A pharmaceutical composition for preventing or treating primary osteoporosis and a preparation method thereof

Abstract

The application provides a medicine composition for treating primary osteoporosis and a preparation method thereof. The medicine composition is composed of Semen Euryae, Herba Epimedii and the like. The application uses the theory of meridian diseases in traditional Chinese medicine to discuss the etiology and pathogenesis of primary osteoporosis. The medicine composition has the effects of tonifying kidney and filling essence, nourishing yin and supporting yang, strengthening muscles and bones.

Description

TECHNICAL FIELD

[0001] The present application relates to a traditional Chinese medicine composition for preventing or treating primary osteoporosis and a preparation method thereof, and belongs to the field of Chinese herbal medicine applications. The primary osteoporosis is mainly for postmenopausal osteoporosis patients, and the basic pathogenesis is kidney essence deficiency. The present application can effectively improve bone density, improve clinical symptoms and quality of life of patients, and also has significant effects on the whole body syndrome manifestations caused by kidney essence deficiency, such as low back pain, soreness of waist and knees, difficulty in holding heavy objects, cramps in lower extremities, dizziness and tinnitus, fatigue, frequent nocturia, etc. The present application is simple and effective, and shows the characteristics and innovations of the present prescription. BACKGROUND

[0002] Osteoporosis (OP) is a systemic bone disease characterized by low bone mass, microarchitectural deterioration of bone tissue, and increased bone fragility and susceptibility to fracture. In 2001, the National Institutes of Health (NIH) proposed that osteoporosis is a skeletal system disease characterized by decreased bone strength and increased risk of fracture. Bone strength reflects two main aspects of the skeleton, namely bone mineral density and bone mass. Osteoporosis can occur in different genders and ages, but is more common in postmenopausal women and elderly men. Osteoporosis is divided into two categories: primary and secondary. Primary osteoporosis is further divided into three categories: postmenopausal osteoporosis (type I), senile osteoporosis (type II), and idiopathic osteoporosis (including juvenile type). Postmenopausal osteoporosis generally occurs within 5-10 years after menopause in women and is the most common bone disease in women during the menopausal stage. Its incidence is related to estrogen deficiency, genetic factors, menstrual cycle disorders, and early menopause. Senile osteoporosis generally refers to osteoporosis occurring after the age of 70. Secondary osteoporosis refers to osteoporosis caused by any disease and / or drug affecting bone metabolism, which mainly occurs in adolescents and the cause is unknown.

[0003] As a degenerative disease, the risk of osteoporosis increases with age. At present, about 200 million people worldwide suffer from osteoporosis, and its incidence has jumped to the 7th place among various common diseases in the world. With the extension of human lifespan and the arrival of an aging society, osteoporosis has become an important health problem for humans.

[0004] Osteoporotic fracture is one of the most serious consequences of osteoporosis. Bone density and mass decrease, bone strength decreases, and fracture occurs with slight violence, so it belongs to brittle fracture. It is estimated that osteoporotic fracture occurs every 3 seconds in the world. The elderly population has a higher risk of fracture and mortality due to poor bone quality, more severe calcium and vitamin D deficiency, and factors such as easy to fall. It is reported that the incidence of osteoporotic fracture in Chinese women is 40%, which is much higher than the sum of the incidence of breast cancer, endometrial cancer and ovarian cancer. Fracture is a serious consequence of osteoporosis, which can significantly increase the morbidity and mortality of patients. The treatment and care of osteoporosis and osteoporotic fracture require a huge investment of manpower and material resources, high cost, and heavy family, social and economic burden. Osteoporosis is related to endocrine factors, nutritional factors, life habits and exercise load, immune factors and cytokines, genetic factors, etc.

