleviate isoproterenol-induced myocardial inflammation.

[0243] like Figure 15 As shown, isoproterenol significantly decreased myocardial SOD and GSH levels in rats with myocardial infarction, while pre-intervention with Zhenyuan granules upregulated myocardial SOD and GSH levels; isoproterenol significantly increased myocardial TBARS and LOOH levels in rats with myocardial infarction, while pre-intervention with Zhenyuan granules downregulated myocardial TBARS and LOOH levels. These results indicate that Zhenyuan granules (ZYKL) can improve the inflammation level in myocardial infarction model animals; compared with the pre-intervention control group, the improvement effect of Zhenyuan granules (ZYKL) was significantly higher. P <0.05) and positive drug ( P With a value close to <0.05, Zhenyuan granules can alleviate isoproterenol-induced ischemic myocardial infarction by relieving oxidative stress.

[0244] 2.5 Post-intervention (treatment) can reduce cardiac function damage in rats with myocardial infarction.

[0245] Figure 16 The study showed that the protective effect of Zhenyuan granules after intervention (treatment) on cardiac function injury was statistically significant, indicating that Zhenyuan granules have a mitigating effect on cardiac function injury. This was manifested in the clear improvement of ejection fraction (EF) and fractional shortening (FS) indicators after intervention (treatment) with Zhenyuan granules. P <0.05%, its effect is even stronger than that of positive control drugs ( P = 0.078 and P = 0.066).

[0246] 2.6 Post-intervention (treatment) can reduce myocardial pathological damage in rats with myocardial infarction.

[0247] like Figure 17As shown, in the post-intervention normal control group, no cardiomyocyte necrosis occurred, no myofibrosis was observed, no interstitial widening was seen, no increase in adipocytes was observed, no inflammatory cell infiltration was observed, and no vascular congestion was observed. In the post-intervention model control group, myocardial necrosis and dissolution occurred, with necrotic areas replaced by fibroblasts and a large number of proliferating collagen cells visible. Significant inflammatory cell infiltration was observed in the interstitium, and cardiomyocytes around the necrotic areas showed significant atrophy, with myofibroblasts becoming noticeably thinner and interstitial spaces significantly widened. In the post-intervention metoprolol group (positive control), small patches of necrosis were observed in the myocardium, with dissolution of necrotic myocardial fibers and surrounding inflammatory cell infiltration. No new capillaries or fibroblasts were observed, and some myocardial atrophy and widening of interstitial spaces were observed. In the post-intervention Zhenyuan granules group, small patches of necrosis occurred in the cardiomyocytes, with necrotic cardiomyocytes replaced by new collagen fibers. Small capillaries were visible, with surrounding fibroblasts and inflammatory cell infiltration. Some myocardial atrophy and widening of interstitial spaces were observed.

[0248] 2.7 Post-intervention (treatment) can reduce ischemic electrocardiographic abnormalities in rats with myocardial infarction.

[0249] like Figure 18 As shown:

[0250] Post-intervention normal control group: normal electrocardiogram, no significant changes in the ST segment of each lead;

[0251] Post-intervention model control group: No obvious abnormalities were found in the ST segment of lead I; ST segment elevation with convex upward and fusion with T wave was observed in lead II; ST segment elevation with convex upward and fusion with T wave was observed in lead III; T wave inversion was observed in lead aVR; ST segment depression was observed in lead aVL; ST segment elevation with convex upward and fusion with T wave was observed in lead aVF.

[0252] Post-intervention metoprolol group (positive control): mild ST segment depression in lead I; no significant ST segment changes in lead II; no significant ST segment changes in lead III; no significant ST segment changes in lead aVR; significant upward-sloping ST segment depression in lead aVL; no significant ST segment changes in lead aVF.

[0253] Post-intervention Zhenyuan Particle Group: Low voltage in lead I, no obvious ST segment abnormalities; no obvious ST segment abnormalities in lead II; no obvious ST segment abnormalities in lead III; no obvious ST segment abnormalities in lead aVR; mild ST segment depression in lead aVL; no obvious ST segment abnormalities in lead aVF. Compared with the post-intervention model control group, the post-intervention Zhenyuan Particle Group had fewer abnormal leads and a lower degree of abnormal ST segment elevation.

[0254] The results showed that treatment with Zhenyuan granules could reduce ischemic electrocardiogram abnormalities in rats with myocardial infarction.

[0255] 2.8 Post-intervention (treatment) can reduce myocardial inflammation and oxidative stress in rats with myocardial infarction.

[0256] like Figure 19 As shown, isoproterenol can significantly increase the levels of myocardial TNF-α, IL-1β, and IL-6 in rats with myocardial infarction; while post-intervention (treatment) with Zhenyuan granules can downregulate the levels of myocardial TNF-α, IL-1β, and IL-6, indicating that Zhenyuan granules can alleviate isoproterenol-induced myocardial inflammation.

[0257] like Figure 20 As shown, isoproterenol significantly decreased myocardial SOD and GSH levels in rats with myocardial infarction, while post-intervention (treatment) with Zhenyuan granules upregulated myocardial SOD and GSH levels; isoproterenol significantly increased myocardial TBARS and LOOH levels in rats with myocardial infarction, while post-intervention (treatment) with Zhenyuan granules downregulated myocardial TBARS and LOOH levels. These results indicate that Zhenyuan granules can improve the inflammation level in myocardial infarction model animals; compared with the post-intervention model control group, the improvement effect of Zhenyuan granules (… P <0.01 or P <0.05) were all similar to the positive drug ( P <0.01 or P With a value close to <0.05, Zhenyuan granules can alleviate isoproterenol-induced ischemic myocardial infarction by relieving oxidative stress.

