New use of dishen decoction in preparation of drugs for preventing and treating postmenopausal osteoporosis

Abstract

The present application belongs to the technical field of medicine, and relates to application of Deshen decoction in preparation of a medicine for preventing and treating postmenopausal osteoporosis. Deshen decoction comprises the following three traditional Chinese medicine raw materials: 500 parts by weight of yam (i.e. Chinese yam) powder, 200-300 parts by weight of almond (soaked in soup, with skin and tip removed, and with double kernels), and 800-1200 parts by volume of raw cow milk. The present application proves through animal experiments that Deshen decoction can effectively improve the low bone mineral density (BMD) of ovariectomized osteoporosis model mice, improve bone biomechanical properties, and improve bone tissue microstructure, thereby providing a safe and effective new medicine selection for prevention and treatment of postmenopausal osteoporosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine and relates to the application of Dixian Decoction in the preparation of drugs for preventing and treating postmenopausal osteoporosis. Background Art

[0002] Postmenopausal osteoporosis (PMOP) is a systemic metabolic bone disease closely related to menopause, characterized by reduced bone mass, destruction of bone tissue microstructure, increased bone fragility, and elevated fracture risk. With the accelerating aging process of the population in China, PMOP has become a common and frequently-occurring disease that seriously affects the quality of life of elderly women.

[0003] Currently, the clinical treatment of PMOP in Western medicine mainly uses bone resorption inhibitors (such as bisphosphonates) and selective estrogen receptor modulators and other drugs to inhibit bone resorption and improve bone metabolism. However, while these traditional drugs exert their therapeutic effects, they are often accompanied by adverse reactions such as gastrointestinal reactions, osteonecrosis of the jaw, atypical femoral fractures, etc., and patients need to take medicine for a long time, and the compliance is often not ideal. Traditional Chinese medicine has advantages such as good tolerance and low toxicity in the treatment of PMOP. At present, some traditional Chinese medicine compound prescriptions have been reported to be used in PMOP, but their compositions are usually relatively complex, and the therapeutic mechanism still needs to be further studied.

[0004] Dixian Decoction (also known as Dixian Ointment) is a diet therapy and medicinal diet prescription in ancient China, which comes from Volume 186 of "Complete Record of Holy Relief" in the Northern Song Dynasty and is composed of three food and medicine homologous products, namely Chinese yam, almonds, and fresh cow milk. Ancient books record that it has the effect of "making people have a pleasant complexion, firm bones and marrow, and being able to walk as fast as a galloping horse", and is mainly used to treat "waist and knee pain and all cold diseases in the abdomen"; "Essentials of Health Preservation" summarizes its effect as "benefiting body fluids and moistening dryness", and is mainly used to treat "all dryness syndromes"; "Supplements to the Yuan of Life Preservation" records that this prescription "nourishes the spleen yin, benefits the lung qi, and tonifies the five zang-organs", and is applicable to "tiredness and weakness, waist and knee pain and all cold diseases in the abdomen".

[0005] In existing research, only literature reports that Dixian Decoction has a protective effect on the cartilage tissue of mice with knee osteoarthritis (KOA), but there has never been a relevant report on its use for preventing and treating postmenopausal osteoporosis. There are essential differences in the pathological mechanisms between knee osteoarthritis and postmenopausal osteoporosis: the former takes the degenerative change of articular cartilage as the core and belongs to a local degenerative disease; the latter is characterized by bone metabolic imbalance, bone mass loss, and bone microstructure destruction and belongs to a systemic metabolic disease. Summary of the Invention

[0006] The present invention aims to provide a new pharmaceutical use of Dixian Decoction, that is, for the preparation of drugs for preventing and treating postmenopausal osteoporosis. The present invention has confirmed through animal experiments that Dixian Decoction can effectively improve the low bone mineral density (BMD) of ovariectomized osteoporosis model mice, improve bone biomechanical properties, and improve bone tissue microstructure, thus providing a safe and effective new drug option for the prevention and treatment of postmenopausal osteoporosis.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] Dixian Decoction consists of the following three Chinese medicinal ingredients: 500 parts by weight of yam powder, 200-300 parts by weight of almonds (soaked in water, skin and tips removed), and 800-1200 parts by volume of raw milk.