[0005] Currently, the prevention and treatment of osteoporosis covers three aspects: basic measures, drug intervention and rehabilitation treatment. Among them, the basic measures include adjusting lifestyle and bone health basic supplements, namely calcium and vitamin D. At present, the drugs for osteoporosis can be roughly divided into the following types: (1) Bisphosphonates: Bisphosphonates have high affinity with hydroxyapatite in bone, specifically bind to the bone surface with active bone turnover, inhibit the function of osteoclasts, and thus inhibit bone resorption. (2) Calcitonin: a calcium-regulating hormone that can inhibit the biological activity of osteoclasts and reduce the number of osteoclasts, thereby preventing bone loss and increasing bone mass. (3) Estrogen: This drug can inhibit bone turnover and prevent bone loss, including estrogen replacement therapy (ET) and estrogen and progestin replacement therapy (EPT), which can prevent bone loss and reduce the risk of osteoporotic vertebral and non-vertebral fractures. (4) Parathyroid hormone: It has the effect of promoting bone formation. (5) Selective estrogen receptor modulators: They selectively act on the target organs of estrogen and produce different biological effects after binding to different forms of estrogen receptors. For example, raloxifene binds to estrogen receptors in the bone, showing estrogen-like activity and inhibiting bone resorption. (6) Strontium salt: Strontium is one of the essential trace elements in the human body, which is involved in many physiological functions and biochemical effects of the human body. The chemical structure of strontium is similar to that of calcium and magnesium, and a small amount of strontium exists in normal human soft tissue, blood, bone, and teeth. (7) Active vitamin D and its analogues: including 1, 25-dihydroxyvitamin D3 (calcitriol) and 1α-hydroxyvitamin D3 (α-cholecalciferol). (8) Vitamin K2: Vitamin K2 is a coenzyme of γ-carboxylase, which plays an important role in the formation of γ-carboxyglutamate. γ-carboxyglutamate is essential for the normal physiological function of osteocalcin, which can promote bone formation and have a certain inhibitory effect on bone resorption. Although the above drugs can be used for the prevention and treatment of osteoporosis, they also face corresponding barriers and application bottlenecks. For example, estrogen preparations can bind to receptors on osteoblasts, promote the secretion of collagenase, release growth factors and cytokines, and promote the synthesis of bone organic matter and bone remodeling. At the same time, it can directly inhibit osteoclast absorption, but the application of estrogen is prone to side effects, mainly including postmenopausal vaginal bleeding, breast cancer, endometrial cancer, cardiovascular accidents, and thromboembolism, so it is rarely used alone. The combination of estrogen and progestin, combined with progestin, can counteract the endometrial proliferative effect of estrogen, and progestin can inhibit bone resorption and promote bone formation. However, there is still a lack of long-term prospective studies on the role of estrogen in preventing fractures, as well as the effects of estrogen and progestin on many other tissues, including the breast, uterus, cardiovascular system, and the less common adverse reactions of deep vein thrombosis. Oral bisphosphonates all have upper gastrointestinal complications, in addition to toxic and side effects including kidney, blood, liver, and immune suppression.Calcitonin is one of the preferred drugs for treating high conversion osteoporosis, and is suitable for patients who are forbidden to use estrogen or cannot tolerate estrogen or have ostealgia osteoporosis. However, long-term use can cause "escape phenomenon". For patients using bone formation promoters, studies have shown that the application of fluoride can increase the BMD (bone mineral density) of the lumbar spine, but cannot reduce vertebral fractures. A number of clinical studies on the application of parathyroid hormone show that intermittent small-dose application can promote bone formation and increase bone mass, while continuous large-dose application can promote bone resorption and cause bone mass loss. Vitamin K2 is an essential vitamin for bone calcium carboxylation, but there are also adverse reactions such as anemia, liver function damage, and polycythemia. In summary, western medicines for treating osteoporosis have different degrees of side effects, which limit the long-term use of the above drugs by osteoporosis patients.

[0006] Traditional Chinese medicine has accumulated rich theoretical and practical experience in the prevention and treatment of osteoporosis. According to syndrome differentiation and treatment, the viscera are holistically regulated to select specific treatment methods according to the different clinical characteristics and conditions of patients, which can significantly improve the improvement of the patient's whole body symptoms, reduce bone pain, improve the patient's quality of life, and has little side effect, and is easy to be accepted by patients, and has obvious advantages in the treatment of osteoporosis.

