Traditional Chinese medicine composition for treating osteoporosis as well as preparation method and application thereof

Abstract

The invention relates to a traditional Chinese medicine composition for treating osteoporosis. The traditional Chinese medicine composition is prepared from the following raw materials in parts by mass: monarch drug: 8-15 parts of glossy privet fruit; ministerial drugs: 10-20 parts of radix fici simplicissimae, 5-15 parts of herba epimedii, 5-15 parts of radix morindae officinalis and 10-20 parts of radix rehmanniae praeparata; adjuvant drugs: 5-13 parts of rhizoma drynariae, 5-13 parts of flos carthami and 8-15 parts of fermented soybean; and a conductant drug: 3-10 parts of chrysanthemum. The traditional Chinese medicine composition has the effects of remarkably improving osteoporosis symptoms, achieving a multi-target synergistic effect, improving bone metabolism, and being low in side effect and safe.

Description

Technical Field

[0001] The present application relates to the technical field of traditional Chinese medicine, and in particular to a traditional Chinese medicine composition for treating osteoporosis, and a preparation method and application thereof. Background Art

[0002] There are many kinds of Chinese patent medicines and Chinese medicines used in the clinic to treat osteoporosis. These medicines are usually based on the principle of TCM syndrome differentiation and treatment, and personalized treatment is provided according to different types and symptoms of patients. The following are some common Chinese patent medicines and Chinese medicine prescriptions:

[0003] Gushukang Capsule: The main ingredients include epimedium, rehmannia root, drynaria, astragalus and salvia miltiorrhiza. It has the effects of nourishing the kidney and bones, replenishing qi and nourishing blood. It has a significant effect on symptoms such as low back pain and weakness in the waist and knees caused by osteoporosis.

[0004] Gusongbao Granules: It has the effects of strengthening tendons and bones and nourishing the kidneys and activating blood circulation. It is mainly used to prevent menopausal osteoporosis.

[0005] Yougui Pills: Suitable for osteoporosis of kidney yang deficiency type, it can tonify the kidney, strengthen yang, strengthen tendons and bones.

[0006] Buzhong Yiqi Decoction combined with Jinkui Shenqi Pills: used for osteoporosis of spleen and kidney yang deficiency type, it has the function of nourishing the spleen and kidney and strengthening the tendons and bones.

[0007] Liuwei Dihuang Decoction: Suitable for osteoporosis of liver and kidney yin deficiency type, it has the effect of nourishing the liver and kidneys and replenishing essence and strengthening bones.

[0008] Shentong Zhuyu Decoction: It is used for osteoporosis caused by blood stasis and qi stagnation. It has the effects of regulating qi, activating blood circulation, removing blood stasis and relieving pain.

[0009] In the treatment of osteoporosis with Western medicine, the main drugs are divided into two categories: bone resorption inhibitors and bone formation promoters, including bisphosphonates, calcitonin drugs, estrogen drugs, parathyroid hormone analogs, denosumab, vitamin K drugs, calcium and vitamin D. However, these drugs have different degrees of side effects in the treatment of osteoporosis:

[0010] Bisphosphonates: such as alendronate and risedronate, may cause adverse reactions such as abdominal pain, nausea, gastroesophageal reflux, esophagitis, fever, bone pain, etc. Long-term use may cause excessive inhibition of bone resorption, leading to serious complications such as heterotopic ossification and mandibular osteonecrosis.

[0011] Calcitonin drugs: such as calcitonin nasal spray, long-term use may be associated with a slightly increased risk of malignant tumors. In addition, it may also cause side effects such as nasal dryness, nose bleeding, and nausea.

[0012] Estrogen drugs: such as estrogen replacement therapy, may lead to an increased risk of breast hyperplasia, endometriosis, thrombosis, and may be associated with certain types of cancer (such as breast cancer).

[0013] Parathyroid hormone analogs: such as teriparatide, may cause nausea, vomiting, headache, dizziness, lower limb edema, etc. Long-term use may be associated with an increased risk of bone tumors.

[0014] Desuomab: This is a new type of anti-bone resorptive drug that may cause musculoskeletal pain, limb pain, hypocalcemia, increased risk of infection (such as upper respiratory tract infection, urinary tract infection), rash, etc.

[0015] Vitamin K drugs: such as vitamin K1 and K2, are used to promote bone formation, but long-term high-dose use may interfere with anticoagulant therapy and cause blood clotting problems.

[0016] Calcium supplements and vitamin D: They are commonly used to supplement the calcium and vitamin D needed for bone health, but excessive use of calcium supplements may cause hypercalcemia and kidney stones, while excessive vitamin D may cause hypercalcemia, renal damage and other problems.

[0017] The above-mentioned related technologies have the following deficiencies: (1) Although the existing Western medicine treatment methods are effective, their side effects limit their long-term application and patient compliance, and have a certain impact on the patient's quality of life; (2) Although there are many traditional Chinese medicine prescriptions for the treatment of osteoporosis, in the actual treatment of osteoporosis, the compatibility of some prescriptions is not accurate, and it is difficult to take into account the comprehensive conditioning of tonifying the kidney, nourishing the marrow and strengthening the bones. (3) Osteoporosis involves many problems such as insufficient kidney essence and unbalanced bone metabolism, and the treatment is relatively difficult. Existing treatment methods mostly start from a single pathway, which is difficult to comprehensively regulate bone metabolism and improve symptoms. Summary of the invention

[0018] In order to solve the problem of numerous side effects of various osteoporosis treatment drugs currently available in clinical practice and to provide a new, efficient and practical drug for treating osteoporosis, the present application provides a Chinese medicine composition for treating osteoporosis, a preparation method and an application thereof.

