Composition for improving osteothione as well as preparation method and application of composition

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a composition for improving osteothione as well as a preparation method and application thereof. The invention provides a composition for improving osteothione, which is prepared from the following components in parts by mass: 500 to 600 parts of folium cortex eucommiae, 300 to 350 parts of male flower of cortex eucommiae, 300 to 350 parts of wine-processed rhizoma polygonati, 300 to 350 parts of radix fici simplicissimae, 200 to 250 parts of maca, 90 to 120 parts of natto, 60 to 80 parts of calcined oyster shell and 0.5 to 1 part of ossein. The composition can increase the number of bone trabecula, increase bone density, bone volume fraction, the number of bone trabecula, bone trabecula thickness and bone surface area-to-tissue volume ratio, lower bone surface area-to-bone volume ratio and bone trabecula separation degree, enhance osteoblast activity, inhibit osteoclast maturation and differentiation and relieve inflammation, and is high in safety performance. In conclusion, the composition provided by the invention has a certain improvement effect on osteoporosis.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a composition for improving osteoporosis, a preparation method and an application thereof. Background Art

[0002] Osteoporosis (OP) is a metabolic bone disease characterized by systemic bone loss, bone density loss, and bone microarchitecture destruction, which leads to increased bone brittleness, decreased bone strength, and easy fractures. The disease is characterized by pain, shortening, and even spinal deformity or fractures as clinical manifestations. Pain is the main symptom and is common in the lower back. According to the cause of osteoporosis, it can be divided into three categories: primary, secondary, and idiopathic osteoporosis. With the aging of my country's population, osteoporosis has become one of the most serious problems endangering the public health of my country's population. Therefore, the prevention and treatment of osteoporosis has become an important issue.

[0003] Osteoporosis belongs to the category of bone arthritis, bone atrophy and asthenia in traditional Chinese medicine. Its causes are mostly organ deficiency and acquired deficiency of spleen and kidney. Spleen deficiency can cause kidney deficiency, and kidney deficiency can lead to spleen deficiency. If the spleen fails to transport and transform, spleen essence is insufficient, kidney essence is deficient, or kidney essence is inherently deficient, spleen and kidney are both deficient, and bones are not nourished, then bones will be fragile and weak, which will eventually lead to the appearance of osteoporosis.

[0004] At present, the main therapeutic drugs for this disease include basic drugs (calcium, vitamin D), bone conversion inhibitors (bisphosphonates, calcitonin, estrogen, etc.), bone formation promoters (parathyroid hormone, fluoride) and uncoupling agents (strontium salts). Among them, estrogen takes a long time to take medicine, and it may cause breast cancer and endometrial cancer at the same time, and its long-term use has large toxic and side effects. Calcitonin has a certain effect on osteoporosis, but it is expensive and difficult for the majority of patients to accept. Calcium carbonate tablets have certain defects, such as inorganic calcium neutralizes gastric acid, can cause belching, constipation, etc., and long-term use has no obvious effect on bone absorption and bone formation. At present, there is a lack of efficient Chinese medicine compositions for the treatment of osteoporosis. Summary of the invention

[0005] The purpose of the present invention is to provide a composition for improving osteoporosis, a preparation method and an application thereof, which has a good effect on improving osteoporosis.

[0006] In order to solve the above technical problems, the following technical solutions are proposed:

[0007] The invention provides a composition for improving osteoporosis. The composition comprises the following components, measured by mass: 500-600 parts of eucommia leaves, 300-350 parts of eucommia male flowers, 300-350 parts of polygonatum odoratum, 300-350 parts of quinquefolius, 200-250 parts of maca, 90-120 parts of natto, 60-80 parts of calcined oysters and 0.5-1 part of collagen.

[0008] Preferably, the following components are included in parts by mass: 540 parts of eucommia leaves, 324 parts of eucommia male flowers, 324 parts of polygonatum odoratum, 324 parts of quinquefolius, 216 parts of maca, 108 parts of natto, 72 parts of calcined oysters and 0.72 parts of collagen.

[0009] The present invention provides a method for preparing the Chinese medicine composition described in the above technical solution, comprising the following steps:

[0010] Extracting the maca, eucommia leaves, eucommia male flowers, polygonatum sibiricum and quinquefolia to obtain an extract; the extraction method of the maca comprises decocting;

[0011] Concentrating and drying the extract to obtain an extract;

[0012] The extract, calcined oyster, natto and collagen are mixed to obtain an extract.

[0013] Preferably, the extraction solvent includes water; and the extraction includes a first extraction and a second extraction.

[0014] Preferably, the amount of water added in the first extraction is 8 to 12 times the mass of the Chinese medicinal raw material, and the time for the first extraction is 1 to 2 hours; the amount of water added in the second extraction is 6 to 10 times the mass of the Chinese medicinal raw material, and the time for the second extraction is 0.5 to 1 hour.

[0015] Preferably, the extraction further includes soaking, and the soaking time is 0.4 to 1 hour.

[0016] Preferably, the concentration includes reduced pressure concentration, the temperature of the reduced pressure concentration is 65 to 70°C, the vacuum degree of the reduced pressure concentration is -0.08 to -0.06Mpa; the reduced pressure concentration is performed until the relative density of the obtained product at 80°C is 1.20 to 1.25.

[0017] Preferably, the drying comprises vacuum drying, the vacuum drying time is 60 to 70° C., and the vacuum degree is -0.08 to -0.06 Mpa.

[0018] The present invention provides use of the composition described in the above technical solution or the composition prepared by the above technical solution in preparing a drug for preventing and / or treating osteoporosis.

[0019] Preferably, the dosage form of the drug includes one or more of oral dosage forms, injections, suppositories and ointments.

[0020] Beneficial effects of the present invention: The present invention provides a composition for improving osteoporosis, which comprises the following components by mass: 500-600 parts of Eucommia leaves, 300-350 parts of Eucommia male flowers, 300-350 parts of wine Polygonatum, 300-350 parts of five-fingered peach, 200-250 parts of maca, 90-120 parts of natto, 60-80 parts of calcined oysters and 0.5-1 parts of collagen. Eucommia leaves and Eucommia male flowers in the present invention are monarch drugs, Eucommia leaves nourish the liver and kidneys, strengthen tendons and bones, and Eucommia male flowers nourish the liver and kidneys, calm the nerves, and the two drugs are used together to strengthen tendons and bones, and command the whole prescription. Take the function of Polygonatum, five-fingered peach, and maca to invigorate qi, strengthen the spleen, benefit the kidney, and strengthen yang, increase the effect of relaxing tendons and activating collaterals, delay aging, and improve symptoms such as soreness of waist and knees. The three drugs cooperate, complement each other, and are ministerial drugs. With natto, calcined oyster and collagen, it can strengthen the tendons and bones and prevent fractures, effectively preventing osteoporosis symptoms. The whole recipe can strengthen the spleen and kidney, strengthen the tendons and bones, and is good at treating the symptoms of weakness in the waist and knees, limb fatigue, cold hands and feet, etc. caused by deficiency of kidney yang and kidney essence.

[0021] The results of the embodiments show that the composition of the present invention can improve the growth state of osteoporosis model rats and alleviate abnormal weight growth; there is no significant difference in the liver and kidney index of osteoporotic rats and its morphology is not abnormal, the toxic and side effects on rats are small, and the safety performance is high; it can also increase the number of trabeculae in osteoporotic rats, so that the reticular structure is restored to a certain extent, and the bone microstructure destruction of osteoporotic rat models is improved; and bone density, bone volume fraction, trabecular number, trabecular thickness, bone surface area tissue volume ratio are increased, while the bone surface area bone volume ratio, trabecular separation, and structural index model are lowered, and there is a certain reversal effect on osteoporotic rat femur bone turnover markers, and at the same time, serum CTX-I and TRAP metabolic levels can be reduced, serum ALP, BALP, BGP, PICP metabolic levels can be increased, serum inflammatory factors IL-6, IL-1β, TNF-α levels are significantly reduced, osteoblast activity is enhanced, osteoclast maturation and differentiation are inhibited, and inflammation is relieved. In summary, the composition provided by the present invention has a certain improvement effect on osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 This is the effect of the Eucommia mixture on the body weight of osteoporotic rats in Example 4;

[0024] Figure 2 This is the effect of the Eucommia mixture of Example 4 on the renal index of osteoporotic rats;

[0025] Figure 3 This is the effect of the Eucommia mixture on the liver and kidney indexes of osteoporotic rats in Example 4;

[0026] Figure 4 This is the effect of the Eucommia mixture on bone microstructure in osteoporotic rats in Example 4;

[0027] Figure 5 The effect of the Eucommia mixture in Example 4 on the bone mineral density (BMD) in the femur bone turnover marker of osteoporotic rats;

[0028] Figure 6 The effect of the Eucommia mixture in Example 4 on the bone volume fraction (BV / TV) in the femur bone turnover marker of osteoporotic rats;

[0029] Figure 7 The effect of the Eucommia mixture in Example 4 on the bone surface area to bone volume ratio (BS / BV) in the femur bone turnover marker of osteoporotic rats;

[0030] Figure 8 The effect of the Eucommia mixture in Example 4 on trabecular bone thickness (Tb.Th), a bone turnover marker in the femur of osteoporotic rats;