[0007] Most of the currently marketed Chinese medicine products for treating osteoporosis are targeted at osteoporosis caused by blood stasis, kidney yang deficiency, liver and kidney deficiency, etc., and mostly use the methods of promoting blood circulation and removing blood stasis, tonifying kidney and strengthening bone, warming and tonifying kidney yang, and nourishing liver and kidney. Among them, the drugs for warming and tonifying kidney yang or tonifying kidney and nourishing yin are mostly used, which inevitably causes the disadvantages of partial tonification of kidney yang and easy heat generation, and the methods and drugs for filling kidney essence and regulating yin and yang are rarely used. SUMMARY

[0008] The drug composition of the application is a compound traditional Chinese medicine developed by exploring the TCM pathogenesis and treatment of osteoporosis based on the theory of traditional Chinese medicine and combined with years of clinical practice. The drug composition fully grasps the syndrome characteristics of kidney essence deficiency in osteoporosis, and focuses on tonifying kidney and filling essence in treatment, while tonifying yin and supporting yang. The previous pharmacological, pharmacological and toxicological experiments have proved that the drug has the effects of treating osteoporosis, analgesia, anti-inflammatory and immune regulation. The clinical application also shows good efficacy in improving the clinical symptoms of patients and improving the quality of life, and is safe to take. It is intended to further evaluate the clinical effectiveness and safety of the drug on the basis of the above research, and to provide safe and effective traditional Chinese medicine preparations for patients with osteoporosis.

[0009] The medicine composition of the present application is a traditional Chinese medicine preparation, which is composed of salted Semen Euryae, Herba Epimedii, Radix Rehmanniae, Radix Salviae Miltiorrhizae, calcined Ostreae Concha and salted Psoraleae Fructus, has the functions of "tonifying kidney and filling essence, nourishing yin and supporting yang", and is suitable for primary osteoporosis (kidney essence deficiency type). Compared with similar Chinese medicine products, the medicine composition of the present application is innovative in the cubic principle and prescription composition, and is different from the previous researches which adopt medicine for warming and tonifying kidney yang to treat osteoporosis. The present application is aimed at kidney essence deficiency, the basic pathological change, and balances yin and yang, thereby avoiding the disadvantages of partial tonification of yin and yang. Semen Euryae in the prescription tonifies kidney and fills essence, strengthens muscles and bones, Herba Epimedii warms and tonifies kidney yang and strengthens muscles and bones, and Radix Rehmanniae nourishes kidney yin, so as to achieve the effects of tonifying kidney and filling essence, and tonifying yin and yang. Meanwhile, Ostreae Concha is used to tonify kidney and benefit essence, nourish yin and hide yang, and strengthen bone joints, Psoraleae Fructus is used to warm kidney and assist yang, and Radix Salviae Miltiorrhizae is used to activate blood, dredge collaterals and relieve pain. The present application is aimed at postmenopausal osteoporosis patients with primary osteoporosis, and can effectively improve bone density, improve clinical symptoms and quality of life of the patients, and has significant curative effects on the symptoms of kidney essence deficiency, such as back pain, soreness of waist and knees, difficulty in carrying heavy objects, cramp of lower limbs, dizziness, tinnitus, fatigue, frequent urination at night and other systemic symptoms. The medicine is simple and effective, and shows the characteristics and innovation of the present application.

[0010] The medicine composition for preventing or treating primary osteoporosis of the present application is characterized in that the composition comprises the following components in the weight parts: Semen Euryae 200-600 parts, Herba Epimedii 100-300 parts, Radix Rehmanniae 100-300 parts, Radix Salviae Miltiorrhizae 80-240 parts, Ostreae Concha 25-80 parts, and Psoraleae Fructus 80-240 parts.