[0258] Example 7: The therapeutic effect of Zhenyuan granules on osteoporosis caused by estrogen deficiency

[0259] 1. Experimental Materials and Methods

[0260] 1.1 Experimental Apparatus

[0261] Micro-CT (SkyScan 1276, Bruke GmbH, Germany); Multi-functional microplate reader (MULTISKANSky, Thermo Fisher Scientific, USA); High-speed centrifuge (5424R, Eppendorf GmbH, Germany); Chemiluminescence analyzer (ChemiDoc, BioRad Laboratories, USA); Real-time PCR instrument (CFX96, BioRad Laboratories, USA).

[0262] 1.2 Experimental Materials

[0263] RNA reverse transcription kit (catalog number AG21102) was purchased from Hunan Aikerui Biotechnology Co., Ltd.; BCA kit (catalog number P0009) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; ECL chemiluminescence kit (catalog number P10100) was purchased from Suzhou Xinsaimei Biotechnology Co., Ltd.

[0264] 1.3 Animal grouping and treatment

[0265] Thirty 8-week-old SPF-grade female C57BL / 6J mice, weighing 20 - 24 g, were purchased from the Guangdong Provincial Medical Laboratory Animal Center (Quality Certificate No.: No.44825400001796, Laboratory Animal Production License: SYXK (Guangdong) 2022 - 0002).

[0266] This experiment was reviewed and approved by the Laboratory Animal Ethics Committee of Guangzhou University of Chinese Medicine (Ethical Approval No.: 20220722002). It was completed in the Laboratory Animal Center of Guangzhou University of Chinese Medicine, and the Laboratory Animal Use License No.: SYXK (Guangdong) 2018 - 0001.

[0267] The mice were housed in the SPF-grade barrier environment of the Laboratory Animal Center of Guangzhou University of Chinese Medicine, at a temperature of 23 ± 2 °C, with a light / dark cycle of 12 h / 12 h, and a humidity of 55% - 65%. During the feeding period, the mice were given free access to food and water. After one week of adaptive feeding, the mice were randomly divided into the following 5 groups, with 6 mice in each group, and housed separately in cages:

[0268] (1) Sham operation control group (equal volume of normal saline);

[0269] (2) Model control group (equal volume of normal saline);

[0270] (3) Low-dose Zhenyuan Granule group (1.6 g / kg);

[0271] (4) Medium-dose Zhenyuan Granule group (3.2 g / kg);

[0272] (5) High-dose Zhenyuan Granule group (6.4 g / kg).

[0273] Except for the sham operation control group, ovariectomy was performed on the remaining groups to establish an osteoporosis model caused by estrogen deficiency. After one week of postoperative recovery, the mice were administered by gavage at the above doses once a day for 10 consecutive weeks, and the administration volume ratio was 10 mL / kg. After the last gavage, the mice were first fasted for 12 h without water deprivation, then blood was collected by eye socket puncture and centrifuged (3000 r / min, 15 min, 4 °C), and the supernatant was taken and stored in a -80 °C refrigerator; the femurs on both sides were taken, and the connective tissue and muscle were completely removed. The right side was fixed with 4% paraformaldehyde, and the left side was stored in a -80 °C refrigerator for later use.

[0274] 1.4 Efficacy experiment and index determination

[0275] 1.4.1 Bone tissue parameters

[0276] Femoral bone samples fixed with 4% paraformaldehyde were collected and examined using Micro-CT. Bone parameters included bone mineral density (BMD), bone volume (BV), bone volume fraction (BV / TV), bone surface area (BS), bone cross-sectional area (iS), bone surface area to tissue ratio (BS / TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and number of trabeculae (Tb.N).

[0277] 1.4.2 Bone Microstructure

[0278] To further determine the protective effect of Zhenyuan granules against bone loss in mice, the microstructure within the cancellous bone was reconstructed and analyzed by scanning the lower femur of each group of mice using Micro-CT.

[0279] 1.4.3 Femoral Histopathology

[0280] Femurs fixed with 4% paraformaldehyde were harvested, rinsed with phosphate buffer, and decalcified in 10% EDTA solution for 4 weeks. The decalcified specimens were rinsed with phosphate buffer, dehydrated, cleared, paraffin-embedded, embedded, sectioned, stained with hematoxylin and eosin (HE), and stained with TRAP. The morphology of the bone tissue of each group of mice was observed under a microscope.

[0281] 1.4.4 Serum Biochemical Indicators

[0282] Serum samples were collected, and the levels of ALP, SOD, MDA, and GPX in the serum were detected using alkaline phosphatase (ALP / AKP) assay kits, superoxide dismutase (SOD) kits, malondialdehyde (MDA) kits, and glutathione peroxidase (GSH-PX) kits.

[0283] 1.4.5 Expression of bone resorption-related gene mRNAs in bone tissue

[0284] RNA was extracted from femur bones using Trizol. Total RNA was reverse transcribed into cDNA using an M-MLV reverse transcriptase assay kit, and then detected by quantitative real-time PCR using the TB Green Master Mix fluorescent dye kit. Car2 , Ctsk , MMP9 and TRAF6 Gene expression levels, using 2 （-△△T） The relative expression of each gene was calculated using the method. The primer sequences for real-time PCR are shown in Table 2.