[0031] Fig. 9 The effect of the Eucommia mixture in Example 4 on the number of trabecular bones (Tb.N) in the femoral bone turnover marker of osteoporotic rats;

[0032] Fig.10 The effect of the Eucommia mixture in Example 4 on the trabecular separation (Th.Sp) in the femoral bone turnover markers of osteoporotic rats;

[0033] Fig.11 The effect of the Eucommia mixture in Example 4 on the bone structure index model (SMI) in the femoral bone turnover markers of osteoporotic rats;

[0034] Fig.12 This is a graph showing the effect of the Eucommia mixture on the bone surface area to tissue volume ratio (BS / TV) in the femur bone turnover marker of osteoporotic rats in Example 4;

[0035] Fig.13 This is the effect of the Eucommia mixture in Example 4 on the serum bone metabolism index BALP in osteoporosis model rats;

[0036] Fig.14 This is the effect of the Eucommia mixture in Example 4 on the serum bone metabolism index TRAP in osteoporosis model rats;

[0037] Fig.15 This is the effect of the Eucommia mixture in Example 4 on the serum bone metabolism index ALP in osteoporosis model rats;

[0038] Fig.16This is the effect of the Eucommia mixture in Example 4 on the serum bone metabolism index CTX-1 in osteoporosis model rats, where CTX stands for CTX-1;

[0039] Fig.17 This is the effect of the Eucommia mixture in Example 4 on the serum bone metabolism index BGP in osteoporosis model rats;

[0040] Fig.18 This is the effect of the Eucommia mixture in Example 4 on the serum bone metabolism index PICP in osteoporosis model rats;

[0041] Fig.19 This is the effect of the Eucommia mixture in Example 4 on the serum inflammatory index IL-1β in osteoporosis model rats;

[0042] Fig. 20 This is the effect of the Eucommia mixture in Example 4 on the serum inflammatory index IL-6 in osteoporosis model rats;

[0043] Fig.21 This is the effect of the Eucommia mixture in Example 4 on the serum inflammatory index TNF-α in osteoporosis model rats, where TNF stands for TNF-α;

[0044] Fig. 22 The survival rate of MC3T3-E1 cells in different ratios of Eucommia compound drug-containing serum (n=3);

[0045] Fig.23 The effect of different ratios of Eucommia compound serum on ALP activity (n=3);

[0046] Fig.24 Alizarin red staining of serum containing different proportions of Eucommia compound;

[0047] Note: Figures 5 to 12 All plotted as mean SD of parallel experiments. Figures 13 to 21 All plots are based on the SEM mean of parallel experiments. DETAILED DESCRIPTION

[0048] The invention provides a composition for improving osteoporosis. The composition comprises the following components, measured by mass: 500-600 parts of eucommia leaves, 300-350 parts of eucommia male flowers, 300-350 parts of polygonatum odoratum, 300-350 parts of quinquefolius, 200-250 parts of maca, 90-120 parts of natto, 60-80 parts of calcined oysters and 0.5-1 part of collagen.

[0049] The composition provided by the present invention comprises 500-600 parts of Eucommia ulmoides leaves, preferably 520-560 parts, and more preferably 540 parts by weight. In the present invention, the Eucommia ulmoides leaves can have the effects of nourishing the liver and kidney and strengthening the tendons and bones.

[0050] Based on the mass of Eucommia leaves, the composition provided by the present invention comprises 300-350 parts of Eucommia male flowers, preferably 310-330 parts, and more preferably 324 parts. In the present invention, the Eucommia male flowers have the effects of nourishing the liver and kidneys and calming the nerves.

[0051] Based on the mass fraction of Eucommia ulmoides leaves, the composition provided by the present invention includes 300-350 parts of Polygonatum odoratum, preferably 305-332 parts, and more preferably 324 parts. In the present invention, the Polygonatum odoratum has the effects of invigorating qi and nourishing yin, tonifying spleen and moistening lungs, and benefiting kidneys; the preparation method of the Polygonatum odoratum can be found in the Pharmacopoeia of the People's Republic of China (2020 Edition).

[0052] Based on the mass of Eucommia ulmoides leaves, the composition provided by the present invention comprises 300-350 parts of Eucommia ulmoides, preferably 312-341 parts, and more preferably 324 parts. In the present invention, the Eucommia ulmoides has the effects of replenishing qi, tonifying deficiency, promoting qi and relieving depression, and strengthening tendons and activating collaterals.

[0053] Based on the mass of Eucommia ulmoides leaves, the composition provided by the present invention includes 200-250 parts of maca, preferably 205-230 parts, and more preferably 216 parts. The maca of the present invention is preferably prepared into maca powder and then used; in the present invention, the maca powder has the effects of nourishing the kidney and strengthening the essence and strengthening the body.

[0054] Based on the mass of Eucommia ulmoides leaves, the composition provided by the present invention includes 90 to 120 parts of natto, preferably 100 to 110 parts, and more preferably 108 parts. The natto of the present invention is preferably prepared into natto powder and then used. In the present invention, the natto can increase bone density and strengthen bones.

[0055] Based on the mass of Eucommia ulmoides leaves, the composition provided by the present invention comprises 60 to 80 parts of calcined oyster, preferably 68 to 76 parts, and more preferably 72 parts. In the present invention, the calcined oyster as an excipient also has the effect of promoting bone health.

[0056] Based on the mass of Eucommia ulmoides leaves, the composition provided by the present invention comprises 0.5 to 1 part of collagen, preferably 0.6 to 0.8 part, and more preferably 0.72 part. In the present invention, the collagen can strengthen tendons and bones and prevent fractures.

[0057] In the present invention, the sources and preparation methods of the above-mentioned components are not particularly limited, as long as they meet the corresponding requirements of the 2020 edition of the "Chinese Pharmacopoeia".

[0058] The composition provided by the present invention can improve osteoporosis, increase the number of trabeculae, and improve the bone microstructure destruction of osteoporosis; and increase the bone density, bone volume fraction, trabeculae number, trabeculae thickness, bone surface area to tissue volume ratio, and simultaneously decrease the bone surface area to bone volume ratio, trabecular separation degree, and structural index model, and has a certain reversal effect on femoral bone turnover markers of osteoporotic rats; and at the same time, it can also reduce the serum type I collagen cross-linked C-terminal peptide (CTX-I) and tartrate-resistant acid phosphatase (TRAP) metabolic levels, increase the serum alkaline phosphatase (ALP), serum bone-specific alkaline phosphatase (BALP), serum osteocalcin (BGP), and serum type I procollagen C-terminal propeptide (PICP) metabolic levels, enhance osteoblast activity, inhibit osteoclast maturation and differentiation, significantly reduce the serum inflammatory factors IL-6, IL-1β, and TNF-α levels, relieve inflammation, and has no adverse reactions, small toxic and side effects, and high safety performance.

[0059] The present invention provides a method for preparing the composition described in the above technical solution, comprising the following steps:

[0060] Extracting the maca, eucommia leaves, eucommia male flowers, polygonatum sibiricum and quinquefolia to obtain an extract; the extraction method of the maca comprises decocting;

[0061] The extract is concentrated and dried to obtain an extract;

[0062] The extract, calcined oyster, natto and collagen are mixed to obtain an extract.

[0063] The present invention extracts the maca, eucommia leaves, eucommia male flowers, polygonatum sibiricum and quinquefolia to obtain an extract; the maca of the present invention is preferably crushed into maca powder before extraction, and the extraction method of the maca of the present invention includes decocting; the decocting is preferably placing the maca powder in a bag for decocting; the bag is preferably a non-woven bag.

[0064] Before the extraction, the present invention preferably soaks the maca, eucommia leaves, eucommia male flowers, Polygonatum sibiricum and Prunus mume. The soaking time of the present invention is preferably 0.4 to 1 hour, more preferably 0.5 hours; the soaking temperature is preferably room temperature, and the soaking allows water to infiltrate the inside of the medicinal materials, which is conducive to complete extraction.

[0065] After soaking, the present invention extracts the soaked product; the extraction preferably includes water extraction; the water extraction preferably includes a first extraction and a second extraction; based on the mass of the raw material, the amount of water added in the first extraction is preferably 8 to 12 times the mass of the Chinese medicine raw material, and more preferably 10 times; the time of the first extraction is preferably 1 to 2 hours, and more preferably 1 to 1.5 hours, and more preferably 1 hour; the amount of water added in the second extraction is based on the mass of the raw material, and the amount of water added in the second extraction is preferably 6 to 10 times the mass of the Chinese medicine raw material, and more preferably 8 times; the time of the second extraction is preferably 0.5 to 1 hour, and more preferably 0.5 hours. After the first extraction is completed, it is preferably included to filter the first extraction to obtain a first filtrate; after the second extraction is completed, it is preferably included to filter the second extraction to obtain a second filtrate; the extraction method of the present invention preferably includes water decoction. The water decoction of the present invention can be performed using conventional parameters without special restrictions. The filtration of the present invention is preferably completed using a filter, and the mesh number of the filter is preferably 200.

[0066] After obtaining the first filtrate and the second filtrate, the present invention preferably combines the first filtrate and the second filtrate to obtain an extract.