[0011] Preferably, the composition comprises the following components in the weight parts: Semen Euryae 200 parts, Herba Epimedii 300 parts, Radix Rehmanniae 100 parts, Radix Salviae Miltiorrhizae 240 parts, Ostreae Concha 25 parts, and Psoraleae Fructus 240 parts.

[0012] Preferably, the composition comprises the following components in the weight parts: Semen Euryae 600 parts, Herba Epimedii 100 parts, Radix Rehmanniae 300 parts, Radix Salviae Miltiorrhizae 80 parts, Ostreae Concha 80 parts, and Psoraleae Fructus 80 parts.

[0013] Preferably, the composition comprises the following components in the weight parts: Semen Euryae 400 parts, Herba Epimedii 200 parts, Radix Rehmanniae 200 parts, Radix Salviae Miltiorrhizae 160 parts, Ostreae Concha 53 parts, and Psoraleae Fructus 160 parts.

[0014] More preferably, the composition comprises the following components in the weight parts: Semen Euryae 380 parts, Herba Epimedii 220 parts, Radix Rehmanniae 200 parts, Radix Salviae Miltiorrhizae 155 parts, Ostreae Concha 55 parts, and Psoraleae Fructus 155 parts.

[0015] In the composition, Semen Euryae is preferably salted Semen Euryae, Ostreae Concha is preferably calcined Ostreae Concha, and Psoraleae Fructus is preferably salted Psoraleae Fructus.

[0016] The preparation dosage form of the composition can be prepared into capsule, tablet, pill, oral liquid, granule, injection or powder.

[0017] The active component of the composition is prepared by the following steps:

[0018] A. The Cuscuta chinensis Lam and Psoralea corylifolia L. are weighed according to the prescription amount, and extracted twice with 50-70% ethanol, each time for 1-3 hours, and the amount of ethanol added is 6-10 times the amount of medicinal materials, the extract is filtered, combined, and concentrated under reduced pressure to a clear paste with a relative density of 1.10±0.05 at 60°C, and standby;

[0019] B. The Herba Epimedii, Rehmannia glutinosa and Salvia miltiorrhiza are weighed according to the prescription amount, and extracted three times with 8-12 times the amount of water, and the extraction time is 1-3 hours, the extract is filtered, concentrated under reduced pressure to a clear paste with a relative density of 1.10±0.05 at 60°C, and the three concentrated liquids are combined, and combined with the alcohol extract clear paste obtained in step A, and concentrated to a thick paste with a relative density of 1.20±0.05 at 60°C, dried, and pulverized to obtain fine powder;

[0020] C. The Ostrea gigas Thunb is pulverized into the finest powder, and sterilized by 60Co irradiation;

[0021] The fine powder obtained by mixing step B and step C together constitutes the active component of the pharmaceutical composition of the present application.

[0022] The preparation process of the tablet of the composition is:

[0023] A. The Cuscuta chinensis Lam and Psoralea corylifolia L. are weighed according to the prescription amount, and extracted twice with 50-70% ethanol, each time for 1-3 hours, and the amount of ethanol added is 6-10 times the amount of medicinal materials, the extract is filtered, combined, and concentrated under reduced pressure to a clear paste with a relative density of 1.10±0.05 at 60°C, and standby;

[0024] B. The Herba Epimedii, Rehmannia glutinosa and Salvia miltiorrhiza are weighed according to the prescription amount, and extracted three times with 8-12 times the amount of water, and the extraction time is 1-3 hours, the extract is filtered, concentrated under reduced pressure to a clear paste with a relative density of 1.10±0.05 at 60°C, and the three concentrated liquids are combined, and combined with the alcohol extract clear paste obtained in step A, and concentrated to a thick paste with a relative density of 1.20±0.05 at 60°C, dried, and pulverized to obtain fine powder;

[0025] C. The Ostrea gigas Thunb is pulverized into the finest powder, and sterilized by 60Co irradiation;

[0026] D. The Ostrea gigas Thunb powder obtained in step C and the fine powder obtained in step B are combined, granulated according to the conventional process, and the granules are pressed into tablets.