[0019] In the first aspect, the present application provides a Chinese medicine composition for treating osteoporosis, which adopts the following technical scheme:

[0020] A Chinese medicine composition for treating osteoporosis, comprising the following raw materials in parts by weight:

[0021] Main drug: 8-15 parts of Ligustrum lucidum fruit;

[0022] Minister drugs: 10-20 parts of Prunus mume, 5-15 parts of Epimedium, 5-15 parts of Morinda officinalis and 10-20 parts of Rehmannia glutinosa;

[0023] Adjuvants: 5-13 parts of Drynaria fortunei, 5-13 parts of safflower and 8-15 parts of light fermented black beans;

[0024] Adjuvant: 3-10 parts of chrysanthemum.

[0025] Optionally, the following raw materials are included in parts by weight:

[0026] Main drug: Ligustrum lucidum 10-13 parts;

[0027] Minister drugs: 12-18 parts of Prunus mume, 8-11 parts of Epimedium, 8-10 parts of Morinda officinalis and 12-17 parts of Rehmannia glutinosa;

[0028] Adjuvants: 8-11 parts of Drynaria fortunei, 8-11 parts of safflower and 10-14 parts of light fermented black beans;

[0029] Adjuvant: 5-7 parts of chrysanthemum.

[0030] By adopting the above technical scheme, the present application uses Ligustrum lucidum as the main medicine. Ligustrum lucidum is sweet in taste and mild in nature. It enters the liver and kidney meridians and has the functions of nourishing the liver and kidney, clearing away heat and improving eyesight, moisturizing the skin and nourishing hair, and strengthening tendons and bones.

[0031] With five-fingered ginseng, epimedium, Morinda officinalis, and Rehmannia glutinosa as the assistant medicine, it has the functions of warming the kidney and strengthening yang, dispelling wind and dampness, nourishing yin and blood, and enhancing bone density; five-fingered ginseng, also known as "Southern Astragalus", is sweet and warm in nature, and belongs to the liver and kidney meridians. It has the functions of nourishing the kidney and replenishing essence, promoting blood circulation and removing blood stasis, reducing swelling and relieving pain, and strengthening tendons and bones; epimedium and Morinda officinalis, when used in combination, have a better effect of warming the kidney and strengthening yang, and strengthen tendons and bones, and dispel wind, cold and dampness. Rehmannia glutinosa is sweet and slightly warm in nature, and has the functions of nourishing yin and blood, replenishing essence and filling marrow. It is an important medicine for nourishing the liver and kidney. The three medicines are used together to strengthen yang and nourish yin at the same time, and have the effect that yin and yang are helped by yin and the biochemical is endless, and yin is raised by yang and the source is inexhaustible.

[0032] With bone-setting herb, safflower and light fermented black beans as adjuvants, it has the effects of tonifying kidney and strengthening bones, strengthening yang and consolidating essence, dispersing wind and relieving exterior symptoms, and harmonizing the properties of the medicine. Bone-setting herb is bitter and warm, and mainly enters the liver and kidney meridians. "Compendium of Materia Medica" says: "It is a medicine for the foot Shaoyin meridian. Therefore, it can enter bones, teeth, and chronic diarrhea." It is good at promoting blood circulation, healing injuries and relieving pain, and repairing tendons and bones. It is a good product commonly used in traumatology; combined with epimedium, it warms and tonifies kidney yang, strengthens tendons and bones, and is a good medicine for orthopedics. Safflower is pungent, dispersing, warming and unblocking. Less use of blood can produce blood, and more use has the function of blood circulation. It is used together with the minister Rehmannia glutinosa to nourish yin and activate blood circulation. Light fermented black beans are cool in nature. When used in combination with the monarch and minister medicines, it can relieve pungent and warm and clear away heat, making this prescription nourishing but not depressed.

[0033] Chrysanthemum is used as the guiding medicine. Chrysanthemum is pungent, bitter, slightly cold in nature. Its sweet and cold properties can nourish yin, while its bitter and cold properties can purge and descend.

[0034] The above raw materials are used together to form a prescription for nourishing the kidney and strengthening bones, nourishing yin and activating blood circulation, namely "Yimui Qianggu Prescription". Compared with traditional drug treatment methods represented by parathyroid hormone as an osteogenic promoter and denosumab as an osteoclast inhibitor, the prescription of the present application has the advantages of more moderate efficacy, obvious effect and fewer side effects in the treatment of osteoporosis.

[0035] The prescription for replenishing marrow and strengthening bones uses Ligustrum lucidum as the main medicine, which can replenish essence and fill marrow. The applicant has shown through research that Ligustrum lucidum can regulate bone metabolism and enhance bone density by promoting the proliferation and function of osteoblasts. Ligustrum lucidum is rarely used in existing osteoporosis prescriptions. On the one hand, it is because its traditional application is more targeted at liver and kidney yin deficiency, and the pathogenesis of osteoporosis often involves kidney yang deficiency, so it is underestimated; on the other hand, the extraction and standardization of the active ingredients of Ligustrum lucidum are difficult, and pharmacological research is still insufficient, which affects its widespread use in the field of bone metabolism. Through modern pharmacological verification, the applicant found that Ligustrum lucidum can effectively regulate bone metabolism-related pathways (Runx2 / OCN), and scientifically designed the compatibility according to the "four qi and five flavors" theory to solve the problem of drug conflicts, so that it can not only play the role of nourishing the kidney and marrow, but also significantly enhance bone density.

[0036] In this application, Ligustrum lucidum is used as the main medicine, and its dosage setting is at the upper limit of its conventional dosage, which can fully play its core role of nourishing the kidney and marrow and regulating bone metabolism. Although the dosage is not the highest, its focus determines that it is the main medicine, while the dosage of Prunus mume and Radix Rehmanniae Preparata is slightly higher than that of Ligustrum lucidum, firstly because their conventional dosage range is wider; secondly, these two medicines can consolidate the effect of Ligustrum lucidum in nourishing the kidney and marrow while expanding the treatment range of the entire prescription, achieving the effect of dispelling wind and dampness, nourishing yin and blood, and playing an important supplementary role in the overall treatment mechanism of osteoporosis.