[0285] Table 2 Primer Sequences for Quantitative Real-Time PCR

[0286]

[0287] 1.4.6 Expression of bone resorption-related proteins (CTSK, Trap, c-fos, GAPDH) and oxidative stress-related proteins (Nrf2, HO-1, NQO1) in bone tissue

[0288] 35 mg of femoral bone protein was extracted and transferred to a PVDF membrane via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membrane was blocked at room temperature for 2 h with TBST solution containing 5% skim milk powder. CTSK antibody (1:1000), Trap antibody (1:1000), c-fos antibody (1:1000), GAPDH antibody (1:10000), Nrf2 antibody (1:1000), HO-1 antibody (1:1000), and NQO1 antibody (1:1000) were added, and the membrane was incubated overnight at 4°C. The membrane was washed 4 times with TBST for 5 min each time, and HRP-labeled antibody (1:20000) was added. The membrane was incubated at room temperature for 1 h. After washing with TBST, ECL chemiluminescence solution was added for development, and the grayscale value was analyzed using ImageJ software.

[0289] 1.5 Statistical Analysis

[0290] IBM SPSS Statistics 25 software was used for statistical analysis of the data. One-way ANOVA and LSD tests were used for multiple group comparisons, and the Dunnett test was used for unequal variances. Graphpad Prime 9.0 was used to plot the statistical data. All data are expressed as mean ± standard error (Mean ± SEM). P <0.05 indicates that the difference is statistically significant.

[0291] 2 Experimental Results

[0292] 2.1 Zhenyuan granules significantly inhibit bone loss in estrogen-deficient mice, enhance bone density, and improve bone microstructure.

[0293] 2.1.1 Changes in bone tissue parameters

[0294] CT scan parameters of the femur of each group of mice are as follows: Figure 21 As shown, compared with the sham-operated control group, the bone mineral density (BMD), bone volume (BV), bone volume fraction (BV / TV), bone surface area (BS), bone surface area to tissue ratio (BS / TV), bone cross-sectional area (iS), and trabecular bone number (Tb.N) of the femur in the model control group mice were significantly reduced. P <0.001, trabecular bone thickness Tb.Th was significantly reduced ( P <0.01, the trabecular separation Tb.Sp increased extremely significantly ( P<0.001); Compared with the model control group, the bone tissue parameters such as BMD of the femur of mice in the low, medium and high dose groups of Zhenyuan granules were improved to varying degrees, and there was a dose-response relationship, indicating that Zhenyuan granules have a significant effect on improving bone loss in mice with osteoporosis caused by estrogen deficiency.

[0295] 2.1.2 Changes in bone microstructure

[0296] Representative images of femoral CT scans from each experimental group of animals are shown below. Figure 22 As can be visually observed from the bone microstructure diagram, the sham-operated control group exhibited a higher number and denser density of trabeculae, with lower separation between trabeculae. In contrast, the model control group showed a significant reduction in the number of trabeculae, with larger and sparser intertrabecular gaps. Compared to the model control group, the low, medium, and high doses of Zhenyuan granules all increased the number of trabeculae and reduced the separation between them, demonstrating a dose-response relationship. These conclusions indicate that Zhenyuan granules can improve the trabecular microstructure in estrogen-deficient osteoporotic mice, alleviate bone loss and decline, and have a protective effect against bone loss.

[0297] 2.1.3 Pathological changes in bone tissue

[0298] Depend on Figure 23 Bone tissue pathology sections showed that in the sham-operated control group, the trabeculae were large, neatly arranged, and had good continuity, interconnecting to form a network structure. No increase in osteoclasts was observed around the trabeculae, nor in the lacunae on the trabecular surface, nor in the medullary cavity. In the model control group, the trabeculae were significantly thinner, the number of trabeculae decreased, the number of osteoclasts in the medullary cavity increased significantly, the continuity of trabeculae was significantly interrupted, and large areas of trabecular-free medullary cavity appeared. Obvious lacunae were visible on the trabecular surface, surrounded by osteoclasts. Compared with the model control group, the low, medium, and high dose groups of Zhenyuan granules showed larger, more neatly arranged trabeculae with good continuity, almost indistinguishable from normal trabeculae. The trabeculae interconnected to form a network structure, and a few osteoclasts were visible around the trabeculae. No increase in lacunae on the trabecular surface, nor in the medullary cavity. Figure 24 Histopathological examination of bone tissue sections (TRAP staining) revealed that in the sham-operated control group, the trabeculae were thick, continuous, and without interruption. No osteoclasts stained purple-red were observed around the trabeculae or in the medullary cavity. In the model control group, the trabeculae were significantly thinner, with lacunae forming around the trabeculae, and an increased number of osteoclasts stained purple-red around them. Trabeculae showed interruption. In the low, medium, and high-dose groups of Zhenyuan granules, the trabeculae were somewhat thinner, but no interruption was observed, and no osteoclasts stained purple-red were observed around the trabeculae. These results indicate that Zhenyuan granules have a protective effect against bone tissue damage caused by estrogen deficiency-induced osteoporosis.

[0299] 2.1.4 Changes in serum biochemical indicators

[0300] The results of serum alkaline phosphatase (ALP) and oxidative stress markers superoxide dismutase (SOD), glutathione peroxidase (GPX), and malondialdehyde (MDA) levels in each group of mice are as follows: Figure 25 As shown, compared with the sham-operated control group, the serum levels of SOD and GPX in the model control group mice were significantly reduced ( P <0.01), MDA levels increased significantly ( P <0.001), indicating an increase in oxidative products, enhanced oxidative stress, and increased oxidation. Compared with the model control group, the low, medium, and high dose groups of Zhenyuan granules showed significantly increased SOD and GPX levels and significantly decreased MDA levels. The experimental results suggest that Zhenyuan granules exert their anti-osteoporosis effect by reducing the production of oxidative products, weakening the oxidative stress effect in estrogen-deficient mice, and thus improving bone antioxidant levels. Compared with the sham-operated control group, the serum ALP level in the model control group mice was significantly increased; however, compared with the model control group, the ALP levels in all dose groups of Zhenyuan granules were significantly decreased, with the high dose group showing a significant decrease in ALP levels. P The result was <0.001, indicating that Zhenyuan granules can improve bone metabolism in estrogen-deficient osteoporotic mice.