[0027] The preparation process of the tablet of the composition is:

[0028] A. Weigh the Cuscuta and Psoralea according to the prescription amount, crush the Cuscuta first, extract twice with 60% ethanol, 1.5 hours each time, add 10 times the amount of alcohol for the first time and 8 times the amount of alcohol for the second time, filter the extract, combine, and concentrate under reduced pressure to a clear extract with a relative density of 1.10±0.05 at 60°C, and reserve;

[0029] B. Weigh the Epimedium, Rehmannia and Salvia miltiorrhiza according to the prescription amount, add 10-12 times the amount of water to extract three times, 1-2 hours each time, filter the extract, concentrate under reduced pressure to a clear extract with a relative density of 1.10±0.05 at 60°C, combine the three concentrated liquids, and combine with the alcohol extract clear extract obtained in step A, concentrate to a thick paste with a relative density of 1.20±0.05 at 60°C, dry and crush to obtain a fine powder;

[0030] C. Crush the Ostrea into the finest powder and sterilize with 60Co irradiation;

[0031] D. Combine the Ostrea powder obtained in step C and the fine powder obtained in step B, granulate according to the conventional process, sieve, and press into tablets to obtain the product.

[0032] The application of the pharmaceutical composition is preferably the application in the preparation of postmenopausal osteoporosis drugs.

[0033] The application of the pharmaceutical composition is preferably the application of the pharmaceutical composition in the preparation of drugs for reducing β-catenin degradation product β-CTX.

[0034] The application of the pharmaceutical composition is preferably the application of the pharmaceutical composition in the preparation of drugs for treating kidney essence deficiency syndrome.

[0035] The application of the pharmaceutical composition is preferably the application of the pharmaceutical composition in the preparation of drugs for increasing the content of serum Ca and P, increasing the content of bone formation marker PINP, reducing the content of bone formation marker BGP, reducing the content of bone absorption markers ALP and TRACP, or increasing the content of CT and PTH.

[0036] The application of the pharmaceutical composition is preferably the application of the pharmaceutical composition in the preparation of drugs for increasing the percentage of tibial trabecular bone volume, increasing the maximum load, bending strength and elastic modulus of the femur, and improving the bone morphology and bone biomechanical properties.

[0037] The pharmaceutical composition of the present application is based on the theory of traditional Chinese medicine to explore the pathogenesis and treatment of osteoporosis from "kidney essence deficiency", and is created through many years of clinical application. The prescription has achieved obvious curative effect in the treatment of postmenopausal osteoporosis, can significantly relieve the symptoms of back pain, soreness of waist and knees, cramps in lower limbs, dizziness, tinnitus, fatigue, frequent urination at night, etc. in postmenopausal osteoporosis patients, and has no obvious adverse reactions.

[0038] According to the clinical manifestations of osteoporosis, it should belong to the category of "bone flaccid", "bone paralysis", "bone dry", "bone extreme", "bone shrinkage" and so on. The clinical manifestations of osteoporosis are described by the medical experts in history. It is said in "Su Wen•Flaccidity Theory" that "if the kidney qi is hot, the waist and back will not lift, the bone will be dry and the marrow will decrease, and it will be flaccid." It is also proposed in "Difficult Questions•Fourteenth Difficulty" that "five losses will damage the bone, and the bone flaccid will not get up from the bed." It is recorded in "Su Wen•Bi Theory" that "bone paralysis will not be cured, and it will be affected by evil, and it will be in the kidney, which is kidney paralysis. The symptoms are swelling, and the buttocks will replace the heels, and the spine will replace the head." It is recorded in "Su Wen•Long Sha Jie Theory" that "the disease is in the bone, the bone is heavy and cannot be lifted, the bone marrow is painful, and the cold air will come, which is called bone paralysis." It is said in "Difficult Questions•Twenty-fourth Difficulty" that "the foot shao yin qi is exhausted, and the bone is dry." The above records in ancient medical books in China are similar to the symptoms of osteoporosis such as waist and back pain, limited limb function and tortoise back, which provides a basis for exploring the etiology and pathogenesis of osteoporosis.