[0009] The term "volume part" is equivalent to "milliliters," meaning that 1 volume part corresponds to 1 milliliter.

[0010] The formula's ingredients are arranged as follows: Yam is the principal ingredient, tonifying the kidneys and replenishing essence, strengthening the spleen and boosting qi, and fortifying muscles and bones, addressing the root cause of "bone atrophy" due to kidney deficiency; Apricot kernel is the assistant ingredient, moistening the lungs to promote fluid production; Raw milk is the adjuvant ingredient, generating fluids and blood to moisten dryness. Together, these three ingredients tonify the spleen and kidneys, nourish yin and blood, and strengthen muscles and bones. This formula is based on the principle of food and medicine sharing the same origin, making it highly safe and particularly suitable for the long-term prevention and treatment of chronic osteoporosis in postmenopausal women.

[0011] The preparation method of Dixian Decoction includes the traditional preparation process recorded in Volume 186 of Shengji Zonglu: grind yam powder, almonds (soaked in soup, peeled, tips and double kernels removed), and raw milk together, put them into a porcelain container, cook them in soup to make a paste, and store them in a sealed porcelain bottle.

[0012] Alternatively, modern preparation processes can be used: yam is pulverized into fine powder, almonds are pulverized or ground after removing the tips and skins, mixed with raw milk, and then homogenized and dried to produce solid dosage forms (such as powders, granules, tablets, capsules, etc.), or directly produced into liquid preparations (such as oral liquids, emulsions, etc.).

[0013] Dixian decoction can be prepared into any dosage form suitable for oral administration, including but not limited to: oral liquid, emulsion, powder, granules, tablets, capsules, pills, ointment, etc.; pharmaceutically acceptable excipients may also be added to the dosage forms.

[0014] Preliminary pharmacodynamic studies have shown that Dixian Decoction exerts its anti-PMOP effect through the following mechanisms: (1) Promotes bone formation, inhibits bone resorption, and improves bone metabolism balance; (2) Increase bone density and enhance bone biomechanical properties; (3) Improves bone tissue microstructure and increases the number of trabeculae; (4) Inhibit osteoblast apoptosis and related signaling pathways.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0016] 1. New Use Revealed for the First Time: The novel use of Dixian Decoction in preventing and treating postmenopausal osteoporosis has not been previously reported, and this invention represents a first-time discovery. While the traditional effects of Dixian Decoction ("strengthening bone marrow" and "relaxation and knee pain") are related to bone health, "strengthening bone marrow" traditionally refers to increased toughness of articular cartilage and bones, which is not equivalent to the specific bone metabolic disease "osteoporosis" in modern medicine. This invention applies to postmenopausal osteoporosis (a systemic bone metabolic disease characterized by bone loss and destruction of bone microstructure), which differs from the pathological mechanism of knee osteoarthritis (the former is mainly characterized by cartilage degeneration, while the latter is mainly characterized by an imbalance between bone resorption and bone formation). Those skilled in the art would not readily associate its novel use in preventing and treating PMOP with the known therapeutic uses for knee osteoarthritis.

[0017] 2. Food and Medicine from the Same Source, High Safety: Dixian Decoction is composed of three ingredients that are both food and medicine, namely yam, almond, and raw milk, all of which are commonly used food ingredients and medicinal materials. During animal experiments, mice in all groups treated with Dixian Decoction showed normal activity, appetite, and defecation, and no obvious toxic side effects were observed. Compared with existing Western medicines, Dixian Decoction has a higher safety profile for long-term use, making it particularly suitable for postmenopausal osteoporosis patients who require long-term medication, and helping to improve patient compliance.