[0067] The present invention concentrates and dries the extract to obtain an extract; the concentration method of the present invention preferably includes reduced pressure concentration, and the temperature of the reduced pressure concentration is preferably 65 to 70°C, more preferably 66 to 69°C, and more preferably 68°C; the vacuum degree of the reduced pressure concentration is preferably -0.08 to -0.06Mpa, more preferably -0.078Mpa to -0.062Mpa, and more preferably -0.07Mpa; the reduced pressure concentration of the present invention is preferably until the relative density of the obtained product at 80°C is about 1.20 to 1.25. The drying of the present invention preferably includes vacuum drying; the temperature of the vacuum drying is preferably 60 to 70°C, more preferably 62 to 69°C, and more preferably 66 to 68°C; the vacuum degree is preferably -0.08 to -0.06Mpa, more preferably -0.075Mpa to -0.063Mpa, and more preferably -0.07Mpa.

[0068] After obtaining the extract, the present invention mixes the extract, calcined oyster, natto and collagen to obtain an extract, and more preferably, the extract, calcined oyster, natto powder, collagen and auxiliary materials are mixed to obtain an extract. The extract of the present invention preferably includes crushing before mixing; the particle size of the mixture after crushing is preferably 80 meshes; the calcined oyster of the present invention is preferably crushed into calcined oyster powder before mixing; the crushing method is not particularly limited, and a conventional method can be used. The particle size of the calcined oyster powder of the present invention is preferably 200 meshes. The present invention preferably mixes the extract, calcined oyster, natto powder, collagen and auxiliary materials to obtain a mixture; the mixture is granulated and dried after a second mixing with a binder to obtain an extract. The auxiliary materials of the present invention preferably include dextrin, and the addition amount of dextrin is preferably 25% to 30% of the mass of the mixture, more preferably 28%; the binder preferably includes ethanol, more preferably 85% ethanol by volume. The addition amount of ethanol of the present invention is preferably 15% to 20% of the mass of the mixture. The granulation of the present invention is preferably completed using a 14-mesh sieve; the drying method preferably includes baking; the baking temperature is preferably 48-52° C., more preferably 50° C.; the baking is used to remove moisture and prevent the particles from getting moldy and deteriorating; the present invention has no special restrictions on the parameters of the mixing and the second mixing, as long as uniform mixing is ensured.

[0069] The extract prepared by the invention (i.e. the composition for improving osteoporosis) has high chlorogenic acid content and high extract yield.

[0070] The present invention provides use of the composition described in the above technical solution or the composition prepared in the above technical solution in preparing a drug for preventing and / or treating osteoporosis.

[0071] The effective dosage of the extract in the medicine prepared by the extract of the present invention is preferably 23-28 g / d, more preferably 25-27 g / d, and more preferably 26.51 g / d. The administration frequency of the medicine of the present invention is preferably once a day. The medicine of the present invention preferably also includes a pharmaceutically acceptable excipient, and the dosage form of the medicine of the present invention includes one or more of an oral dosage form, an injection, a suppository and an ointment.

[0072] The drug of the present invention is preferably used for alleviating abnormal weight gain, improving bone microstructure, reversing bone turnover markers, enhancing bone metabolism indicators and alleviating inflammation, and more preferably alleviating abnormal weight gain, improving bone microstructure, reversing bone turnover markers, enhancing bone metabolism indicators and alleviating inflammation. The improvement of bone microstructure of the present invention preferably includes increasing the number of trabeculae; the reversal of bone turnover markers preferably includes simultaneously upregulating one or more of bone density, bone volume fraction, trabecular number, trabecular thickness and bone surface area to tissue volume ratio, and downregulating one or more of bone surface area to bone volume ratio, trabecular separation and structural index model; more preferably, simultaneously upregulating bone density, bone volume fraction, trabecular number, trabecular thickness and bone surface area to tissue volume ratio, and downregulating bone surface area to bone volume ratio, trabecular separation and structural index model.

[0073] The enhanced bone metabolism index of the present invention preferably includes reducing the metabolic level of serum CTX-I and / or TRAP, and increasing the metabolic level of one or more of serum ALP, BALP, BGP and PICP; more preferably includes reducing the metabolic level of serum CTX-I and TRAP, and increasing the metabolic level of serum ALP, BALP, BGP and PICP. The relieving inflammation of the present invention preferably includes reducing the level of one or more of IL-6, IL-1β and TNF-α in serum inflammatory factors, and more preferably reducing IL-6, IL-1β and TNF-α in serum inflammatory factors. After experimental verification, the prepared composition of the present invention has a certain improvement effect on osteoporosis.

[0074] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0075] Example 1

[0076] Compound recipe: 540g of Eucommia leaves, 324g of Eucommia male flowers, 324g of Polygonatum sibiricum, 324g of Eleutherodactyla quinquefolia, 216g of maca powder, 108g of natto powder, 72g of calcined oyster and 0.72g of collagen.

[0077] Preparation method: Among the eight herbs in the compound, the calcined oyster is crushed into fine powder for later use; among them, maca powder (packaged decoction), eucommia leaves, eucommia male flowers, wine polygonatum, and five-fingered ginseng are decocted twice with water; maca powder (packaged decoction), eucommia leaves, eucommia male flowers, wine polygonatum, and five-fingered ginseng are recorded as mixture 1, and 10 times the weight of the raw materials of mixture 1 are added to the first decoction, soaked at room temperature for 0.5h, decocted for 1h, filtered through 200 mesh, and the filtrate is used for later use; 8 times the weight of the raw materials of mixture 1 are added to the second decoction, decocted for 0.5h, filtered through 200 mesh, and the filtrate is combined; the obtained filtrate is concentrated under reduced pressure (65℃~70℃, vacuum degree -0.08~- 0.06Mpa) to a thick paste with a relative density of about 1.20 to 1.25 (80°C), vacuum drying (60°C to 70°C, vacuum degree -0.08 to -0.06Mpa), crushing, and then adding the above-mentioned calcined oyster powder, natto powder, collagen and dextrin in appropriate amounts, mixing to obtain mixture 2, the amount of dextrin added is 25% to 30% of the mass of mixture 2; then adding 85% ethanol by volume, the amount of 85% ethanol by volume is 15 to 20% of the mass of mixture 2, and then granulating through a 14-mesh sieve, drying at 50°C, 14-mesh granules, and making 1000g granules, that is, compound Eucommia ulmoides granules. 85% ethanol is a binder. The range value of dextrin addition of 25% to 30% is to ensure that 1000g granules are prepared.

[0078] Example 2 Process research of anti-osteoporosis samples

[0079] 1. Optimization of decoction conditions for water-decocted drugs

[0080] In order to explore the process conditions of water extraction and decoction of maca powder (wrapped and decocted), Eucommia ulmoides leaves, Eucommia ulmoides male flowers, Polygonatum sibiricum and Eleutherodactyla quinquefolia, the extract yield and the extraction amount of chlorogenic acid were taken as the examination indicators. The orthogonal test was used to optimize the soaking time, water addition amount and decoction time during extraction, and the factor level table was designed.

[0081] (1) Factor level table

[0082] The factor level table of the orthogonal experiment of water extraction process conditions is shown in Table 1. The amount of water added in Table 1 is based on the raw material quality and weight of five kinds of Chinese medicines: maca powder (decoction), eucommia leaves, eucommia male flowers, wine polygonatum and five-fingered peach.

[0083] Table 1 Factor level table

[0084]

[0085] (2) Test methods and data

[0086] Use L 9 (3 4 )Orthogonal table is used to arrange the experiment. The specific experimental conditions are as follows:

[0087] Weigh 96g of water-extracted Chinese medicine (12g of maca powder, 30g of eucommia leaves, 18g of eucommia male flowers, 18g of wine polygonatum, and 18g of five-fingered peach), wherein the maca powder is placed in a non-woven bag for decocting (the volume ratio of maca powder is 1 / 4), and decocted in water according to the scheme in Table 1, and the extract is collected, and the extract is concentrated under reduced pressure to 250mL to obtain a sample solution; the temperature of the reduced pressure concentration is 65°C to 70°C, and the vacuum degree is -0.08 to -0.06Mpa, and the extract yield and the extraction amount of chlorogenic acid are determined.

[0088] Extract yield determination method:

[0089] Accurately pipette 50 mL of the extract into an evaporating dish that has been dried to constant weight, evaporate to dryness in a water bath, dry at 105°C for 3 h, cool in a desiccator for 0.5 h, weigh quickly, and calculate the extract yield.

[0090] Extract yield (%) = (W×V) / (50×W1)×100%

[0091] Note: W—extract weight (g), W1—medicinal material weight (g), V—fixed volume (mL). In this example, V is the volume of the sample solution.

[0092] Determination method of chlorogenic acid content:

[0093] (1) Materials and instruments

[0094] The decoction pieces were provided by Hunan Zhenxing Chinese Medicine Co., Ltd., chlorogenic acid reference substance (provided by China Food and Drug Inspection Institute, batch number: 110753-202018); high performance liquid chromatography (Agilent 1200), AE240 electronic analytical balance.

[0095] (2) Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 General Chapter 0512).