[0039] According to traditional Chinese medicine, the kidney is the root of life and the root of nature, and the kidney stores essence. It is said in "Su Wen•Ancient and Natural Theory" that "the kidney is the master of water, and it receives the essence of five internal organs and six viscera and stores it." The kidney has the characteristics of hibernation, sealing and closing. Essence is the basic material of the human body and the material basis for growth and development and various functional activities. The essence stored in the kidney does not remain unchanged, but changes regularly with the different stages of human life, such as "women at the age of seven, kidney qi is full, teeth change and hair grows……five and seven, yangming meridians decline, face begins to be dry, hair begins to fall. Six and seven, three yang meridians decline, face is dry, hair begins to be white. Seven and seven, the ren meridian is empty, the taichong meridian is weak, the tianqi is exhausted, the earth channel is not connected, so the body is broken and there is no child." The point of view of "seven and seven……tianqi is exhausted" in the article is consistent with the menopausal age in modern medical research. The close physiological relationship between the kidney, bone and marrow is fully reflected in the whole process of bone growth and development. With the increase of age, the kidney essence is full and the bone growth also changes. Especially in the middle and old age, the kidney essence is deficient, the bone marrow is insufficient, the bone is weak and easy to break, and the whole body is prone to osteoporosis. For women, the kidney essence determines the strength of the bone through the action of "tianqi". With the increase of age, the kidney essence is gradually deficient, the tianqi is gradually exhausted after menopause, the kidney essence is gradually exhausted, the bone marrow is insufficient, and the bone marrow is empty, which is prone to osteoporosis such as waist and back pain.

[0040] In summary, the disease is located in the bone, the main organ is in the kidney, and the main pathogenesis is kidney essence deficiency and bone marrow malnutrition. The main causes of the disease are old age, physical weakness, overwork, and improper treatment, which lead to bone marrow malnutrition, resulting in weak and weak bones, and the bone marrow is empty, and the disease is caused. According to the main pathogenesis of kidney essence deficiency and bone marrow malnutrition, the treatment should be to fill the kidney essence and benefit the bone marrow as the primary task, supplemented by nourishing kidney yin and warming kidney yang, and assisted by astringent and astringent to prevent excessive nourishment, kidney essence, yin and yang balance, kidney qi, and bone marrow, so the treatment principle of the disease is to fill the kidney essence and benefit the bone marrow, and the treatment of the disease will achieve good effect.

[0041] The drug composition of the present application is based on the pathogenesis of postmenopausal osteoporosis and kidney essence deficiency, and establishes the treatment method of filling kidney essence and nourishing yin and yang, which aims to fill the kidney essence to consolidate the root, and to balance yin and yang to assist the effect. The kidney stores essence, and the kidney essence generates kidney qi, kidney yin and kidney yang. Kidney yin and kidney yang are rooted in kidney essence, so the treatment of kidney essence deficiency in osteoporosis needs to be based on kidney essence, while considering the balance of yin and yang, supporting yang and nourishing yin. The kidney yin is generated by the evaporation of yang, and the source is endless. On the contrary, the isolated yin does not grow. The product of filling essence and nourishing yin has yang in yin, and the kidney yin is generated, so it is supplemented with kidney yin drugs.

[0042] Around the treatment method of "filling kidney essence and nourishing yin and yang", the inventors establish the composition of the drug composition of the present application:

[0043] The monarch drug: cuscuta, sweet and bitter, slightly warm, its function is to benefit essence and marrow, and to strengthen bones. It is used as the monarch, and its effect of warming and filling essence and benefiting marrow and strengthening bones is taken.