[0037] In addition, osteoporosis treatment is mainly based on kidney tonifying and bone strengthening. Although chrysanthemum is known for "clearing the liver and improving eyesight", traditional Chinese medicine emphasizes that "liver and kidney are of the same origin", liver governs tendons, and kidney governs bones. Chrysanthemum clears liver fire and calms liver yang, which can indirectly assist the kidney's function of governing bones. Chrysanthemum, as a guiding drug, guides other kidney tonifying drugs (such as Ligustrum lucidum and Epimedium) to act on the liver and kidney, strengthens the systemic regulatory effect of liver and kidney coordination on bone metabolism, and embodies the concept of "treating both the symptoms and the root causes". The applicant found through research that the combination of Ligustrum lucidum and chrysanthemum can play a synergistic role in the treatment of osteoporosis, which helps to ensure that the active ingredients in Ligustrum lucidum play a better role. The combination of the two can promote osteogenesis and jointly regulate bone metabolism. Based on its characteristics of tonifying the kidney and marrow, clearing the liver and improving eyesight, this combination can greatly improve osteoporosis.

[0038] In a second aspect, the present application provides a method for preparing a Chinese medicine composition for treating osteoporosis, using the following technical scheme:

[0039] A method for preparing a Chinese medicine composition for treating osteoporosis comprises the following steps:

[0040] The raw materials such as Ligustrum lucidum, Prunus quinquefolius, Epimedium, Morinda officinalis, Rehmannia root, Drynaria fortunei, Carthamus tinctorius, Fructus sojae and Chrysanthemum are weighed according to the above proportions, and then washed, soaked and boiled in sequence to obtain the medicinal solution.

[0041] Optionally, include the following steps:

[0042] (1) Weigh Ligustrum lucidum fruit, Prunus mume, Epimedium, Morinda officinalis, Rehmannia root, Drynaria fortunei, Carthamus tinctorius, Fructus sojae and Chrysanthemum in the above proportions;

[0043] (2) Cleaning the raw materials;

[0044] (3) Primary decoction: add purified water to the weighed raw materials, with the weight ratio of raw materials to purified water being 1:(8-10), soak for 25-35 minutes, boil, and then decoct at 60°C-70°C for 1-1.5 hours, filter and collect the decoction for later use;

[0045] (4) Concentrating the decoction at 60° C. to less than one tenth of the volume of the original decoction to obtain a medicinal solution.

[0046] Optionally, a second decoction step is further included, wherein the second decoction is performed after the first decoction, and the preparation steps are as follows:

[0047] (1) Weigh Ligustrum lucidum fruit, Prunus mume, Epimedium, Morinda officinalis, Rehmannia root, Drynaria fortunei, Carthamus tinctorius, Fructus sojae and Chrysanthemum in the above proportions;

[0048] (2) Cleaning the raw materials;

[0049] (3) Primary decoction: add purified water to the weighed raw materials, with the weight ratio of raw materials to purified water being 1:(8-10), soak for 25-35 minutes, boil, and then decoct at 60°C-70°C for 1-1.5 hours, filter and collect the decoction for later use;

[0050] (4) Secondary decoction: adding purified water to the above-mentioned decoction raw material, the weight ratio of the decoction raw material to purified water is 1: (8-10), after boiling, decocting at a temperature of 60° C.-70° C. for 40-50 minutes, filtering the decoction and setting aside;

[0051] (5) combining the two decoctions, and concentrating the combined decoction at 60° C. to less than one tenth of the volume of the original decoction to obtain a medicinal solution.

[0052] In a third aspect, the present application provides an application of a traditional Chinese medicine composition in the preparation of a drug for treating osteoporosis.

[0053] Clinical trials have shown that it can effectively strengthen the kidney and bones, has low side effects, and is suitable for long-term use. The Chinese medicine composition has the following effects: significantly improves osteoporosis symptoms, multi-target synergy, improves bone metabolism, low side effects and safety.

[0054] By adopting the above technical solution, Micro-CT is the gold standard for detecting the effect of drug intervention on osteoporosis animal models. After using the Yisui Qianggu formula to intervene in the ovariectomized osteoporosis mouse model, Micro-CT examination was performed, and the results showed that the formula of this application can effectively increase bone density, the number of trabeculae is more sufficient, the bone tissue is more dense, and the bone strength is significantly improved.

[0055] The formula of the present application achieves comprehensive regulation of osteoporosis through the multi-target synergistic effects of regulating kidney essence, improving microcirculation and promoting bone metabolism. Pharmacological studies have shown that Ligustrum lucidum, Prunus mume, Epimedium and Drynaria can significantly upregulate osteoblast activity and inhibit osteoclast function, ultimately achieving the effect of promoting bone formation and reducing bone resorption.

[0056] Compared with the existing clinically used bone resorption inhibitors and bone formation promoters, the side effects of the present invention are significantly reduced. No obvious adverse reactions were observed during long-term medication, especially no obvious toxic reactions were observed in liver and kidney function monitoring, showing good safety and tolerability.