[0096] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as filler; acetonitrile-0.4% phosphoric acid (10:90) is used as mobile phase; detection wavelength is 327nm. The theoretical plate number calculated based on the chlorogenic acid peak should be no less than 2000.

[0097] Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference substance, weigh it accurately, place it in a brown volumetric flask, and add 50% methanol to make a solution containing 50 μg per 1 mL.

[0098] Preparation of test solution: Measure the fixed volume of sample solution, filter it through a microporous filter membrane (0.45μm), and take the filtrate.

[0099] Determination method: Accurately pipette 10 μL of the test solution and the reference solution respectively, inject into the high performance liquid chromatograph, determine, and calculate the content of chlorogenic acid; set up two parallel experiments, and take the average value of the peak area of ​​the two parallel experiments to calculate the chlorogenic acid extraction amount and extraction rate.

[0100] The chlorogenic acid content in the Eucommia ulmoides leaves and the Eucommia ulmoides male flowers was determined by the above-mentioned high performance liquid chromatography method, and the total chlorogenic acid content in the Eucommia ulmoides leaves and the Eucommia ulmoides male flowers was measured to be 633.4 mg.

[0101] Extraction rate = chlorogenic acid content in the extract / (the sum of chlorogenic acid content in Eucommia ulmoides leaves and Eucommia ulmoides male flowers) × 100%

[0102] The test results are shown in Table 2. D in Table 2 is a blank column, which can reflect the random error or systematic error in the experiment and help analyze the reliability of the experimental results.

[0103] Table 2 Orthogonal test design table and results

[0104]

[0105] The orthogonal test results were subjected to variance analysis, and the results are shown in Tables 3 and 4.

[0106] Table 3 Variance analysis table of extract yield

[0107] Sources of variation SS r MS F P* A 9.4907 2 4.7453 2.7147 >0.05 B 59.4476 2 29.7238 17.0040 >0.05 C 22.7943 2 11.3971 6.5199 >0.05 error 3.4961 2 1.7480

[0108] Note: F 0.05 (2,2)=19, F 0.01 (2,2)=99.

[0109] Table 4 Analysis of variance table of chlorogenic acid extraction rate

[0110] Sources of variation SS r MS F P* A 458.5770 2 229.2885 60.1890 <0.05 B 82.2294 2 41.1147 10.7927 >0.05 C 374.8923 2 187.4461 49.2052 <0.05 error 7.6190 2 3.8095

[0111] Note: F 0.05 (2,2)=19, F 0.01 (2,2)=99.

[0112] Taking the extract yield as the inspection index, the range R value in Table 2 shows that the role of each factor is B>C>A, and the variance analysis results show that none of the three factors are significant (P>0.05); taking the chlorogenic acid extraction rate as the inspection index, the range R value in Table 2 shows that the role of each factor is A>C>B, and the variance analysis results show that factors A and C are significant (P<0.05), the best level of factor A is A2, the best level of factor C is C3, and factors A and B are not significant, so A is the best. 2 B 2 C 3The combination is the best, taking into account the influence of the extract yield and chlorogenic acid content, as well as saving working hours and reducing energy consumption, and A is scheduled 2 B 2 C 3 The best combination is that after the medicinal materials are soaked for 0.5 h, 10 times the amount of water is added for the first time and boiled for 1 h; the second time, 8 times the amount of water is added and boiled for 0.5 h.

[0113] Example 3

[0114] 1. Materials

[0115] 1.1 Animals

[0116] 114 SPF male SD rats and 38 female SD rats, weighing 200±20g, were purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd., with a laboratory animal production license number of SCXK (Xiang) 2019-0004; they were kept in the barrier environment of Hunan New Pilot Testing Technology Co., Ltd., with a laboratory animal use license number of SYXK (Xiang) 2022-0005, with a room temperature of 20.0-26.0℃ and a relative humidity of 40.0-70.0%. All operations in this study were strictly carried out in accordance with the relevant laws and regulations on experimental animal welfare ethics in my country and the relevant standard operating procedures of Hunan New Pilot Testing Technology Co., Ltd. (ethical approval number: IACUC-XLH-2023-107).

[0117] 1.2 Cells

[0118] Mouse embryonic osteoblast precursor cells MC3T3-E1 cells (Mei Lun, CL0729) were cultured in α-MEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 Incubator at 4 ℃ and observe under an inverted microscope that the cell density is 80% to 90%, then subculture. Cryopreservation is performed at the 4th to 5th generation, and cells in logarithmic growth phase are taken for experiments.

[0119] 1.3 Drugs and reagents

[0120] Different mixtures of Eucommia ulmoides are composed of Eucommia ulmoides leaves, Eucommia ulmoides male flowers, Polygonatum sibiricum, Prunus mume, Maca powder, Natto, and collagen; according to the orthogonal experimental table L18 (3 7 ) 18 groups of Eucommia ulmoides compound prescriptions with different mass ratios were obtained, and the ratios are shown in Table 5. There is no calcined oyster in Table 5, and calcined oyster is an excipient, so the dosage was not optimized.

[0121] Table 5 Proportions of different compound prescriptions of Eucommia ulmoides

[0122] Eucommia ulmoides leaves (A) Eucommia ulmoides male flowers (B) Huangjing (C) Five-fingered peach (D) Maca powder(E) Natto(F) Collagen (G) 1 15 9 9 9 6 3 0.02 2 15 6 6 6 3 1 0.03 3 15 12 12 12 9 6 0.06 4 10 9 9 6 3 6 0.06 5 10 6 6 12 9 3 0.01 6 10 12 12 9 6 1 0.03 7 20 9 6 9 9 1 0.06 8 20 6 12 6 6 6 0.01 9 20 12 9 12 3 3 0.03 10 15 9 12 12 3 1 0.01 11 15 6 9 9 9 6 0.03 12 15 12 6 6 6 3 0.06 13 10 9 6 12 6 6 0.03 14 10 6 12 9 3 3 0.06 15 10 12 9 6 9 1 0.01 16 20 9 12 6 9 3 0.03 17 20 6 9 12 6 1 0.06 18 20 12 6 9 3 6 0.01

[0123] Positive drug estradiol valerate tablets (batch number: 20171038), light yellow sugar-coated tablets, 1 mg / tablet, are mainly used to supplement the deficiency of estrogen; they can also be used to prevent osteoporosis, amenorrhea, menstrual abnormalities, functional uterine bleeding, etc. caused by decreased estrogen levels; oral, 1 tablet at a time, once a day; provided by Bayer Healthcare Co., Ltd.

[0124] Dexamethasone (Solebao, ID0170), α-MEM basal medium (Mei Lun, MA0216), mouse MC3T3-E1 osteogenic differentiation kit (Starfish Biotechnology, EOMX-D101R), CCK-8 (Biyuntian, C0037), mineralized nodule staining kit (Biyuntian, C0148S), alkaline phosphatase detection kit (Elabscience, E-BC-K091-M) (Qingdao Huaren Pharmaceutical Co., Ltd.), medical alcohol, physiological saline, 4% paraformaldehyde and other commonly used laboratory reagents are all of analytical grade (Sinopharm Chemical Reagent Co., Ltd.), etc.

[0125] 1.4 Instruments

[0126] PTT-B1000 electronic balance (Huazhi Electronic Technology Co., Ltd.), Sky Scan1276 Micro-CT (Bruker, Germany), Centrifuge 5424R refrigerated centrifuge (Eppendorf), etc.

[0127] 2. Methods

[0128] 2.1 Methods of drug preparation

[0129] Taking the Eucommia compound numbered 1 in Table 5 as an example, the preparation method is described:

[0130] Animal experiment of Eucommia compound: Weigh 540g of Eucommia leaves, 324g of Eucommia male flowers, 324g of Polygonatum sibiricum, 324g of Eleutherodactyla tricholoma, 216g of maca powder, 108g of natto powder, 72g of calcined oyster, and 0.72g of collagen; extract and dry the five herbs of Eucommia leaves, Eucommia male flowers, Polygonatum sibiricum, Eleutherodactyla tricholoma, and maca powder (a total of 1728g) to obtain dry paste powder (540g of dry paste powder, yield of 31.25%), and mix the remaining 3 raw drug powders (a total of 180.72g) with the dry paste powder obtained above to obtain the test sample.

[0131] The specific process of water extraction is as follows: after the medicinal materials are soaked for 0.5 h, 10 times the amount of water of the medicinal materials is added for the first time, decocted for 1 h, filtered through 200 mesh, and the filtrate is reserved; 8 times the amount of water of the medicinal materials is added for the second time, decocted for 0.5 h, filtered through 200 mesh, and the two filtrates are combined to obtain the water extract.

[0132] Drying: The water extract is concentrated under reduced pressure (65°C to 70°C, vacuum degree -0.08 to -0.06Mpa) to a thick paste with a relative density of about 1.20 to 1.25 at 80°C, vacuum dried (60°C to 70°C, vacuum degree -0.08 to -0.06Mpa), and crushed to obtain a dry paste powder.

[0133] Accurately weigh the corresponding amount of dry paste powder obtained from the test sample, add purified water to prepare the test sample solution of corresponding concentration, prepare it on the spot and use it in animal experiments on drug-containing serum.