[0044] The ministerial drug: epimedium, sweet and bitter, warm, and the effect of tonifying kidney and essence, warming kidney yang. Epimedium is used as the minister, which helps the monarch to invigorate kidney yang and strengthen bones.

[0045] The ministerial drug: rehmannia glutinosa, sweet and bitter, cold, heart, liver and kidney channels. Rehmannia glutinosa is used as the minister, and is used with epimedium to supplement kidney yin and yang, and to help the monarch to fill kidney essence, so as to achieve the effect of seeking yin in yang and growing yin in yang.

[0046] The assistant drug: oyster, salty and astringent, cold, liver, gallbladder and kidney channels, oyster can supplement kidney and essence, soften and disperse, nourish yin and yang, and enhance the function of bone quality.

[0047] The assistant drug: psoralea: sweet and bitter, warm, kidney and spleen channels. It has the functions of warming kidney and assisting yang, and filling essence. Psoralea has the functions of warming kidney and assisting yang, and filling essence. It is used with oyster as the assistant drug to supplement kidney and essence, strengthen bones, and help the monarch and minister to supplement kidney and essence.

[0048] The medicine: Danshen, bitter, cold, into the heart, liver, function of activating blood, dredging collaterals, pain. With: "Tong Li Guanmai" function, with Danshen as medicine, can lead the drug into the blood, but also enhance the effect of dredging collaterals and pain relief.

[0049] Throughout the whole prescription, medicinal cuscuta is warm and filling, and can fill essence, strengthen the marrow and bones. Herba epimedii and rehmannia can tonify kidney and yin and yang, and can tonify kidney qi. Concha mallei and psoralea can tonify kidney and fill essence, and can enhance the effect of tonifying yin and yang, and can converge and astringe. The combination of Danshen as medicine can lead the drug into the blood, enhance the effect, and can also activate blood, dredge collaterals, calm and relieve pain, effectively relieve the symptoms of back pain, soreness of waist and knees, difficulty in carrying, cramping of lower limbs, dizziness, tinnitus, fatigue, frequent urination at night and other symptoms, and improve the quality of life.

[0050] In summary, the drug composition of the present application is aimed at the pathogenesis of postmenopausal osteoporosis and kidney essence deficiency, and the treatment on one hand focuses on filling kidney essence, and on the other hand takes into account nourishing yin and supporting yang, regulating kidney yin and yang, and the prescription is simple and effective, which is helpful to improve the symptoms of back pain, soreness of waist and knees, difficulty in carrying, cramping of lower limbs, dizziness, tinnitus, fatigue, frequent urination at night and other symptoms caused by kidney essence deficiency, and improve the quality of life.

[0051] The traditional decoction is troublesome, has poor taste, unstable quality, is inconvenient for patients to carry and take, and is greatly limited in use. Therefore, it is selected to be made into a capsule, a tablet, a granule, an oral liquid or other dosage forms. Compared with other dosage forms, the tablet has less amount of auxiliary materials, is easier to form, has small volume, accurate dosage, and is more convenient to carry, transport and take.

[0052] In addition, the tablet is a dry solid, and the quality is stable and controllable. Some drugs that are easily oxidized and deteriorated and deliquescent can be protected by coating, and light, air and moisture have less effect on the tablet. In addition, the tablet can mask the unpleasant odor of traditional Chinese medicine.

[0053] Furthermore, the tablet production has high degree of mechanization and automation, and is suitable for industrialization and popularization. In order to better exert the social value and economic value of the medicine, the tablet is selected as the dosage form of the prescription in combination with the properties of the medicinal herbs in the prescription.

[0054] Selection of preparation process:

[0055] In the process of preparing the process, we have completed the selection of the process from many aspects,

[0056] I. Salted Cuscuta

[0057] 1. The effect of crushing on the extraction of salted Cuscuta

[0058] (1) Test design

[0059] Seed of Semen Eurycoma is seed class, seed is small, hard texture, in order to extract the effective component completely, the influence of crushing on extraction effect is investigated. Semen Eurycoma is crushed, extracted with 60% ethanol, and compared with Semen Eurycoma without crushing, with hyperoside content as index, the extraction effect is investigated.