[0301] 2.1.5 Effects of Zhenyuan Granules on the mRNA Expression of Bone Resorption-Related Genes

[0302] The results of q-PCR detection of RNA-related genes for bone resorption in mouse femoral tissue are as follows: Figure 26 As shown, compared with the sham surgery control group, the model control group had fewer genes related to bone resorption. Car2 , Ctsk , MMP9 , TRAF6 mRNA expression was significantly increased ( P <0.05); Compared with the model control group, the low, medium and high dose groups of Zhenyuan granules MMP9 and TRAF6 The mRNA expression level of the gene was significantly downregulated. P <0.001), Car 2 and Ctsk The mRNA expression of the target cells was also significantly reduced. The results indicate that Zhenyuan granules can inhibit the expression of genes related to bone resorption in estrogen-deficient osteoporosis mice.

[0303] 2.1.6 Effects of Zhenyuan Granules on the Expression of Bone Resorption-Related Proteins

[0304] Effects on the expression of bone resorption-related proteins: The expression results of bone resorption-related proteins c-fos, Trap, and CTSK in the femoral tissue of mice in each group are shown in the figure. Figure 27The expression levels of bone resorption-related proteins c-fos, Trap, and CTSK in the model control group were significantly higher than those in the sham-operated control group. Compared with the model control group, the expression of bone resorption-related proteins c-fos, Trap, and CTSK in the low, medium, and high dose groups of Zhenyuan granules were reduced to varying degrees, and showed a dose-response relationship. This indicates that Zhenyuan granules can inhibit bone resorption by inhibiting the expression of bone resorption-related proteins in estrogen-deficient mice, thereby exerting an anti-osteoporosis effect.

[0305] 2.1.7 Effects of Zhenyuan Particles on the Expression of Oxidative Stress-Related Proteins

[0306] Effects of Zhenyuan granules on the expression of oxidative stress-related proteins: The expression levels of oxidative stress-related proteins Nrf2, HO-1, and NQO1 in the femoral tissue of mice in each group are shown in the figure. Figure 28 Compared with the sham-operated control group, the expression of oxidative stress-related proteins Nrf2, HO-1, and NQO1 in the femoral tissue of the model control group mice was reduced to varying degrees, with HO-1 expression being significantly reduced. P <0.001); Compared with the model control group, the expression levels of Nrf2, HO-1, and NQO1 proteins in the femoral tissue of mice in the low, medium, and high dose groups of Zhenyuan granules were all increased to varying degrees, with the high dose group showing a highly significant increase in the expression levels of Nrf2, HO-1, and NQO1 proteins. P The value <0.001 indicates that Zhenyuan particles may promote the expression of Nrf2, HO-1, and NQO1 proteins by activating the Nrf2 / HO-1 pathway, thereby protecting the body from oxidative damage and exerting an antioxidant effect, thus enabling the bones of estrogen-deficient osteoporotic mice to defend against oxidative stress.

[0307] Example 8: The therapeutic effect of Zhenyuan granules on obesity and uterine atrophy caused by estrogen deficiency.

[0308] 1. Zhenyuan granules inhibit the adipogenic differentiation of 3T3-L1 preadipocytes.

[0309] 1.1 Experimental Materials

[0310] The mouse 3T3-L1 preadipocyte cell line, derived from the American Type Culture Center (ATCC), was cultured in DMEM high-glucose medium containing 10% newborn calf serum at 37°C, 5% CO2, and saturated humidity. Cells were passaged when confluence reached 70–80%.

[0311] Zhenyuan Granule Stock Solution: Accurately weigh 200 mg of Zhenyuan granules, dissolve in DMSO, filter through a 0.22 µM filter membrane, and prepare a 200 mg / mL Zhenyuan granule stock solution. Store the stock solution at -20°C and bring it to room temperature before use.

[0312] Adipogenic differentiation induction medium: The induction medium for days 1-2 was a growth medium containing 1 μM dexamethasone, 10 µg / mL insulin, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX); the induction medium for days 3-4 was a growth medium containing 10 µg / mL insulin; and the culture medium was used for days 5-8. The culture medium was changed every 2 days.

[0313] 1.2 Preadipocyte-induced adipogenic differentiation experiment

[0314] Mouse 3T3-L1 preadipocytes were seeded into 96-well plates and cultured. The culture medium was changed every 2 days. After the cells reached complete confluence and were exposed to inhibition for 2 days, the cells were divided into 6 groups, with 6 replicates per group. Adipogenic differentiation induction was induced for 8 days using adipogenic differentiation induction medium with or without the test drug.

[0315] (1) Undifferentiated group: cultured in growth medium;

[0316] (2) Differentiation induction control group: cultured using adipogenic differentiation induction solution;

[0317] (3) Zhenyuan Particle 100 µg / mL group: cultured with adipogenic differentiation induction solution containing 100 µg / mL Zhenyuan Particle;

[0318] (4) Zhenyuan Particle 200 µg / mL group: cultured with adipogenic differentiation induction solution containing 200 µg / mL Zhenyuan Particle;

[0319] (5) Zhenyuan Particle 400 µg / mL group: cultured with adipogenic differentiation induction solution containing 400 µg / mL Zhenyuan Particle;

[0320] (6) Lovastatin group: cultured with adipogenic differentiation induction solution containing 10 µmol / L lovastatin.