[0134] The method for preparing dry paste powder of other numbered Eucommia compounds in Table 5 is the same as described above.

[0135] 2.2 Preparation of drug-containing serum

[0136] 114 male SD rats were randomly divided into 19 groups, recorded as 0-18 groups, of which group 0 was the normal control group, and groups 1-18 corresponded to groups 1-18 of Eucommia compound in Table 5. For example, group 1 was given the dry paste powder prepared by Eucommia compound numbered 1 in Table 5, and the dosage of groups 1-18 was 0.74 g / kg. The normal control group was given an equal amount of normal saline, and blood was collected from the abdominal aorta after continuous gavage for 14 days. Three vacuum blood collection tubes were taken from each rat. The collected blood was centrifuged at 13000rpm and 4℃ for 15min, and the supernatant, i.e., the drug-containing serum, was stored in a refrigerator at -80℃ for later use. The drug-containing serum of Eucommia compound numbered 1-18 in Table 5 was obtained, and there were 18 drug-containing serums, recorded as drug-containing serums No. 1-18; the serum obtained from the normal control group was recorded as number "0", i.e., serum No. 0.

[0137] 2.3 Cell culture and drug intervention

[0138] Drug-added group: After MC3T3-E1 cells were cultured to the fourth generation, MC3T3-E1 cells in the logarithmic growth phase were obtained and digested with 0.25% trypsin to prepare a cell suspension. The density was adjusted to 1.0*10 4 / mL was inoculated into a 96-well plate, 100μL per well, and placed in an incubator for culture. After it adhered to the wall, the original culture medium was discarded, and 100μL of complete culture medium was added respectively. The complete culture medium contained serum, and the volume proportion of serum in the complete culture medium was 10%. The serum was the drug-containing serum No. 1 to No. 18 obtained by feeding Eucommia ulmoides with different ratios of the compound in 2.2 and the serum No. 0 obtained by the normal control group.

[0139] The negative control group was the same as the drug-added group, with the only difference being that only 100 μL of complete culture medium was added to the MC3T3-E1 cells and the cells continued to be cultured in the incubator.

[0140] Only 100 μL of complete culture medium was added to the blank control group without MC3T3-E1 cells, and the cells were cultured in the incubator.

[0141] Three parallel experiments were set up for each group.

[0142] After 24 hours of intervention with drug-containing serum, the drug-containing culture medium was discarded, and 100 μL of CCK-8 solution with a mass concentration of 10% prepared with complete culture medium was added to each well, oscillated and mixed, and incubated in the incubator for another 2 hours. The absorbance value (OD value) was detected at a wavelength of 450 nm on a microplate reader to reflect the cell survival rate. Cell survival rate = (OD value of the drug-treated group - OD value of the blank control group) / (OD value of the negative control group - OD value of the blank control group) × 100%.

[0143] 2.4 Alkaline phosphatase activity detection and mineralization analysis

[0144] MC3T3-E1 cells were cultured at a rate of 1×10 5 The cells were inoculated at a density of 100 / mL in a 24-well plate. After they were completely attached to the wall, the original culture medium was removed and 500 μL of osteogenic differentiation medium was added to each well. The cells were then divided into a control group, an experimental group and a positive control group.

[0145] Experimental group: After the osteogenic differentiation solution was added to the cells, the volume of the drug-containing serum in the differentiation solution accounted for 10%. The osteogenic differentiation solution was replaced every 2 to 3 days. After 14 days of induction, the culture medium was aspirated for alkaline phosphatase kit detection. The remaining cells were washed twice with PBS and fixed with fixative for 30 minutes. After washing with PBS, an appropriate amount of alizarin red solution was added for staining. After 30 minutes, the image of the stained cells was captured under an inverted fluorescence microscope. Three parallel experiments were set. The drug-containing serum was the drug-containing serum No. 1 to No. 18 obtained by feeding Eucommia ulmoides with different ratios of the compound in 2.2.

[0146] Positive control group: Same as the experimental group, the only difference is that dexamethasone is added to the osteogenic differentiation solution, and the amount of dexamethasone added is 1 μM. Dexamethasone is an inhibitor. Three parallel experiments are set up.

[0147] Control group: Same as the experimental group, the only difference is that the osteogenic differentiation solution added to the cells contains blank serum No. 0, and the volume proportion of blank serum in the differentiation solution is 10%. Set up 3 parallel experiments.

[0148] 3. Results

[0149] 3.1 Effects of different ratios of Eucommia ulmoides compound on cell activity

[0150] The activity of MC3T3-E1 cells after incubation with different ratios of Eucommia ulmoides compound for 24 hours was detected by CCK-8, and the cell survival rate was calculated by the formula. The results showed that (see Table 6 below, Fig. 22), compared with the control group, after treatment with different ratios of Eucommia compound, except for the decreased activity of MC3T3-E1 cells in groups 6 and 12, the other groups had no significant effect or increased cell activity, and the cell survival rate was the highest in groups 1, 7, and 13. The high activity of MC3T3-E1 cells indicates that Eucommia compound has a stable cell state, high safety performance, and no toxicity.

[0151] Table 6 Effect of different ratios of eucommia compound serum on the survival rate of MC3T3-E1 cells (n=3)

[0152]

[0153] Note: 0 to 18 correspond to the numbers of the serum, for example, number 0 corresponds to the survival rate of MC3T3-E1 cells added with serum No. 0.

[0154] 3.2 Effects of different ratios of Eucommia ulmoides compound on alkaline phosphatase ALP

[0155] The results showed (see Table 7, Fig.23 ), compared with the control group, dexamethasone significantly inhibited the ALP activity (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001), there was no significant difference in ALP in the sera containing different compound ratios of Eucommia ulmoides in groups 2, 3, 11, 13, 14, and 17, the ALP activity in groups 7, 8, 9, 10, 15, and 16 was significantly reduced (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001), and the ALP activity in groups 1, 4, 5, 6, 12, and 18 was significantly increased (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). It can be seen that dexamethasone can significantly inhibit the osteogenic differentiation of MC3T3-E1 cells, and different ratios of sera containing Eucommia compound 1, 4, 5, 6, 12, and 18 can significantly promote the osteogenic differentiation of MC3T3-E1 cells, and the ratios of groups 1, 15, and 18 are the most obvious. Fig.23 "Dexamethasone" in this drug refers to dexamethasone.

[0156] Table 7 Effects of different ratios of Eucommia compound serum on ALP activity (n=3)

[0157] ALP(U / L) Parallel experiment 1 Parallel experiment 2 Parallel experiment 3 0 4.494696 4.355109 4.494696 1 6.867672 7.286432 7.007259 2 4.773869 4.773869 4.355109 3 4.494696 4.634283 4.913456 4 5.332217 5.890564 5.192630 5 7.565606 7.286432 7.007259 6 5.471803 5.471803 5.332217 7 2.400893 2.400893 2.540480 8 2.680067 2.680067 2.819654 9 3.098827 2.959241 3.378001 10 4.215522 3.657175 3.517588 11 4.773869 5.471803 4.773869 12 5.053043 5.192630 4.634283 13 3.936348 4.355109 3.796762 14 5.471803 4.215522 3.936348 15 2.819654 2.540480 2.540480 16 3.238414 2.819654 3.098827 17 4.355109 5.192630 4.773869 18 6.728085 7.286432 6.448911 Dexamethasone 2.457424 2.646445 2.445631

[0158] 3.3 Mineralization analysis of different ratios of Eucommia ulmoides compound

[0159] Mineralized nodules are a sign of osteoblast differentiation and maturation, and are also the main morphological characteristics of osteoblasts in performing their osteogenic function. This study mainly analyzed them using the Alizarin Red S method. Fig.24As shown, compared with the control group, dexamethasone significantly inhibited the osteogenic differentiation of cells. The cells in groups 1, 4, 5, 6, and 18 with different ratios of Eucommia compound all had different degrees of osteogenic differentiation, and the osteogenic differentiation of groups 1, 5, and 18 was the most obvious.

[0160] In summary, in the present invention, the effects of different compound ratios of Eucommia ulmoides on the activity of MC3T3-E1 were first obtained by CCK-8, and it was found that the cell survival rate of the ratios of groups 1, 7, and 13 was the highest. The ALP kit and the Alizarin red method were used to reflect the osteogenic differentiation effect of each ratio. The results showed that the ALP activity of the ratios of groups 1, 15, and 18 increased most significantly, and Alizarin red staining found that the osteogenic differentiation of the ratios of groups 1, 5, and 18 was the most obvious. Comprehensive CCK-8, ALP activity detection and Alizarin red staining found that the ratio of group 1 was the best, that is, Eucommia ulmoides leaves: Eucommia ulmoides male flowers: Polygonatum sibiricum: Five-fingered peach: Maca powder: Natto: Collagen = 15:9:9:9:6:3:0.02. Therefore, this ratio was used for the next invention to explore whether the Eucommia compound (540g of Eucommia leaves, 324g of Eucommia male flowers, 324g of Polygonatum sibiricum, 324g of Prunus mume, 216g of maca powder, 108g of natto powder, 0.72g of collagen, and 72g of calcined oyster) had any pharmacological effect on osteoporotic rats.