[0057] In summary, this application has the following beneficial effects:

[0058] 1. The marrow-benefiting and bone-strengthening prescription composed of raw materials of the present application uses Ligustrum lucidum, which can replenish essence and fill marrow, as the main medicine. The applicant has shown through research that Ligustrum lucidum can regulate bone metabolism and enhance bone density by promoting the proliferation and function of osteoblasts. Ligustrum lucidum is rarely used in existing osteoporosis prescriptions. On the one hand, it is because its traditional application is more targeted at liver and kidney yin deficiency, and the pathogenesis of osteoporosis often involves kidney yang deficiency, so it is underestimated; on the other hand, the extraction and standardization of the active ingredients of Ligustrum lucidum are difficult, and pharmacological research is still insufficient, which affects its widespread use in the field of bone metabolism. The applicant has found through modern pharmacological verification that Ligustrum lucidum can effectively regulate bone metabolism-related pathways (Runx2 / OCN), and scientifically designs the compatibility according to the "four qi and five flavors" theory to solve the problem of drug conflict, so that it can not only play the role of tonifying the kidney and benefiting the marrow, but also significantly enhance bone density. This is also the innovation of this prescription.

[0059] In addition, the treatment of osteoporosis focuses on tonifying the kidney and strengthening bones. Although chrysanthemum is known for "clearing the liver and improving eyesight", traditional Chinese medicine emphasizes that "the liver and kidney are of the same origin", the liver governs the tendons, and the kidney governs the bones. Chrysanthemum clears liver fire and calms liver yang, which can indirectly assist the kidney in governing bones. As a guiding drug, chrysanthemum guides other kidney-tonifying drugs (such as Ligustrum lucidum and Epimedium) to act on the liver and kidney, strengthens the systemic regulatory effect of liver and kidney coordination on bone metabolism, and embodies the concept of "treating both the symptoms and the root causes". The applicant found through research that the combination of Ligustrum lucidum and chrysanthemum can play a synergistic role in the treatment of osteoporosis. The combination of the two can promote osteogenesis and jointly regulate bone metabolism. Based on its characteristics of tonifying the kidney and marrow, clearing the liver and improving eyesight, this combination can greatly improve osteoporosis.

[0060] 2. The Yisui Qianggu prescription of the present application is mainly used to treat osteoporosis, and is particularly suitable for the symptoms of low back and knee weakness, fatigue and pain caused by decreased bone density in middle-aged and elderly people. The prescription can also be used as an auxiliary medicine for post-fracture rehabilitation and prevention of osteoporosis.

[0061] 3. The present invention uses Ligustrum lucidum as the main drug, and is combined with Eleutherococcus chinensis, Epimedium, Morinda officinalis, Rhizoma Drynariae and other drugs to treat osteoporosis. It has the effects of significantly improving the symptoms of osteoporosis, multi-target synergistic effects, improving bone metabolism, and having low side effects and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is a diagram of the Q-PCR experimental test results of Application Example 1.

[0063] Figure 2 This is the ELISA experiment test result diagram of Application Example 2.

[0064] Figure 3 This is a diagram of the Q-PCR experimental test results of Application Example 3.

[0065] Figure 4 This is the Micro-CT test result diagram of Application Example 4.

[0066] Figure 5 This is the Micro-CT test result diagram of Application Example 4.

[0067] Figure 6 This is a test result diagram of the ELISA experiment of Application Example 4.

[0068] Figure 7 This is a test result diagram of the ELISA experiment of Application Example 4.

[0069] Figure 8 ELISA test results for Application Example 5 Figure 1 .

[0070] Fig. 9 ELISA test results for Application Example 5 Figure 2 .

[0071] Fig.10 ELISA test results for Application Example 5 Figure 3 . DETAILED DESCRIPTION

[0072] Example 1

[0073] A Chinese medicine composition for treating osteoporosis, comprising the following raw materials by weight:

[0074] Fructus Ligustri Lucidi 12g, Rhizoma Cibotii 15g, Herba Epimedii 10g, Herba Morindae Officinalis 9g, Herba Rehmanniae 15g, Herba Drynariae 9g, Herba Safflower 9g, Herba Sojae 12g, and Herba Chrysanthemi 6g;

[0075] The preparation method comprises the following steps:

[0076] (1) Weigh Ligustrum lucidum fruit, Prunus mume, Epimedium, Morinda officinalis, Rehmannia root, Drynaria fortunei, Carthamus tinctorius, Fructus sojae and Chrysanthemum in the above proportions;

[0077] (2) Cleaning the raw materials to improve extraction efficiency;

[0078] (3) Primary decoction: add purified water to the symmetrically taken raw materials, with the weight ratio of raw materials to purified water being 1:10, soak for 30 minutes, boil at 100°C, and then decoct at 65°C for 1 hour, filter and take the decoction for later use;

[0079] (4) Secondary decoction: adding purified water to the above-mentioned decoction raw material, the weight ratio of the decoction raw material to purified water is 1:8, boiling at 100° C., and then decocting at 65° C. for 45 minutes, filtering the decoction and setting aside;

[0080] (5) The two decoctions were combined and concentrated to 200 ml at 65° C. to obtain a medicinal solution.

[0081] Example 2

[0082] A Chinese medicine composition for treating osteoporosis, which is different from Example 1 in that it comprises the following raw materials by weight:

[0083] Fructus Ligustri Lucidi 8g, Rhizoma Cibotii 10g, Herba Epimedii 5g, Herba Morindae Officinalis 5g, Herba Rehmanniae 10g, Herba Drynariae 5g, Herba Safflower 5g, Fructus Sojae 8g and Herba Chrysanthemi 3g.

[0084] Example 3

[0085] A Chinese medicine composition for treating osteoporosis, which is different from Example 1 in that it comprises the following raw materials by weight:

[0086] Fructus Ligustri Lucidi 10g, Rhizoma Cibotii 5g, Herba Epimedii 8g, Herba Morindae Officinalis 8g, Herba Rehmanniae 12g, Herba Drynariae 8g, Herba Safflower 8g, Herba Sojae Indici 10g and Herba Chrysanthemi 5g.