[0321] 1.2.1 Effect of Zhenyuan Particles on Adipogenic Differentiation Rate

[0322] After induction, adipocytes were stained with Oil Red O, and intracellular lipid droplet formation was observed under a microscope. Then, 100 µL of isopropanol was added to each well to dissolve Oil Red O, and the OD value was detected at a wavelength of 492 nm.

[0323] The adipogenic differentiation rate under drug action was calculated using the following formula to evaluate whether Zhenyuan granules have an inhibitory effect on adipocyte differentiation:

[0324] Adipogenic differentiation rate (%) = 100 × (OD) 药物 / 样品 -OD 未分化 ) / (OD 对照 -OD 未分化 )

[0325] 1.2.2 Effects of Zhenyuan Granules on the mRNA Expression of Adipogenic Differentiation-Related Genes in Mouse Preadipocytes

[0326] Cells after adipogenic differentiation induction were collected in 1.5 mL RNase-free EP tubes, and Trizol reagent was added for digestion on ice for 5 min. Chloroform was added, followed by vortex extraction and centrifugation (12000 r / min, 4℃, 10 min). The supernatant was collected, and an equal volume of isopropanol was added and mixed thoroughly. After standing, the mixture was centrifuged again, and the supernatant was discarded. The precipitate was washed with 75% ethanol, dried, and dissolved in DEPC water to obtain an RNA solution. The purity and concentration of the RNA solution were determined using a micro-spectrophotometer. Then, 1 µg of RNA was taken and reverse transcribed into cDNA according to the instructions of the PrimeScript RT reagent Kit with gDNA Eraser. Primers and TB Green were then added as a template. Premix Ex Taq II and DEPC water, with β-actin Genes are used as internal controls, according to TB Green Premix Ex Taq II. The instruction manual indicates that qPCR analysis should be performed to detect transcriptional genes regulating adipogenic differentiation. C / EBPα , PPARy , SREBP-1c , ACC1 The mRNA expression level was determined. The primer sequences used in the experiment are shown in Table 3.

[0327] Table 3 Primer Sequences

[0328]

[0329] 1.2.3 Effects of Zhenyuan Granules on the Expression Levels of AMPK Signaling Pathway and Downstream Adipogenic Key Proteins in Mouse Preadipocytes

[0330] After the induction of adipogenic differentiation was completed, 100 µL of lysis buffer (RIPA:PMSF:phosphatase inhibitor = 100:1:1) was added to each well, and the cells were lysed on ice for 30 min. The cells were transferred to a 1.5 mL EP tube using a pipette and centrifuged (12,000 r / min, 4 °C, 15 min), and the supernatant was taken. The total protein concentration was measured using a BCA protein assay kit, with bovine serum albumin (BSA) as the standard. Then, the protein samples containing loading buffer were separated by SDS-PAGE gel, and the separated protein bands were transferred to a polyvinylidene fluoride (PVDF) membrane; blocked with 5% milk for 1-2 h; incubated overnight at 4 °C with the corresponding primary antibodies. The dilution ratios of the primary antibodies corresponding to the proteins were as follows: AMPK antibody (1:1000), p-AMPK antibody (1:500), ACC1 antibody (1:1000), p-ACC1 antibody (1:1000), PPARγ antibody (1:1000), SREBP-1c antibody (1:1000), FAS antibody (1:2000), β-actin antibody (1:3000); then washed 4 times with TBS-Tween20 buffer, 5 min each time; incubated with the secondary antibody (1:2000) for 1 h at room temperature, and then washed 3 times with Tris-buffered saline (TBS)-Tween-20 buffer, 10 min each time; finally, the protein bands were visualized using an enhanced chemiluminescence (ECL) substrate, scanned using an imaging system, and the band optical density was analyzed using image analysis software (ImageJ), with β-actin as the internal reference.

[0331] 2. Effects of Zhenyuan Granules on body weight gain, fat accumulation, and uterus in estrogen-deficient mice

[0332] 2.1 Animal grouping and treatment

[0333] SPF-grade female C57BL / 6J mice (60 mice, 12 weeks old, weighing approximately 20 g) were purchased from Zhuhai Bestong Biotechnology Co., Ltd., with the production license number SCXK (Guangdong) 2020-0051.

[0334] This experiment was conducted at the Experimental Animal Center of Guangzhou University of Chinese Medicine (license number: SYXK (Guangdong) 2018-0001), and the Animal Experiment Ethics Committee of Guangzhou University of Chinese Medicine reviewed and approved the animal experiment ethics (approval number: 20220722002).

[0335] The mice were housed in an SPF-grade barrier environment (light / dark rhythm 12 h / 12 h, temperature 23 ± 2 °C, relative humidity 55-65%), fed a maintenance diet, and allowed free access to food and water. They were adaptively fed for one week.

[0336] After one week of acclimatization, 60 mice were randomly divided into two groups: a sham-operated control group (equal volume of pure water, n=10) and a bilateral ovariectomy model group (n=50). The 50 mice in the bilateral ovariectomy model group underwent surgical modeling. Two weeks post-surgery, the mice with bilateral ovariectomy were randomly divided into the following 5 groups, with 10 mice in each group:

[0337] (1) Model control group (equal volume of pure water);

[0338] (2) Orlistat group (positive weight loss drug, 74 mg / kg);

[0339] (3) Estradiol valerate group (positive estrogen replacement therapy, 0.2 mg / kg);

[0340] (4) Low-dose group of Zhenyuan granules (0.63 g / kg);

[0341] (5) High dose group of Zhenyuan granules (3.77 g / kg).