[0161] Osteoporosis is the most common bone disease associated with aging. As the global population ages, the number of osteoporosis patients is increasing rapidly, and the quality of life is seriously declining. The main manifestations are decreased bone mass, deterioration of bone tissue structure, increased bone brittleness, and thus fractures. Currently, clinical drugs for the treatment of osteoporosis have many side effects, while traditional Chinese medicine has the characteristics of multiple components, multiple targets, and multiple pathways, and its safety can effectively avoid them, so it has great advantages.

[0162] Example 4 Study on the efficacy of Eucommia mixture in improving osteoporosis

[0163] 1. Test samples

[0164] Eucommia compound dry paste powder: Eucommia compound in animal experiments: weigh 540g of Eucommia leaves, 324g of Eucommia male flowers, 324g of Polygonatum sibiricum, 324g of Eleutherodactyla tricholoma, 216g of maca powder, 108g of natto powder, 72g of calcined oyster, and 0.72g of collagen; five herbs (a total of 1728g) including Eucommia leaves, Eucommia male flowers, Polygonatum sibiricum, Eleutherodactyla tricholoma, and maca powder are extracted and dried to obtain powder (540g of dry paste powder, with a yield of 31.25%), and the remaining 3 raw drug powders (a total of 180.72g) are used as medicine to prepare a dry paste powder.

[0165] The specific preparation method of the dry paste powder is as follows: maca powder (wrapped decoction), eucommia leaves, eucommia male flowers, wine polygonatum, and quinquefolia are decocted twice with water, and the maca powder (wrapped decoction), eucommia leaves, eucommia male flowers, wine polygonatum, and quinquefolia are recorded as a mixture. For the first time, 10 times the weight of the raw materials of the mixture are added with water, soaked at room temperature for 0.5h, decocted for 1h, filtered through 200 mesh, and the filtrate is reserved. For the second time, 8 times the weight of the raw materials of the mixture are added with water, decocted for 0.5h, filtered through 200 mesh, and the filtrate is combined; reduced pressure concentration (65°C to 70°C, vacuum degree -0.08 to -0.06Mpa) to a thick paste with a relative density of about 1.20 to 1.25 (80°C), vacuum drying (60°C to 70°C, vacuum degree -0.08 to -0.06Mpa), and crushing to obtain the dry paste powder.

[0166] Estradiol valerate tablets (batch number: 20171038), the source and efficacy information of estradiol valerate tablets are the same as those in Example 3.

[0167] Preparation of test samples: According to the experimental dosage design in Table 8, accurately weigh the corresponding amount of Eucommia compound dry paste powder and estradiol valerate tablets, add purified water to prepare the test sample solutions of corresponding concentrations for gavage, and prepare them before use.

[0168] 2. Experimental Animals

[0169] Thirty-eight female SPF SD rats, weighing 200 ± 20 g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., with experimental animal production license number: SCXK (Xiang) 2019-0004 and experimental animal quality certificate number: No.430727241101707774.

[0170] 3. Test conditions

[0171] The animals were housed in a barrier environment of Hunan Institute of Traditional Chinese Medicine (temperature range: 20.0-26.0°C; relative humidity range: 40.0%-70.0%), with an experimental animal use license number of SYXK(Xiang)2020-0008. Mouse feed was provided by Hunan Jiatai Experimental Animal Co., Ltd. Drinking water: tap water was filtered and sterilized and placed in high-pressure sterilized drinking bottles, with free access to food and water.

[0172] 4. Main reagents and instruments

[0173] 4.1 Main Reagents

[0174] Physiological saline (Qingdao Huaren Pharmaceutical Co., Ltd.), medical alcohol, 4% paraformaldehyde and other commonly used laboratory reagents were all of analytical grade (Sinopharm Chemical Reagent Co., Ltd.).

[0175] 4.2 Main instruments

[0176] PTT-B1000 electronic balance (Huazhi Electronic Technology Co., Ltd.), Sky Scan1276 Micro-CT (Bruker, Germany), etc.

[0177] 5. Experimental grouping and dosage design

[0178] After 5 days of adaptive feeding, 30 rats were randomly selected and anesthetized for bilateral ovariectomy to induce osteoporosis model. Specifically, all instruments were completely disinfected, clean foam boards were prepared for disinfection, sodium pentobarbital (0.2mL / 100g) was injected into rats intraperitoneally, and the rats were anesthetized and cut from the middle of the abdomen. The uterus was found along the fat. The ovaries were above the uterus. The ovaries were ligated with 3.0 silk thread, and the ovaries were removed with scissors. The left and right ovaries were removed and sutured. After suturing, 2 to 3 drops of antibiotics were dripped on the wound. The state and wound of the rats were observed every day after surgery. After four weeks, 30 rats were randomly divided into model group (N=8), estradiol valerate group (N=8), Eucommia mixture high-dose group (N=8), and Eucommia mixture low-dose group (N=6). The other 8 rats did not undergo surgery as a normal control group.

[0179] The clinical dosage of Eucommia mixture is 26.51 g per day, using mg / m 2 The equivalent dose for rats was calculated by the body surface area method: 26.51*31.2%*0.018 / 0.2=0.74 g / kg. In this study, the equivalent dose was set as the low-dose group of Eucommia mixture (0.74 g / kg) and the high-dose group of Eucommia mixture (1.49 g / kg), and the drug was administered once a day.

[0180] The recommended clinical dosage of estradiol valerate tablets for adults is 1 mg. The equivalent dose for rats is calculated using the body surface area method: 1*0.018 / 0.2=0.09 mg / kg. This study set the equivalent dose to the dosage of estradiol valerate tablets (0.09 mg / kg), which is administered once a day. See Table 8.

[0181] Table 8 Experimental grouping and dosage design

[0182]

[0183] 6. Test methods

[0184] 6.1 Administration of drugs to osteoporosis rat model

[0185] The day of surgery was recorded as the first day of surgery. Four weeks after surgery, each medication group was gavaged with the corresponding test drug according to the dosage in Table 8 above, and the normal group and model group were gavaged with purified water once a day for 8 consecutive weeks.

[0186] 6.2 Indicator Observation

[0187] (1) Observation of the general condition of rats: observe and record the rats' mental state, activity, fur gloss, and excretion of feces and urine every day; weigh the rats every 4 days after the start of drug administration;

[0188] (2) Major organ index: Weigh the liver and kidneys, and calculate the liver and kidney index of the rat based on the rat's body weight: organ / body weight.

[0189] (3) Micro-CT analysis of femoral tissue: The left femur removed from the experimental rats was subjected to micro-CT scanning and the region of interest was analyzed using CTAnalyser. Uniform parameters were set and the software calculated parameters such as bone density, bone volume ratio, trabecular number, trabecular thickness, and trabecular separation of bone tissue.

[0190] (4) Serum biochemical index detection: ELISA kits were used to detect the levels of serum ALP, BALP, TRAP, BGP, PICP, CTX-I and other bone metabolism indexes, and the levels of inflammatory factors such as TNF-α, IL-6, and IL-1β.

[0191] 7. Statistics

[0192] The experimental results are expressed as mean ± standard deviation. Graphpadprism 8.0 software was used to process all data of each group. One-way ANOVA test was used for analysis of multiple groups. * indicates P < 0.05 for significant difference, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001 for extremely significant difference. # P < 0.05 was used for significant difference. ## Indicates PP<0.01, ### Indicates P < 0.001, #### P < 0.0001 was used to indicate extremely significant differences.

[0193] 8. Test results

[0194] 8.1 General Observations

[0195] The weight of all experimental animals increased normally. There were no obvious abnormalities in the mental state, behavioral activities, food and water intake, feces, urine and cavity secretions of the rats in each group before modeling. Compared with the control group, the rats gained weight significantly after modeling, became fatter, moved more slowly, and had dull fur, indicating that the osteoporosis rat model was successfully replicated.

[0196] 8.2 Effect of Eucommia mixture on body weight of rats in each group

[0197] like Figure 1As shown in the figure, compared with the control group, the body weight of the rats in the model group increased significantly (P<0.0001); after administration, the body weight of the estradiol valerate group (P<0.01) and the high-dose and low-dose groups of Eucommia mixture (P<0.0001) decreased significantly, with statistical differences. The above results show that Eucommia mixture and estradiol valerate have a certain effect on alleviating the abnormal weight gain of osteoporotic rats.

[0198] 8.3 Effects of Eucommia mixture on liver and kidney indexes of rats in each group

[0199] like Figure 2 and Figure 3 As shown, compared with the control group, except for the model group and the estradiol valerate group, there was no significant difference between the low-dose and high-dose groups of the Eucommia mixture, and the liver and kidney were observed during the rat dissection process, and their morphology was found to be normal. The above results show that the Eucommia mixture has low toxicity and side effects in rats, high safety performance, and does not cause secondary damage to organs.