[0087] Example 4

[0088] A Chinese medicine composition for treating osteoporosis, which is different from Example 1 in that it comprises the following raw materials by weight:

[0089] Fructus Ligustri Lucidi 13g, Rhizoma Cibotii 18g, Herba Epimedii 11g, Herba Morindae Officinalis 10g, Herba Rehmanniae 17g, Herba Drynariae 11g, Herba Safflower 11g, Herba Sojae Indici 14g and Herba Chrysanthemi 7g.

[0090] Example 5

[0091] A Chinese medicine composition for treating osteoporosis, which is different from Example 1 in that it comprises the following raw materials by weight:

[0092] Fructus Ligustri Lucidi 15g, Rhizoma Cibotii 50g, Herba Epimedii 15g, Herba Morindae Officinalis 15g, Herba Rehmanniae 20g, Herba Drynariae 13g, Herba Safflower 13g, Herba Sojae Indici 15g and Herba Chrysanthemi 10g.

[0093] Performance testing experiment

[0094] 1. Q-PCR Experiment

[0095] The main process of Q-PCR experiment includes sample processing, RNA extraction, reverse transcription and fluorescence quantitative PCR detection. First, after the cell or tissue sample is frozen and ground with liquid nitrogen, the total RNA is extracted using reagents such as TRIzol, and its concentration and purity are determined to ensure that the A260 / A280 ratio is between 1.8-2.0. Subsequently, 1 μg of RNA is reverse transcribed into cDNA using a reverse transcription reagent, and the reaction conditions are 42°C for 30 minutes and 85°C for 5 minutes. Next, the amplification reaction is performed on a fluorescence quantitative PCR instrument. The 20 μL system includes cDNA template, SYBR Green dye and upstream and downstream primers. The reaction program is set to 95°C for 3 minutes for pre-denaturation, followed by 40 cycles (95°C for 10 seconds, 60°C for 30 seconds). Finally, the specificity is verified by the melting curve, and the relative gene expression is calculated using the 2^(-ΔΔCt) method.

[0096] 2.ELISA experiment

[0097] ELISA experiments are used to detect the level of target proteins, and the steps include sample processing, sample addition, washing, secondary antibody incubation, color development, and termination of the reaction. The sample is a cell culture supernatant, tissue homogenate, or serum, and the supernatant needs to be centrifuged to collect the supernatant or diluted to an appropriate concentration. Using a commercial ELISA kit, add the standard and sample to the ELISA plate, 50 μL per well, set a blank control, and incubate at room temperature for 2 hours. After incubation, wash the ELISA plate 4 times with washing solution, add 200 μL washing solution each time, dry it, add 50 μL of HRP-labeled enzyme-labeled secondary antibody, and continue to incubate at room temperature for 60 minutes. Then add 50 μL of TMB substrate solution, react in the dark for 15 minutes, and terminate the reaction with 2M sulfuric acid. Finally, read the OD value with an ELISA reader at a wavelength of 450 nm, and calculate the concentration of the target substance using the standard curve.

[0098] 3. Micro-CT test

[0099] Micro-CT testing uses ScancoμCT 50 instrument, with tube voltage set to 50-70kV, tube current set to 100-200μA, resolution set to 9-18μm / voxel, 360° full-view scanning mode, rotation step length set to 0.5° / step, exposure time set to 200-300ms, and equipped with 0.5mm aluminum filter to reduce low-energy X-ray interference. The samples are processed C57 mouse bones (distal femur, upper tibia), which are scanned after fixation and decalcification. The region of interest (ROI) is usually selected as the tibial plateau to the 50th layer below the growth plate. Analysis indicators include bone volume fraction (BV / TV), trabecular number (Tb.N) and trabecular separation (Tb.Sp). The experiment was carried out in an environment of 22-25℃ and humidity of 50%-60%, and three-dimensional image reconstruction and quantitative data analysis were completed by Scanco Analysis dedicated software to ensure the accuracy and reliability of the results.

[0100] Application Example 1

[0101] Test on the effect of Chinese herbal medicine combination on improving osteoporosis

[0102] Experimental methods:

[0103] 1. Animal Model Preparation

[0104] The modeling method of the OVX group (ovarian castration mouse model group) is as follows: 8-week-old healthy female C57BL / 6 mice weighing about 18-22g were selected for the experiment. They were fasted for 12 hours before surgery and anesthetized by intraperitoneal injection of sodium pentobarbital (40mg / kg) 30 minutes before surgery. The surgery was performed after complete anesthesia. The dorsal midline incision method was used to cut the skin on both sides of the spine of the mouse back to expose the ovaries and fallopian tubes on both sides. The ovaries were removed after the ovarian pedicles were ligated with sterile surgical thread, and the incision was then disinfected and sutured. Penicillin (4000U / mouse) was given intramuscularly for 3 consecutive days after surgery to prevent infection, and the weight, activity status and wound healing of the mice were monitored daily. A stable osteoporosis model was formed within 6 weeks after surgery to ensure the accuracy and consistency of the experiment.

[0105] Modeling method of sham operation group (NC group): the same surgical method as above was used, but only the ovaries were exposed and sutured without removing the ovaries.

[0106] Drug group (Ligustroside group): This group is a drug intervention group using the efficacy-related ingredient "Ligustroside" in Ligustrum lucidum. On the basis of the OVX group mice, only Ligustroside was used to intervene them. The intervention method was: use a 1ml syringe with an 8# gavage needle to gavage the OVX mice once a day, 0.2ml / mouse.

[0107] 2. Detection Experiment

[0108] Q-PCR experiments were performed on mice in the OVX group (ovarian castration mouse model group), sham operation group (NC group) and drug group (Ligustroside group).