[0018] 3. Simple formulation and easy quality control: Compared with the traditional Chinese medicine compound formulas for PMOP reported in the existing technology (usually composed of multiple, a dozen or even dozens of Chinese medicines), Dixian Decoction is composed of only three herbs. The formulation is concise, the herbs are clearly defined, and each component is a food and medicine homology variety with stable source. The quality control is easy and standardized production and quality traceability are easy to achieve.

[0019] 4. Synergistic effect of multiple targets: By leveraging the TCM theory of "tonifying the kidney and spleen, replenishing qi and nourishing blood, and strengthening muscles and bones", it exerts an overall regulatory effect and can intervene in the occurrence and development of PMOP from multiple aspects.

[0020] 5. Prevention and Treatment: Experimental results show that Dixian Decoction can not only improve existing osteoporosis (therapeutic effect), but also effectively prevent further bone loss during administration (preventive effect). Therefore, Dixian Decoction can be used for early prevention in high-risk groups of PMOP, as well as for clinical treatment of confirmed patients, and has a wider range of application prospects. Attached Figure Description

[0021] Figure 1 Micro-CT three-dimensional reconstruction image of the distal femur of a mouse.

[0022] Figure 2 Changes in morphometric parameters of mouse bone tissue.

[0023] Figure 3 HE staining of the distal femur of mice (scale bar = 300 μm).

[0024] Figure 4 The results are from mouse serum ELISA assays.

[0025] Figure 5 Protein expression in mouse femoral tissue.

[0026] Figure 6 Normalized statistics for Western blot (WB) results of mouse femur. Detailed Implementation

[0027] The present invention will now be described in detail with reference to specific embodiments. The following embodiments will help to understand the present invention, but these embodiments are only for illustrative purposes, and the present invention is not limited thereto. The operating methods in the embodiments are all conventional operating methods in this technical field.

[0028] Example 1.

[0029] Preparation of Dixian Decoction: Take 500g of yam, 200g of almonds, and 800ml of raw milk. Grind the naturally dried yam and almonds together in a mortar and pestle. Add the ground mixture to the raw milk, place in an earthenware pot, and simmer over low heat until the broth thickens into a paste. Stir constantly to prevent sticking to the bottom of the pot or browning. When the paste becomes non-flowable, stop heating and allow it to air dry naturally. Grind it into powder using a mortar and pestle. Do not add any preservatives and immediately seal and store.

[0030] Example 2: Pharmacodynamic experimental verification.

[0031] 1. Experimental Materials (1) Test drug: Dixian decoction prepared according to the method in Example 1; (2) Experimental animals: Forty 6-week-old SPF-grade nulliparous female C57BL / 6J mice, weighing (20±2g), were purchased from Liaoning Changsheng Biotechnology Co., Ltd., which has obtained the corresponding animal production license, batch number SCXK(Liaoning)2020-0001. Animal use license number: SYXK(Liaoning)2024-0006. They were fed standard nutrient pellet feed and distilled water for 7 days for acclimatization. (3) Main reagents and instruments: small animal CT scanner, multifunctional enzyme-linked immunosorbent assay (ELISA) reader, pathological slide cutter, fully automated immunohistochemical staining machine, optical microscope, etc.

[0032] 2. Experimental Methods 2.1 Animal grouping and administration regimen Forty C57BL / 6J mice were randomly divided into four groups of 10 mice each: Normal control group (Blank group): No surgery was performed, and the same volume of distilled water was administered by gavage; Sham group: An equal amount of fat was removed by sham surgery, and an equal volume of distilled water was administered by gavage; Model group (OVX group): Ovaries removed for modeling, and equal volume of distilled water was administered by gavage; Dixian Decoction Group (DXJ Group): Ovaries were removed to create the model, and Dixian Decoction solution (3.38 g / kg / day) was administered by gavage. Except for the Sham group, mice in all other groups underwent bilateral ovariectomy (OVX) to establish a postmenopausal osteoporosis model. The specific modeling method was as follows: after anesthesia, the skin and muscles were incised on both sides of the spine under sterile conditions to locate and remove the ovaries, and the incisions were sutured. The mice were then fed for 12 weeks post-surgery to confirm successful osteoporosis model establishment. Starting from the second week post-surgery, each group received the medication once daily via gavage according to the above protocol for 12 consecutive weeks. All animals had free access to food and water during the experiment.