[0200] 8.4 Effect of Eucommia mixture on bone microstructure of rat femoral tissue

[0201] Micro-CT images of rat femur tissue are shown in Figure 2. Figure 4 As shown in the figure, the trabeculae of the rats in the normal group were complete in shape, orderly connected, and dense in structure; compared with the control group, the number of trabeculae in the rats in the model group was significantly reduced, and the network structure was severely damaged, indicating that the bearing capacity of the bones was significantly weakened, and deformation and fracture were prone to occur; after administration, compared with the model group, the low-dose group of estradiol valerate and Eucommia ulmoides mixture and the high-dose group of Eucommia ulmoides mixture had a certain degree of improvement on the reduction of the number of trabecular structures, and the network structure was partially restored. This result suggests that the Eucommia ulmoides mixture can improve the adverse effects of osteoporosis on the trabecular structure of rats.

[0202] 8.5 Effect of Eucommia mixture on bone turnover markers in rat femoral tissue

[0203] The parameters related to rat femoral bone turnover markers were analyzed by CT-Analyser, as shown in Tables 9 to 16. Figure 5 to Figure 12As shown in the figure, compared with the control group, all the indicators in the model group changed significantly. Except for the significant increase in bone surface area to bone volume ratio (BS / BV), trabecular separation (Th.Sp) and structural index model (SMI) (P<0.001, P<0.0001), bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Trabecular Number, Tb.N), trabecular thickness (Tb.Th) and bone surface area to tissue volume ratio (BS / TV) all decreased significantly (P<0.001, P<0.0001). After administration, bone surface area to bone volume ratio, trabecular separation and structural index model were significantly decreased (P<0.01, P<0.001, P<0.0001), while bone mineral density, bone volume fraction, trabecular number, trabecular thickness and bone surface area to tissue volume ratio were significantly increased (P<0.01, P<0.001, P<0.0001). The above results indicate that the Eucommia mixture has a certain reversal effect on femoral bone turnover markers in osteoporotic rats.

[0204] Table 9 Bone density values ​​of rats in each group (g / cm 3 , n=5)

[0205] Conrtol Model Estradiol ZL ZH Parallel experiment 1 0.4343922 0.1182935 0.3173119 0.2208546 0.4549472 Parallel experiment 2 0.4409399 0.1339751 0.4049508 0.5185459 0.5168996 Parallel experiment 3 0.2970832 0.1372412 0.2492494 0.2553146 0.4647328 Parallel experiment 4 0.4494459 0.1379138 0.1836675 0.4809554 0.3817863 Parallel experiment 5 0.3751404 0.1318431 0.3907873 0.582091 0.3640231 Mean 0.39940032 0.13185334 0.30919338 0.4115523 0.4364778

[0206] Table 10 Bone volume fraction of rats in each group (%, n = 5)

[0207] Conrtol Model Estradiol ZL ZH Parallel experiment 1 50.19559941 4.77119908 29.90993566 19.8184134 54.11001661 Parallel experiment 2 51.16716386 8.25873348 44.63511589 62.70346921 62.02332621 Parallel experiment 3 26.99843229 5.1054047 20.83179153 20.85222855 54.64477269 Parallel experiment 4 51.53661252 6.82487769 12.34703987 57.37965869 39.78621249 Parallel experiment 5 40.04915204 5.27736776 42.0465075 70.63078495 38.87149618 Mean 43.98939202 6.047516542 29.95407809 46.27691096 49.88716484

[0208] Table 11 Bone surface area to bone volume ratio of rats in each group (1 / mm, n=5)

[0209] Conrtol Model Estradiol ZL ZH Parallel experiment 1 22.55635503 37.19454633 26.94216285 29.91728432 21.1547169 Parallel experiment 2 20.34852026 36.04348206 25.64858702 16.72188897 18.66830179 Parallel experiment 3 28.97935587 40.92637735 29.39772465 34.8053751 20.76325635 Parallel experiment 4 20.92099663 42.09051331 34.48021674 19.70722642 25.37567557 Parallel experiment 5 26.07039609 39.56762886 22.85805003 15.29654371 27.80302627 Mean 23.77512478 39.16450958 27.86534826 23.2896637 22.75299538

[0210] Table 12 Trabecular bone thickness of rats in each group (mm, n=5)

[0211] Conrtol Model Estradiol ZL ZH Parallel experiment 1 0.14322059 0.1089194 0.13206865 0.11670206 0.15069223 Parallel experiment 2 0.15040365 0.10873587 0.12623376 0.21931364 0.15661511 Parallel experiment 3 0.12334029 0.10361281 0.1259746 0.10705509 0.14926854 Parallel experiment 4 0.15209851 0.09498772 0.11521663 0.160484 0.13358533 Parallel experiment 5 0.12378769 0.10398974 0.14519553 0.19617614 0.12445792 Mean 0.138570146 0.104049108 0.128937834 0.159946186 0.142923826

[0212] Table 13 Number of trabecular bone in each group of rats (1 / mm, n=5)

[0213] Conrtol Model Estradiol ZL ZH Parallel experiment 1 3.50477546 0.43804861 2.26472636 1.69820589 3.59076349 Parallel experiment 2 3.40198949 0.75952245 3.53590962 2.85907744 3.96023888 Parallel experiment 3 2.18893856 0.49273873 1.65365017 1.9478036 3.66083652 Parallel experiment 4 3.38837062 0.71850106 1.07163692 3.575413 2.97833693 Parallel experiment 5 3.23530965 0.50748926 2.89585409 3.60037587 3.12326417 Mean 3.143876756 0.583260022 2.284355432 2.73617516 3.462687998

[0214] Table 14 Trabecular separation of rats in each group (mm, n=5)

[0215] Conrtol Model Estradiol ZL ZH Parallel experiment 1 0.19127756 1.52766878 0.33258876 0.81942613 0.17486687 Parallel experiment 2 0.21358361 1.28940375 0.190358 0.17321007 0.14951596 Parallel experiment 3 0.32711739 1.34109479 0.55394347 0.40389913 0.1765188 Parallel experiment 4 0.19724043 1.15862036 0.8492805 0.16823536 0.23546647 Parallel experiment 5 0.22762177 1.25432196 0.25927818 0.13388697 0.21592385 Mean 0.231368152 1.314221928 0.437089782 0.339731532 0.19045839

[0216] Table 15 Structural pattern index of rats in each group (n=5)

[0217]

[0218]

[0219] Table 16 Bone surface area to tissue volume ratio of each group of rats (n=5)

[0220] Conrtol Model Estradiol ZL ZH Parallel experiment 1 11.32229761 1.77462585 8.05838357 5.92913108 11.44682083 Parallel experiment 2 10.4117607 2.97673512 11.44827654 10.4852045 11.57870172 Parallel experiment 3 7.82397177 2.08945719 6.12407271 7.25769636 11.34603424 Parallel experiment 4 10.78197297 2.87262605 4.25728611 11.30793925 10.0960202 Parallel experiment 5 10.44097257 2.08812929 9.61101172 10.80406889 10.8074523 Mean 10.15619512 2.3603147 7.89980613 9.156808016 11.05500586

[0221] 8.6 Effects of Eucommia mixture on serum biochemical parameters in rats

[0222] ELISA kits were used to measure serum bone metabolism indicators (see Tables 17-22 and Figure 13 to Figure 18 ), inflammatory factors (see Tables 23-25 ​​and Figure 19 to Figure 21 ) were detected. Compared with the normal control group, all indicators in the model group showed significant changes. TRAP, CTX-I, IL-6, IL-1β, and TNF-α levels were significantly increased (P<0.05, P<0.01, P<0.001); BALP, ALP, BGP, and PICP levels were significantly decreased (P<0.05, P<0.001, P<0.0001). * represents comparison with the normal control group.

[0223] After administration, compared with the model group, the levels of TRAP, CTX-I, IL-6, IL-1β, and TNF-α were significantly decreased (P<0.01, P<0.001, P<0.0001), and the levels of BALP, ALP, BGP, and PICP were significantly increased (P<0.01, P<0.001, P<0.0001). The above results show that the Eucommia mixture can significantly enhance the activity of osteoblasts, inhibit the maturation and differentiation of osteoclasts, and relieve inflammation in osteoporotic rats. # represents comparison with the model group.

[0224] Table 17 Rat serum bone alkaline phosphatase levels (n=2)

[0225] Control Model Estradiol ZL ZH Parallel experiment 1 96.15963 30.16571 46.87685 82.53544 132.96563 Parallel experiment 2 88.29247 28.57609 47.74644 85.39646 180.44845 Mean 92.22605 29.37090 47.311645 83.96595 156.70704

[0226] Table 18 Rat serum tartrate-resistant acid phosphatase levels (n=3)

[0227] Control Model Estradiol ZL ZH Parallel experiment 1 7.78769 12.9684 3.21306 7.07871 10.0087 Parallel experiment 2 8.01863 12.85368 3.34856 9.52769 10.95748 Parallel experiment 3 8.11681 13.04721 3.54261 10.01742 11.66147 Mean 7.97438 12.95643 3.36808 8.87461 10.87588

[0228] Table 19 Rat serum alkaline phosphatase levels (n=3)

[0229]

[0230]

[0231] Note: “-” means no measured data.