[0109] Experimental results:

[0110] like Figure 1 The q-PCR results shown show that:

[0111] Runx2 (osteogenesis-related gene) and OCN (osteocalcin) were expressed most in the NC group, indicating that osteoblast activity and bone mineralization ability were strong under normal physiological conditions; Ligustroside group was second, indicating that Ligustroside can partially restore the expression of Runx2 and OCN, and has the effect of promoting osteoblast activity and mineralization; OVX group was the lowest, reflecting that estrogen deficiency caused by ovariectomy significantly inhibited osteoblast activity and bone mineralization ability. The difference was statistically significant (P < 0.05).

[0112] Application Example 2

[0113] Test on the effect of Chinese medicine composition on improving osteoporosis

[0114] Experimental methods:

[0115] 1. Animal Model Preparation

[0116] The modeling method of the OVX group (ovarian castration mouse model group) is as follows: 8-week-old healthy female C57BL / 6 mice weighing about 18-22g were selected for the experiment. They were fasted for 12 hours before surgery and anesthetized by intraperitoneal injection of sodium pentobarbital (40mg / kg) 30 minutes before surgery. The surgery was performed after complete anesthesia. The dorsal midline incision method was used to cut the skin on both sides of the spine of the mouse back to expose the ovaries and fallopian tubes on both sides. The ovaries were removed after the ovarian pedicles were ligated with sterile surgical thread, and the incision was then disinfected and sutured. Penicillin (4000U / mouse) was given intramuscularly for 3 consecutive days after surgery to prevent infection, and the weight, activity status and wound healing of the mice were monitored daily. A stable osteoporosis model was formed within 6 weeks after surgery to ensure the accuracy and consistency of the experiment.

[0117] Modeling method of sham operation group (NC group): the same surgical method as above was used, but only the ovaries were exposed and sutured without removing the ovaries.

[0118] In the YSQG-1 group (the castrated osteoporosis mouse model was intervened by the Chinese herbal composition without the addition of Ligustrum lucidum and Chrysanthemum), the OVX mice were gavaged with the medicinal solution made from the Chinese herbal composition without the addition of Ligustrum lucidum and Chrysanthemum using a 1ml syringe and a 8# gavage needle, once a day, 0.2ml / mouse.

[0119] In the YSQG-2 group (the Chinese medicine composition of Example 1 intervened in the castrated osteoporosis mouse model), the drug solution prepared in Example 1 was gavaged into OVX mice using a 1 ml syringe and a 8# gavage needle, once a day, 0.2 ml / mouse.

[0120] 2. Detection Experiment

[0121] ELISA experiments were performed on mice in the sham operation group (NC group), YSQG-1 group (castrated osteoporosis mouse model intervened by the Chinese herbal composition without adding Ligustrum lucidum and Chrysanthemum) and YSQG-2 group (castrated osteoporosis mouse model intervened by the Chinese herbal composition of Example 1).

[0122] Experimental results:

[0123] like Figure 2 As shown, mouse serum was extracted for ELISA experiment, and the results showed that the promoting effect on osteogenesis of the group without adding "Ligustrum lucidum and Chrysanthemum" (YSQG-1 group) was lower than that of the group with adding "Ligustrum lucidum and Chrysanthemum" (YSQG-2 group).

[0124] After using the Chinese medicine composition of Example 1 of the present application to intervene in the ovariectomized osteoporosis mouse model, the results showed that it can effectively improve bone density, the number of trabeculae is more sufficient, the bone tissue is denser, and the bone strength is significantly improved.

[0125] Application Example 3

[0126] Multi-target synergistic effect of traditional Chinese medicine combination and test of its effect on improving bone metabolism

[0127] Experimental methods:

[0128] 1. Animal Model Preparation

[0129] The modeling method of the OVX group (ovarian castration mouse model group) is as follows: 8-week-old healthy female C57BL / 6 mice weighing about 18-22g were selected for the experiment. They were fasted for 12 hours before surgery and anesthetized by intraperitoneal injection of sodium pentobarbital (40mg / kg) 30 minutes before surgery. The surgery was performed after complete anesthesia. The dorsal midline incision method was used to cut the skin on both sides of the spine of the mouse back to expose the ovaries and fallopian tubes on both sides. The ovaries were removed after the ovarian pedicles were ligated with sterile surgical thread, and the incision was then disinfected and sutured. Penicillin (4000U / mouse) was given intramuscularly for 3 consecutive days after surgery to prevent infection, and the weight, activity status and wound healing of the mice were monitored daily. A stable osteoporosis model was formed within 6 weeks after surgery to ensure the accuracy and consistency of the experiment.

[0130] Modeling method of sham operation group (NC group): the same surgical method as above was used, but only the ovaries were exposed and sutured without removing the ovaries.

[0131] The drug intervention group (YSQG group) was treated with the Chinese medicine composition prepared in Example 1 to treat OVX mice. The specific method was as follows: the drug solution prepared in Example 1 was gavaged into OVX mice using a 1 ml syringe and a 8# gavage needle, once a day, 0.2 ml / mouse.

[0132] The denosumab control group (Denosumab group) used denosumab solution to intervene in OVX mice. The specific method was: subcutaneous injection of 5 mg / ml denosumab solution (diluted with PBS) was performed on OVX mice once every 4 weeks, 0.1 ml / mouse.

[0133] 2. Detection Experiment

[0134] mRNA was extracted from the bone tissues of mice in the OVX group, NC group, YSQG group, and Denosumab group, and Q-PCR experiments were performed.

[0135] Experimental results:

[0136] like Figure 3 As shown, with denosumab as the control group, it is shown that the Chinese medicine composition of Example 1 of the present application has a good ability to inhibit osteoclastogenesis.