[0342] The mice were administered the above-mentioned doses via gavage to the sham-operated control group and each model group for 14 consecutive weeks, once daily, at a volume ratio of 15 mL / kg. The weight of the mice was measured periodically during the administration period.

[0343] After drug administration, mice were fasted but allowed free access to water overnight (12 h). Mice were then euthanized under sodium pentobarbital anesthesia, and blood was collected from the orbital rim. The blood was allowed to stand at room temperature for 30 min, then centrifuged (3000 r / min, 15 min, 4℃) to separate serum, which was then stored at -80℃ for later use. Gonadal and inguinal adipose tissue, as well as the uterus, were collected, weighed, and a portion of the adipose tissue was stored at -80℃. The remaining tissue was fixed with 4% paraformaldehyde solution for subsequent analysis.

[0344] 2.2 Histopathological Analysis

[0345] Adipose tissue was collected from the uterus, gonads, and inguinal adipose tissue after 48 hours of fixation. Routine pathological sections were prepared and stained with hematoxylin and eosin (HE). The morphology of the uterus and the morphology of adipocytes in the adipose tissue were observed under a microscope. Adipocyte size analysis was performed using ImageJ software. Adipocyte size was expressed as the average diameter (μm) and average cross-sectional area (μm) of each cell in the tissue sample. 2 ).

[0346] 2.3 Serum Biochemical Analysis

[0347] Serum TC levels were measured using a total cholesterol (TC) assay kit (Nanjing Jiancheng Bioengineering Institute). Serum leptin levels were measured using a leptin enzyme-linked immunosorbent assay (ELISA) kit (Wuhan Huamei Bioengineering Co., Ltd.).

[0348] 2.4 Effects of Zhenyuan Granules on the Expression Levels of AMPK Signaling Pathway and Downstream Key Adipogenic Proteins in Adipose Tissue of Estrogen-Deficient Mice

[0349] A certain amount of mouse white adipose tissue was weighed and mixed with RIPA lysis buffer and a protease-phosphatase inhibitor mixture (50:1) at a ratio of 1:10. The mixture was ground in an ice bath and centrifuged (15000 r / min, 4℃, 10 min). The supernatant was collected. The total protein concentration was determined using a BCA protein assay kit, with bovine serum albumin (BSA) as the standard. The protein sample containing loading buffer was then separated using an SDS-PAGE gel, and the separated protein bands were transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% milk for 1–2 minutes. h; Incubate overnight at 4°C with the corresponding primary antibodies. The primary antibody dilution ratios for the proteins were as follows: AMPK antibody (1:1000), p-AMPK antibody (1:500), ACC1 antibody (1:1000), p-ACC1 antibody (1:1000), PPARγ antibody (1:1000), SREBP-1c antibody (1:1000), FAS antibody (1:2000), and β-actin antibody (1:3000). Then wash 4 times with TBS-Tween 20 buffer for 5 min each time. Incubate with secondary antibody (1:2000) at room temperature for 1 h, and then wash 3 times with Tris salt buffer (TBS)-Tween-20 buffer for 10 min each time. Finally, visualize the protein bands using enhanced chemiluminescence (ECL) substrate, scan using an imaging system, and analyze the band optical density using image analysis software (ImageJ). β-actin was used as an internal control.

[0350] 2.5 Statistical Analysis

[0351] SPSS 22.0 statistical analysis software was used to analyze the experimental data, and the results are expressed as mean ± SD. One-way ANOVA was used for statistical analysis; the LSD test was used for comparisons when variances were homogeneous, and Dunnett's test was used when variances were unequal. P A value <0.05 indicates that the data difference is statistically significant. Image output was processed using GraphPad Prism 9.5 software.

[0352] 3. Experimental Results

[0353] 3.1 Zhenyuan Granules Inhibit Adipogenic Differentiation of Mouse Preadipocytes

[0354] This experiment investigated the effects of different concentrations of Zhenyuan particles (100, 200, and 400 µg / mL) on adipogenic differentiation of 3T3-L1 preadipocytes. The results are as follows: Figure 29As shown: Undifferentiated 3T3-L1 preadipocytes were barely stained with Oil Red O, while in the differentiation control group, the vast majority of cells became round with prominent intracellular oil droplets, staining red with Oil Red O, indicating they had differentiated into mature adipocytes. Compared to the differentiation control group, the number of intracellular lipid droplets was significantly reduced in the positive control lovastatin group and all Zhenyuan granule groups, with varying degrees of reduction among the Zhenyuan granule groups, showing a dose-response relationship.

[0355] The adipogenic differentiation rate was quantified by dissolving Oil Red O in isopropanol, and the results are shown in Table 4. The adipogenic differentiation rates of both the control drug lovastatin and the Zhenyuan granules groups decreased to varying degrees. The degree of decrease in adipogenic differentiation rate varied among the Zhenyuan granule groups, showing a dose-response relationship. These results indicate that Zhenyuan granules significantly inhibited adipocyte formation.

[0356] Table 4 Effects of Zhenyuan granules on adipogenic differentiation of mouse preadipocytes (mean ± SD, n = 3)

[0357]

[0358] Compared with the differentiation-inducing control group, *** P <0.001.

[0359] 3.2 Zhenyuan particles inhibit the mRNA expression of adipogenic differentiation-related genes.