[0232] Table 20 Cross-linked C-terminal peptide of type I collagen in rat serum (n=2)

[0233] Control Model Estradiol ZL ZH Parallel experiment 1 0.9864 2.89222 1.08485 1.10449 1.00612 Parallel experiment 2 0.90732 3.16581 1.10449 1.16333 1.02583 Mean 0.94686 3.02922 1.09467 1.13391 1.01598

[0234] Table 21 Serum osteocalcin in rats of each group (n=2)

[0235] Control Model Estradiol ZL ZH Parallel experiment 1 1.20276 0.47226 1.257 1.20276 0.86871 Parallel experiment 2 1.24148 0.45151 1.24148 1.257 0.92848 Mean 1.22212 0.46189 1.24924 1.22988 0.89860

[0236] Table 22 Rat serum type I procollagen carboxyl terminal peptide (n=3)

[0237] Control Model Estradiol ZL ZH Parallel experiment 1 1.41115 0.79327 1.58349 1.03035 2.10055 Parallel experiment 2 1.59802 0.65682 1.51564 1.22147 1.53156 Parallel experiment 3 1.30915 1.0563 1.5041 1.0072 2.35814 Mean 1.43944 0.83770 1.53441 1.08634 1.99675

[0238] Table 23 Rat serum interleukin-1β levels (n=2)

[0239] Control Model Estradiol ZL ZH Parallel experiment 1 12.43791 193.9358 29.24975 41.55022 54.05725 Parallel experiment 2 17.17579 244.5309 21.97175 44.03121 41.55022 Mean 14.80685 219.23335 25.61075 42.79072 47.80373

[0240] Table 24 Rat serum interleukin-6 levels (n=2)

[0241] Control Model Estradiol ZL ZH Parallel experiment 1 10.54629 27.86495 9.10252 29.46463 7.49887 Parallel experiment 2 11.87767 28.26858 9.10252 33.68983 9.84077 Mean 11.21198 28.06677 9.10252 31.57723 8.66982

[0242] Table 25 Rat serum tumor necrosis factor α (n=3)

[0243] Control Model Estradiol ZL ZH Parallel experiment 1 22.91927 31.08119 17.60787 20.14821 14.3892 Parallel experiment 2 21.93363 28.4753 20.0484 20.7484 15.76789 Parallel experiment 3 20.66815 26.08351 21.83682 21.09544 13.00592 Mean 21.84035 28.54667 19.83103 20.66402 14.38767

[0244] Conclusion: Bilateral ovariectomy was used to successfully induce osteoporosis in rats:

[0245] The Eucommia mixture improves the growth status of osteoporosis model rats and alleviates the abnormal weight gain of osteoporosis rats.

[0246] Eucommia mixture had no significant difference on the liver and kidney index of osteoporotic rats and its morphology was not abnormal, with low toxicity and side effects on rats and high safety performance;

[0247] Eucommia mixture increased the number of trabeculae in osteoporotic rats, restored the reticular structure to a certain extent, and improved the bone microstructure destruction in osteoporotic rat models;

[0248] The Eucommia mixture up-regulated bone density, bone volume fraction, trabecular number, trabecular thickness, and bone surface area to tissue volume ratio, while down-regulated bone surface area to bone volume ratio, trabecular separation, and structural index model, and had a certain reversal effect on femoral bone turnover markers in osteoporotic rats.

[0249] The Eucommia mixture can reduce the metabolic levels of serum CTX-I and TRAP, increase the metabolic levels of serum ALP, BALP, BGP, and PICP, significantly reduce the levels of serum inflammatory factors IL-6, IL-1β, and TNF-α, enhance osteoblast activity, inhibit osteoclast maturation and differentiation, and relieve inflammation.

[0250] The present invention establishes an osteoporosis model by bilateral ovariectomy OVX. The OVX rat model is an animal model that simulates the physiological characteristics of women after menopause. Its bone loss characteristics are similar to those of postmenopausal women. After ovarian removal, fat accumulation will form in the abdomen of rats, causing abnormal weight growth. By weighing every 4 days during the administration period, it is found that the weight of osteoporotic rats increases significantly, indicating that the model is initially successfully established. After the intervention of Eucommia ulmoides, it has a certain alleviating effect on the abnormal weight growth, indicating that the Eucommia compound improves the abnormal weight changes of osteoporosis model rats. By weighing the collected liver and kidneys and calculating their liver and kidney indexes, it is found that the Eucommia compound has no significant difference in the liver and kidney index of OVX rats and their morphology is not abnormal, indicating that the Eucommia compound has little toxic and side effects on rats and has high safety performance. Bone density and bone microstructure indicators are important indicators for judging the phenotype of osteoporosis. The present invention detected the femoral tissue of the left leg of each group of rats through Micro-CT. The results showed that the Eucommia compound increased the number of trabeculae in osteoporotic rats, restored the reticular structure to a certain extent, and increased bone density, bone volume fraction, trabecular number, trabecular thickness, and bone surface area to tissue volume ratio, while downregulated the bone surface area to bone volume ratio, trabecular separation, and structural index model. It had a certain reversal effect on the femoral bone turnover markers of osteoporotic rats and improved the bone microstructure destruction of the osteoporotic rat model.

[0251] Under normal physiological conditions, bone resorption and bone formation are in a dynamic equilibrium state, and the activities of osteoclasts and osteoblasts can respectively reflect the states of the two. When bone resorption is greater than bone formation, that is, osteoclast activity increases and osteoblast activity decreases, it will lead to osteoporosis, and the activities of osteoblasts and osteoclasts can be reflected by bone turnover markers (BTM). The present invention mainly detects the serum bone formation markers ALP, BALP, PICP, BGP and the bone resorption markers TRAP and CTX-I of each group of rats. The results show that compared with the normal group, the levels of ALP, BALP, PICP and BGP in the model group are significantly reduced (*P<0.05, **P<0.01, ***P<0.001, ****P<0.001), and the levels of TRAP and CTX-I are significantly increased (*P<0.05, **P<0.01, ***P<0.001, ****P<0.001). After the intervention of Eucommia ulmoides, the levels of ALP, BALP, PICP and BGP are significantly increased and the levels of TRAP and CTX-I are significantly reduced. The results show that inflammation is a key factor in maintaining bone balance. When the content of pro-inflammatory factors such as tumor necrosis factor (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6) is increased, macrophage differentiation into osteoblasts is inhibited, and osteoclasts increase. The results of the present invention show that the Eucommia compound can significantly reduce the levels of serum inflammatory factors IL-6, IL-1β, and TNF-α by reducing the metabolic levels of serum CTX-I and TRAP, increasing the metabolic levels of serum ALP, BALP, BGP, and PICP, enhancing the activity of osteoblasts, inhibiting the maturation and differentiation of osteoclasts, and relieving inflammation.

[0252] In summary, the results of osteoporosis model rats verified that the Eucommia mixture had a certain improvement effect on osteoporosis. It can be seen that the Eucommia compound has a certain pharmacological effect on osteoporosis rats.

[0253] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A composition for improving osteoporosis, characterized in that: The invention comprises the following components by weight: 500-600 parts of eucommia leaves, 300-350 parts of eucommia male flowers, 300-350 parts of polygonatum sibiricum, 300-350 parts of quinquefolius, 200-250 parts of maca, 90-120 parts of natto, 60-80 parts of calcined oysters and 0.5-1 part of collagen.

2. The composition according to claim 1, characterized in that The invention comprises the following components by weight: 540 parts of eucommia leaves, 324 parts of eucommia male flowers, 324 parts of polygonatum sibiricum, 324 parts of quinquefolius, 216 parts of maca, 108 parts of natto, 72 parts of calcined oyster and 0.72 parts of collagen.

3. The method for preparing the Chinese medicine composition according to claim 1 or 2, characterized in that: The steps include: Extracting the maca, eucommia leaves, eucommia male flowers, polygonatum sibiricum and quinquefolia to obtain an extract; the extraction method of the maca comprises decocting; Concentrating and drying the extract to obtain an extract; The extract, calcined oyster, natto and collagen are mixed to obtain an extract.

4. The preparation method according to claim 3, characterized in that: The extraction solvent includes water; and the extraction includes a first extraction and a second extraction.

5. The preparation method according to claim 4, characterized in that: The amount of water added in the first extraction is 8 to 12 times the mass of the Chinese medicine raw material, and the time of the first extraction is 1 to 2 hours; the amount of water added in the second extraction is 6 to 10 times the mass of the Chinese medicine raw material, and the time of the second extraction is 0.5 to 1 hour.

6. The preparation method according to claim 3, characterized in that: The extraction further includes soaking, and the soaking time is 0.4 to 1 hour.

7. The preparation method according to claim 3, characterized in that: The concentration includes reduced pressure concentration, the temperature of the reduced pressure concentration is 65 to 70° C., the vacuum degree of the reduced pressure concentration is -0.08 to -0.06 Mpa; the reduced pressure concentration is performed until the relative density of the obtained product at 80° C. is 1.20 to 1.

25.

8. The preparation method according to claim 3, characterized in that: The drying includes vacuum drying, the vacuum drying time is 60 to 70° C., and the vacuum degree is -0.08 to -0.06 Mpa.

9. Use of the composition according to claim 1 or 2 or the composition prepared according to claims 3 to 8 in the preparation of a medicament for preventing and / or treating osteoporosis.

10. The use according to claim 9, characterized in that: The dosage form of the drug includes one or more of oral dosage form, injection, suppository and ointment.

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