[0137] Application Example 4

[0138] Multi-target synergistic effect of traditional Chinese medicine combination and test of its effect on improving bone metabolism

[0139] Experimental methods:

[0140] 1. Animal Model Preparation

[0141] The modeling method of the OVX group (ovarian castration mouse model group) is as follows: 8-week-old healthy female C57BL / 6 mice weighing about 18-22g were selected for the experiment. They were fasted for 12 hours before surgery and anesthetized by intraperitoneal injection of sodium pentobarbital (40mg / kg) 30 minutes before surgery. The surgery was performed after complete anesthesia. The dorsal midline incision method was used to cut the skin on both sides of the spine of the mouse back to expose the ovaries and fallopian tubes on both sides. The ovaries were removed after the ovarian pedicles were ligated with sterile surgical thread, and the incision was then disinfected and sutured. Penicillin (4000U / mouse) was given intramuscularly for 3 consecutive days after surgery to prevent infection, and the weight, activity status and wound healing of the mice were monitored daily. A stable osteoporosis model was formed within 6 weeks after surgery to ensure the accuracy and consistency of the experiment.

[0142] Modeling method of sham operation group (NC group): the same surgical method as above was used, but only the ovaries were exposed and sutured without removing the ovaries.

[0143] The Yisui Qianggu prescription group (drug group), i.e., the Chinese medicine composition prepared in Example 1, was used to intervene in OVX mice. The specific method was as follows: the medicinal solution prepared in Example 1 was gavaged into OVX mice using a 1 ml syringe and a 8# gavage needle, once a day, 0.2 ml / mouse.

[0144] 2. Detection Experiment

[0145] The mice in the OVX group, sham operation group and Yisui Qianggu prescription group were subjected to Micro-CT test and ELISA experiment.

[0146] Experimental results:

[0147] The experimental results of Micro-CT test are shown in Table 1 and Figure 4 , Figure 5

[0148] The experimental results of ELISA experiment are as follows Figure 6-Figure 7 .

[0149] Table 1

[0150] Group Number BV / TV TBSP TBN Sham operation group (NC group) 4 4.148±0.456 56.018±4.693 0.00746±0.00139 OVX group (ovarian castration mouse model group) 6 3.222±0.644 74.828±6.429 0.00563±0.00068 Yisui Qianggu Fang Group (Drug Group) 7 4.299±0.919 65.095±7.055 0.00733±0.00112

[0151] The results of Micro-CT are shown in Table 1. After medication, the key bone density indicators (BV / TV, TBSP, TBN) of the drug group mice were close to those of the sham operation group mice. BV / TV reflects the overall level of bone mass and macroscopically evaluates bone density. Tb.Sp and Tb.N reflect the microstructure of trabeculae and provide an assessment of the mechanical strength and stability of bone tissue. Bone quality assessment: In the study of diseases such as osteoporosis, a decrease in BV / TV, an increase in Tb.Sp, and a decrease in Tb.N usually mean that the bone microstructure is damaged and the risk of fracture is increased.

[0152] like Figure 4 As shown: the BV / TV and TbN of the Yisui Qianggu Fang group of the Chinese medicine composition for treating osteoporosis in the embodiment of the present application are significantly higher than those in the OVX group, and there is a significant difference with the OVX group, proving that the Yisui Qianggu Fang helps to promote the growth of trabeculae and the recovery of bone mass. In addition, the TbSp of the Yisui Qianggu Fang group is significantly lower than that of the OVX group, indicating that the Yisui Qianggu Fang group of the Chinese medicine composition for treating osteoporosis in the embodiment of the present application has the effect of restoring the spacing of trabeculae, and the difference is statistically significant (P < 0.05).

[0153] like Figure 5 As shown, after treatment with the Yisui Qianggu Fang group of the traditional Chinese medicine composition for treating osteoporosis in the embodiment of the present application, there was no significant difference in the recovery of bone mass and bone morphology under CT scan between the mice and the sham operation group, which proves that the Yisui Qianggu Fang helps promote the growth of trabeculae and the reconstruction of their structure.

[0154] like Figure 6 As shown, after treatment with the Yisui Qianggu Fang group of the traditional Chinese medicine composition for treating osteoporosis in the embodiment of the present application, the transcription level of RUNX2 in the bone marrow of the drug group was significantly different from that of the OVX group, which proves that the Yisui Qianggu Fang group of the traditional Chinese medicine composition for treating osteoporosis in the embodiment of the present application has the effect of promoting the expression of RUNX2 transcription level in the bone marrow to promote the formation of endochondral bone and inhibit bone absorption, and the difference is statistically significant (P < 0.01).

[0155] like Figure 7 As shown, after treatment with the Yisui Qianggu Fang group of the traditional Chinese medicine composition for treating osteoporosis in the embodiment of the present application, the E2 level in the serum of the drug group was significantly different from that of the OVX group, which proves that the Yisui Qianggu Fang group of the traditional Chinese medicine composition for treating osteoporosis in the embodiment of the present application has the effect of promoting bone growth and accelerating bone fusion by promoting the secretion of E2 levels in serum, and the difference is statistically significant (P>0.05).

[0156] Application Example 5

[0157] Low side effects and safety effect test of traditional Chinese medicine combination

[0158] Experimental methods:

[0159] The mouse models prepared by the OVX group, sham operation group and Yisui Qianggu prescription group in Application Example 4 were subjected to ELISA experiments. The test results are shown in Tables 2-3 and Figure 8-Figure 10 .

[0160] Experimental results:

[0161] Table 2

[0162] Group Number AST ALT Sham operation group (NC group) 9 51.444±13.593 33±11.672 OVX group (ovarian castration mouse model group) 8 46.375±13.287 26.875±8.967 Yisui Qianggu Fang Group (Drug Group) 8 42.375±12.861 32±11.747

[0163] Note: AST (Aspartate Aminotransferase): It exists in the mitochondria and cytoplasm of cells and is mainly distributed in tissues such as myocardium, hepatocytes, skeletal muscle and kidney. It is not a liver-specific enzyme and myocardial and skeletal muscle diseases can also cause an increase in AST.