[0360] Key transcription genes for adipogenesis in cells of each group at the end of differentiation induction were detected using real-time quantitative PCR. PPARγ and C / EBPα Key genes for fat synthesis SREBP - 1c The mRNA levels, the results are as follows Figure 30 As shown: Compared with the undifferentiated group, the differentiated control group PPARγ , C / EBPα , SREBP - 1c and ACC1 The mRNA level of the gene was significantly upregulated; compared with the differentiation-inducing control group, the levels of the Zhenyuan granules intervention in each group were significantly higher. PPARγ , C / EBPα , SREBP-1 and ACC1 The mRNA expression level of the gene was significantly downregulated, and the difference was statistically significant. P <0.05, P <0.01 or P <0.001). The results confirmed that Zhenyuan particles can directly inhibit adipocyte differentiation and lipid synthesis, and this effect is related to the inhibition of the expression of key transcription factors for adipocyte differentiation and key genes for lipid synthesis.

[0361] 3.3 Zhenyuan particles activate the AMPK signaling pathway and inhibit the expression of downstream key adipogenic proteins.

[0362] This experiment examined the expression of the AMPK / ACC signaling pathway and key proteins in downstream adipogenic differentiation and lipid synthesis in cells of each group after differentiation induction. The results are as follows: Figure 31 As shown: Compared with the differentiation-inducing control group, p-AMPK / AMPK and p-ACC1 / ACC1 were significantly upregulated in the Zhenyuan Granules group. Figure 31 In Figures B-C, the expression of ACC1, SREBP-1c, PPARγ, and FAS was significantly downregulated. Figure 31 (See Figures D-G in the original text). The results of this experiment further confirm that the Zhenyuan particles have an inhibitory effect on adipocyte differentiation and adipogenesis, and that the adipogenic inhibition is related to the activation of AMPK / ACC signal transduction.

[0363] 3.4 Zhenyuan granules inhibited weight gain and fat accumulation in estrogen-deficient mice.

[0364] This experiment evaluated the inhibitory effect of Zhenyuan granules on estrogen deficiency-induced weight gain and fat accumulation in a bilateral ovariectomized mouse model, using body weight and the size of subcutaneous and visceral adipocytes as indicators. The results showed that Zhenyuan granules significantly inhibited weight gain and adipocyte hypertrophy in ovariectomized mice, with effects comparable to the weight-loss drug orlistat and estrogen replacement therapy. Figure 32 The trend of mouse weight gain during treatment ( Figure 32 Figure A) and net weight gain ( Figure 32 Figure B shows the following: Compared with the sham-operated control group, the model control group mice showed a faster rate of weight gain and a significant net weight gain. Compared with the model control group, the high-dose Zhenyuan granule group, low-dose Zhenyuan granule group, orlistat group, and estradiol valerate group showed a slower rate of weight gain and a significant decrease in net weight gain. In the experiment, the weight gain trends of the high-dose and low-dose Zhenyuan granule groups were basically consistent with those of the orlistat group or estradiol valerate group, and the differences in net weight gain were not significant.

[0365] Figure 33 The net weight, histopathological images, and average cross-sectional area and diameter of adipocytes of gonadal adipose tissue (pWAT) (visceral fat) and inguinal adipose tissue (ingWAT) (subcutaneous fat) of mice in each group were recorded. Compared with the sham-operated control group, the gonadal adipose tissue of the model control group mice (pWAT) was significantly different. Figure 33 (Figure A in the image) and groin fat ( Figure 33(Figure B) The weight of adipocytes increased, and the average cross-sectional area and diameter of adipocytes increased significantly, indicating excessive intracellular fat accumulation. Compared with the model control group, the weight of gonadal and inguinal fat decreased in the orlistat group, estradiol valerate group, and Zhenyuan granule group, and the average diameter and average cross-sectional area of ​​adipocytes were significantly reduced. The effects of Zhenyuan granules were similar to those of orlistat and estradiol valerate. These results indicate that Zhenyuan granules can inhibit adipocyte hypertrophy.

[0366] 3.5 Zhenyuan granules improve uterine atrophy in estrogen-deficient mice

[0367] Figure 34 The results of HE staining histopathological analysis of the uterus tissue of mice in each group are shown. Compared with the sham-operated control group, the uterus of the model control group mice was significantly atrophied. As expected, uterine atrophy in the estradiol valerate group was significantly improved, and estradiol valerate could significantly inhibit uterine atrophy in mice. No improvement was observed in uterine atrophy in the orlistat group, and orlistat could not inhibit uterine atrophy. The low- and high-dose groups of Zhenyuan granules showed varying degrees of significant improvement in uterine atrophy, indicating that Zhenyuan granules have a certain protective effect on the uterus of ovariectomized mice.

[0368] 3.6 Zhenyuan granules improved serum leptin and total cholesterol levels in estrogen-deficient mice.

[0369] Serum biochemical analysis results as follows Figure 35 As shown in the figure, compared with the sham-operated control group, the serum total cholesterol and leptin levels in the model control group were significantly increased. Compared with the model control group, the serum total cholesterol levels in all drug treatment groups decreased, with the orlistat group and the high-dose Zhenyuan granules group showing the most significant decreases; the serum leptin levels in all drug treatment groups also decreased significantly. This indicates that Zhenyuan granules can improve adipocyte dysfunction and cholesterol metabolism abnormalities caused by estrogen deficiency.

[0370] 3.7 Zhenyuan granules activate the AMPK signaling pathway in adipose tissue of estrogen-deficient mice and inhibit the expression of downstream key adipogenic proteins.