[0033] 2.2 Observation Indicators and Detection Methods (1) General condition observation: Observe the animal's coat color, spirit, activity, diet, defecation, etc. every day. Weigh the animal weekly.

[0034] (2) Bone mineral density (BMD) measurement: Animals were sacrificed 12 weeks after drug administration, and the right femur was harvested. Soft tissue was removed, and the femoral BMD value was measured using a dual-energy X-ray absorptiometry (unit: g / cm³). 2 ).

[0035] (3) Observation of bone tissue morphology: The proximal end of the right tibia was taken, fixed with 4% paraformaldehyde, decalcified with EDTA, dehydrated, embedded in paraffin, sectioned (thickness 5μm), stained with hematoxylin-eosin (HE), and the pathological morphological changes of bone tissue were observed under a light microscope. Parameters such as the percentage of trabecular area (%Tb·Ar), the thickness of trabecular bone (Tb·Th, μm), and the separation degree of trabecular bone (Tb·Sp, μm) were measured.

[0036] (4) Detection of biochemical indicators of bone metabolism: Animal serum was collected and estradiol (E2), tumor necrosis factor α, type I procollagen amino-terminal propeptide (PINP), type I collagen cross-linked carboxyl-terminal peptide (CTX-1), and tartrate resistant acid phosphatase (TRAP) were measured by enzyme-linked immunosorbent assay (ELISA).

[0037] (5) Signal pathway detection: Total protein was extracted from bone tissue, and the expression levels of key molecules in bone metabolism-related signal pathways such as Bcl-2, Bax, Caspase-3 and its activated form cleaved Caspase-3 were detected by Western Blot.

[0038] 3. Experimental Results 3.1 Micro-CT Three-Dimensional Reconstruction of Distal Femur in Mice like Figure 1 As shown, compared with Sham, the OVX group mice showed significant deterioration in the distal femoral bone microstructure, specifically manifested as a decrease in the number and density of trabeculae, a decline in trabecular connectivity, and an overall typical osteoporotic change. After intervention with Dixian Decoction, the distal femoral bone microstructure of mice was significantly improved, including a reduction in the number and spacing of trabeculae, and enhanced bone structural integrity.

[0039] 3.2 Bone histomorphometric parameters like Figure 2 As shown ( (±S, n=10) Compared with the Sham group, the OVX group mice showed a significant decrease in femoral bone mineral density (BMD) and bone microstructure parameters, including trabecular bone number (Tb.N), bone volume fraction (BV / TV), and bone surface area to tissue volume ratio (BS / TV) (P<0.001), indicating successful establishment of the osteoporosis model. After DXJ intervention, compared with the OVX group, Tb.N, BV / TV, and BS / TV were all significantly improved (P<0.05), indicating that the administration of Dixian Decoction can effectively inhibit the degeneration of trabecular bone structure.

[0040] 3.3 Results of bone tissue morphology observation like Figure 3 As shown, the HE staining results indicate that: Sham group: The trabecular structure is intact, regularly arranged, and tightly connected. The number of trabeculae is abundant, the thickness is uniform, and the size of the medullary cavity is normal.

[0041] OVX group: The number of trabeculae was significantly reduced, the arrangement was sparse and irregular, the connection was incomplete, the trabeculae were thinner, the medullary cavity was significantly enlarged, and a large amount of fibrous tissue and fat cell infiltration were visible, presenting typical osteoporosis.

[0042] The number of trabeculae in the Dixianjian group increased compared to the OVX group, their arrangement became more regular, the thickness of the trabeculae was relatively uniform, and the morphology of the bone tissue was significantly improved compared to the OVX group.