[0164] ALT (Alanine Aminotransferase): Mainly distributed in the cytoplasm of hepatocytes, it is a more liver-specific enzyme. Elevated ALT usually indicates hepatocyte damage.

[0165] Table 3

[0166] Group Number weight E2 Sham operation group (NC group) 9 23.744±1.081 76.637±9.748 OVX group (ovarian castration mouse model group) 8 25.475±1.27 59.553±16.079 Yisui Qianggu Fang Group (Drug Group) 8 23.212±1.218 66.734±10.172

[0167] Note: E2: estradiol, can inhibit osteoclast activity, reduce bone resorption and maintain bone density.

[0168] As shown in Table 2, the results of AST and ALT in the ELISA experiment of the mouse serum in the sham operation group, bilateral ovariectomy group and drug group indicate that this prescription has no hepatotoxicity.

[0169] From Table 3, it can be seen that the weight of mice in the drug group was close to that of normal mice after medication. The serum ELISA results showed that the estradiol level of mice in the drug group rebounded and was close to the normal level, which could inhibit osteoclast activity, reduce bone resorption, and maintain bone density.

[0170] like Figure 8 , Fig. 9 As shown, after treatment with the Yisui Qianggu Fang group of the traditional Chinese medicine composition for treating osteoporosis in the embodiment of the present application, there was no significant difference in the AST content in the serum of the drug group and the sham operation group, and the ALT content in the drug group was lower than that in the sham operation group, which proved that the Yisui Qianggu Fang group of the traditional Chinese medicine composition for treating osteoporosis in the embodiment of the present application had no obvious liver damage effect, and the difference was statistically significant (P>0.05).

[0171] like Fig.10 As shown, after treatment with the traditional Chinese medicine composition for treating osteoporosis in Example 1 of the present application, there was no significant difference in the body weight of mice between the drug group and the sham operation group, and the difference was statistically significant (P<0.05).

[0172] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A Chinese medicine composition for treating osteoporosis, characterized in that: Including the following raw materials by mass: Main drug: 8-15 parts of Ligustrum lucidum fruit; Minister drugs: 10-20 parts of Prunus mume, 5-15 parts of Epimedium, 5-15 parts of Morinda officinalis and 10-20 parts of Rehmannia glutinosa; Adjuvants: 5-13 parts of Drynaria fortunei, 5-13 parts of safflower and 8-15 parts of light fermented black beans; Adjuvant: 3-10 parts of chrysanthemum.

2. A Chinese medicine composition for treating osteoporosis according to claim 1, characterized in that: Including the following raw materials by mass: Main drug: Ligustrum lucidum 10-13 parts; Minister drugs: 12-18 parts of Prunus mume, 8-11 parts of Epimedium, 8-10 parts of Morinda officinalis and 12-17 parts of Rehmannia glutinosa; Adjuvants: 8-11 parts of Drynaria fortunei, 8-11 parts of safflower and 10-14 parts of light fermented black beans; Adjuvant: 5-7 parts of chrysanthemum.

3. A method for preparing a Chinese medicine composition for treating osteoporosis according to any one of claims 1 to 2, characterized in that: The following steps are involved: After weighing Ligustrum lucidum fruit, Prunus quinquefolia, Epimedium, Morinda officinalis, Rehmannia root, Drynaria fortunei, Carthamus tinctorius, Fructus sojae and Chrysanthemum according to the above proportions, they are washed, soaked and boiled in sequence to obtain a medicinal solution.

4. The method for preparing a Chinese medicine composition for treating osteoporosis according to claim 3, characterized in that: The following steps are involved: (1) Weigh Ligustrum lucidum fruit, Prunus mume, Epimedium, Morinda officinalis, Rehmannia root, Drynaria fortunei, Carthamus tinctorius, Fructus sojae and Chrysanthemum in the above proportions; (2) Cleaning the raw materials; (3) Primary decoction: add purified water to the weighed raw materials, with the weight ratio of raw materials to purified water being 1:(8-10), soak for 25-35 minutes, boil, and then decoct at 60°C-70°C for 1-1.5 hours, filter and collect the decoction for later use; (4) Concentrating the decoction at 60° C. to less than one tenth of the volume of the original decoction to obtain a medicinal solution.

5. The method for preparing a Chinese medicine composition for treating osteoporosis according to claim 4, characterized in that: The second decoction step is also included, and the second decoction is performed after the first decoction. The preparation steps are as follows: (1) Weigh Ligustrum lucidum fruit, Prunus mume, Epimedium, Morinda officinalis, Rehmannia root, Drynaria fortunei, Carthamus tinctorius, Fructus sojae and Chrysanthemum in the above proportions; (2) Cleaning the raw materials; (3) Primary decoction: add purified water to the weighed raw materials, with the weight ratio of raw materials to purified water being 1:(8-10), soak for 25-35 minutes, boil, and then decoct at 60°C-70°C for 1-1.5 hours, filter and collect the decoction for later use; (4) Secondary decoction: adding purified water to the above-mentioned decoction raw material, the weight ratio of the decoction raw material to purified water is 1: (8-10), after boiling, decocting at a temperature of 60° C.-70° C. for 40-50 minutes, filtering the decoction and setting aside; (5) combining the two decoctions, and concentrating the combined decoction at 60° C. to less than one tenth of the volume of the original decoction to obtain a medicinal solution.

6. Use of the Chinese medicine composition according to any one of claims 1 to 2 in the preparation of a drug for treating osteoporosis.

7. A drug for treating osteoporosis, characterized in that: The invention comprises the Chinese medicine composition according to any one of claims 1 to 2 and pharmaceutically acceptable excipients.

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