[0371] This experiment examined the expression of the AMPK / ACC signaling pathway and downstream adipogenic differentiation and adipogenesis-related proteins in the adipose tissue of mice in each group. The results showed that compared with the sham-operated control group, the levels of p-AMPK / AMPK and p-ACC1 / ACC1 in the model control group mice were significantly reduced. Figure 36 (Figures B-C in the table), while the levels of ACC1 and adipogenic differentiation-related proteins SREBP-1c, PPARγ, and adipogenesis-related protein FAS were significantly upregulated ( Figure 36Figures D-G in the table illustrate that estrogen deficiency inhibits AMPK activity in adipocytes, leading to increased lipogenesis and decreased fatty acid metabolism. Compared with the model control group, the levels of p-AMPK / AMPK and p-ACC1 / ACC1 in the adipose tissue of mice in each group treated with Zhenyuan granules were significantly upregulated, while the expression of ACC1, SREBP-1c, PPARγ, and FAS was significantly downregulated. These results indicate that at the animal level, intervention with Zhenyuan granules activates the AMPK pathway in adipose tissue, thereby inhibiting lipogenesis, enhancing fatty acid metabolism, and suppressing the rapid weight gain and fat accumulation in estrogen-deficient mice.

[0372] This invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of this invention shall be considered equivalent substitutions and shall be included within the protection scope of this invention.

Claims

1. A traditional Chinese medicine composition, characterized in that, It is composed of the following components in parts by weight: Epimedium 2-8 parts, dried ginger 2-8 parts, licorice 2-8 parts, ginseng 1-4 parts, Poria cocos 1-4 parts, and Salvia miltiorrhiza 1-4 parts.

2. A traditional Chinese medicine extract, characterized in that, The traditional Chinese medicine composition of claim 1 is extracted by water decoction extraction method.

3. The herbal extract according to claim 2, characterized in that, The water decoction extraction method includes the following steps: S1. Water extraction: Take the raw material and decoct it 3-4 times with water; each time add 8-20 times the weight of the raw material with water, decoct for 2-3 hours, cool, and filter; combine the filtrates; S2. Concentration: Centrifuge the filtrate, collect the supernatant and concentrate it under reduced pressure to obtain the water extract concentrate.

4. A traditional Chinese medicine oral preparation, characterized in that, An oral preparation is made from the traditional Chinese medicine composition of claim 1 or the traditional Chinese medicine extract and excipients of claim 2 or 3.

5. The use of the traditional Chinese medicine composition of claim 1, the traditional Chinese medicine extract of claim 2 or 3, or the traditional Chinese medicine oral preparation of claim 4 in the preparation of a drug having the effects of preventing and treating impotence, anti-fatigue, preventing and treating myocardial infarction, or preventing and treating estrogen deficiency-related diseases; wherein the estrogen deficiency-related diseases refer to osteoporosis and obesity caused by estrogen deficiency after menopause in women.

6. The application of the traditional Chinese medicine composition of claim 1, the traditional Chinese medicine extract of claim 2 or 3, or the traditional Chinese medicine oral preparation of claim 4 in the preparation of a drug that simultaneously has multiple effects such as preventing and treating impotence, anti-fatigue, preventing and treating myocardial infarction, and preventing and treating estrogen deficiency-related diseases; wherein the estrogen deficiency-related diseases refer to osteoporosis and obesity caused by estrogen deficiency after menopause in women.

7. The application of the traditional Chinese medicine composition of claim 1, the traditional Chinese medicine extract of claim 2 or 3, or the traditional Chinese medicine oral preparation of claim 4 in the preparation of health food, wherein the health food has the effect of relieving male physical fatigue.

8. The use of the traditional Chinese medicine composition of claim 1, the traditional Chinese medicine extract of claim 2 or 3, or the traditional Chinese medicine oral preparation of claim 4 in the preparation of health food, wherein the health food helps control body fat caused by estrogen deficiency in postmenopausal women.

9. The use of the traditional Chinese medicine composition of claim 1, the traditional Chinese medicine extract of claim 2 or 3, or the traditional Chinese medicine oral preparation of claim 4 in the preparation of health food, wherein the health food helps to improve osteoporosis caused by estrogen deficiency in postmenopausal women.

10. The method for establishing the fingerprint spectrum of the oral preparation of traditional Chinese medicine according to claim 4, characterized in that, Includes the following steps: S1. Reference solution: methanol solution of icariin, icariin A, icariin B, icariin C, glycyrrhizin, ammonium glycyrrhizate, quercetin, and salvianolic acid C; S2. Test solution: Add the oral Chinese medicine preparation to 50%–80% ethanol at a ratio of (0.1–2) g: 100 mL, sonicate, centrifuge and take the supernatant, which is the test solution; S3. Perform HPLC chromatographic analysis. The test solution was analyzed by HPLC, with the reference solution as a control. The HPLC chromatographic conditions were as follows: The chromatographic column was a Kromasil 100-5-C18 column, with dimensions of 250 mm × 4.6 mm and a diameter of 5 µm. Mobile phase A is acetonitrile, and mobile phase B is a 0.1% formic acid aqueous solution. The detection wavelength was 254 nm, the column temperature was 30℃, and the flow rate was 1 mL / min. The mobile phase elution gradient is as follows: Continue elution until the end; thus obtaining the fingerprint spectrum of the oral Chinese medicine preparation.

11. The quality testing method for oral Chinese medicine preparations according to claim 4, characterized in that, The method includes the following steps: comparing the HPLC chromatogram of the sample to be tested with the fingerprint chromatogram obtained in claim 10, and determining whether the sample is qualified based on the retention time, peak area ratio and peak order of the HPLC chromatogram of the sample to be tested and the reference standard; wherein, the method for obtaining the HPLC chromatogram of the sample to be tested is the same as the method for establishing the fingerprint chromatogram of the oral Chinese medicine preparation described in claim 10.

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