[0043] 3.4 Results of Bone Metabolic Biochemical Indicators like Figure 4 As shown ( (±S, n=10) Compared with the model group, the bone metabolism biochemical indicators of the Dixianjian group were significantly restored compared with the OVX group.

[0044] 3.5 Signal Path Detection Results like Figure 5 and Figure 6 ( As shown in the figure (±S, n=10), compared with the Sham group, the expression levels of pro-apoptotic proteins Bax, Caspase-3 and their activated form cleaved Caspase-3 in the femoral tissue of OVX group mice were significantly upregulated (P<0.05), while the expression of anti-apoptotic protein Bcl-2 was significantly downregulated (P<0.05). This indicates that estrogen deficiency induces an imbalance in the expression of key proteins in the apoptosis signaling pathway, leading to overactivation of the apoptosis signaling pathway.

[0045] After intervention with Dixian Decoction, compared with the OVX group, the protein expression of both Caspase-3 and cleaved Caspase-3 in the DXJ group was significantly reduced (P<0.05), and the ratio of cleaved Caspase-3 to Caspase-3 was also significantly decreased (P<0.05), indicating that the terminal stage of apoptosis execution may be inhibited. Although the individual expression levels of Bax and Bcl-2 in the DXJ group did not show statistical differences compared with the OVX group, the Bcl-2 / Bax ratio increased significantly (P<0.05), indicating that the abnormal enhancement of apoptosis activity was alleviated.

[0046] 3.7 Safety Observation No mice died during the experiment. The mice in each group were in normal condition in terms of activity, eating, and defecation, and no obvious toxic side effects were observed.

[0047] 4. Experimental Conclusions (1) The ovariectomized mouse model is a recognized animal model for studying postmenopausal osteoporosis. The OVX group mice showed typical osteoporosis characteristics: decreased bone density, reduced bone biomechanical properties, destruction of bone tissue microstructure, and increased bone resorption. The model was successfully constructed.

[0048] (2) Compared with the OVX group, the number of bone trabeculae in mice treated with Dixian Decoction was significantly increased, the bone tissue microstructure was significantly repaired, and the level of bone resorption markers decreased. All of the above indicators were better than those in the model group (P<0.05 or P<0.01).

[0049] (3) The anti-PMOP effect of Dixianjian may be related to its bone metabolism signaling pathways, such as promoting bone formation, inhibiting osteoblast apoptosis, and improving bone metabolism.

[0050] (4) The above experimental results show that Dixian Decoction has a significant preventive and therapeutic effect on postmenopausal osteoporosis caused by ovariectomy and can be prepared into a drug for the prevention and treatment of postmenopausal osteoporosis.

Claims

1. Application of Dixian Decoction in the preparation of drugs for the prevention and treatment of postmenopausal osteoporosis.

2. The application according to claim 1, characterized in that, The Dixian Decoction comprises the following ingredients: 500 parts by weight of yam, 200-300 parts by weight of almonds and 800-1200 parts by volume of raw milk.

3. The application according to claim 2, characterized in that, The yam is finely powdered yam; the almonds are double kernels that have been soaked in broth and have had their skin and tips removed.

4. The application according to claim 1, characterized in that, Postmenopausal osteoporosis is characterized by at least one of the following pathological features: decreased bone density, decreased bone biomechanical properties, destruction of bone tissue microstructure, reduced number and continuity of trabeculae.

5. The application according to claim 1, characterized in that, The drug is an oral preparation.

6. The application according to claim 5, characterized in that, The oral formulation also contains pharmaceutically acceptable excipients.

7. The application according to claim 1, characterized in that, The preparation method of the Dixian Decoction includes the following steps: pulverizing yam into fine powder, soaking almonds in soup, removing the tips and skins, pulverizing or grinding them, grinding them together with raw milk, putting them into a porcelain container, cooking them into a paste with soup, and storing them in a sealed porcelain bottle.

8. The application according to any one of claims 1-7, characterized in that, The drug is intended for women diagnosed with postmenopausal osteoporosis or at risk of developing postmenopausal osteoporosis.

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