[0060] (2) Test method

[0061] Take Semen Eurycoma, crush, take 30g of Semen Eurycoma and crushed Semen Eurycoma, extract with 60% ethanol for 1.5 hours, add 10 times solvent, filter the extract, combine, appropriately concentrate, transfer to 500ml volumetric flask, add 60% ethanol to the mark, shake well, and reserve.

[0062] (3) Test method

[0063] Determination of hyperoside

[0064] Preparation of test solution: take the above extract, filter, take the filtrate, and get it.

[0065] Preparation of control solution: accurately weigh hyperoside control product, add methanol to prepare a solution containing 0.04968mg per 1ml, and get it.

[0066] Chromatographic conditions: octadecylsilane bonded silica gel as filler; mobile phase: acetonitrile-0.1% phosphoric acid solution (16:84); detection wavelength: 360nm; column temperature: 30℃; flow rate: 1.0ml / min.

[0067] Determination method: accurately take 10ul of control solution and test solution respectively, inject into liquid chromatograph, determine, and get it.

[0068] (4) Test results are shown in Table 1.

[0069]

[0070] The test results show that whether Semen Eurycoma is crushed or not has great influence on extraction effect, Semen Eurycoma should be crushed before extraction.

[0071] 2, Process research of extraction solvent optimization

[0072] (1) Test design

[0073] Single factor investigation method is adopted, with hyperoside content and extract yield as index, the extraction effect of water, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol and 90% ethanol is investigated.

[0074] (2) Test method

[0075] Take the broken salt Cuscuta six parts, 30 g each, under the condition of parallel operation, heated to reflux extraction with different solvents twice, each add 10 times the amount of solvent, extraction 1.5 hours, the filtrate, combined, concentrated, transfer to 500 ml volumetric flask, add the corresponding solvent to the mark, shake, ready for use.

[0076] (3) test method

[0077] Determination of hyperoside

[0078] Preparation of test solution: take the above extract, filter, take the filter, that is.

[0079] Preparation of reference solution and chromatographic conditions with "broken salt Cuscuta extraction effect" test.

[0080] Determination method: respectively, 10 μl of reference solution and test solution were injected into liquid chromatograph, and the determination was made.

[0081] Determination of extract yield

[0082] Take the above extract, 200 ml each, in the evaporating dish, first concentrated in the water bath, then transferred to the vacuum drying oven, weighed, calculated, and obtained.

[0083] (4) test results are shown in table 2.

[0084]

[0085] The test results show that the content of hyperoside and the extract yield are higher when using 50%-70% ethanol as the solvent for extraction, so the extraction solvent of salt Cuscuta is initially determined as 50%-70% ethanol.

[0086] II. Process research on the optimization of extraction solvent of Epimedium

[0087] 1. Test design

[0088] Single factor test was used to investigate the extraction effect of water, 40% ethanol, 50% ethanol, 60% ethanol and 70% ethanol with the content of icariin and extract yield as indexes.

[0089] 2. Test method

[0090] Take Epimedium five parts, 15 g each, under the condition of parallel operation, reflux extraction with different solvents twice, each add 12 times the amount of solvent, extraction 1.5 hours, filter, transfer to 1000 ml volumetric flask, add the corresponding solvent to the mark, shake, ready for use.

[0091] 3. Test method

[0092] Determination of Icaritin

[0093] Preparation of test solution: Take the above extract, filter, and take the filtrate, which is the test solution.

[0094] Preparation of reference solution: Accurately weigh a certain amount of icaritin reference substance, add methanol to make a solution containing 0.1 mg per 1 ml, and you get the reference solution.

[0095] Chromatographic conditions: octadecylsilane-bonded silica gel as the filler; mobile phase: acetonitrile-water (30:70); detection wavelength: 270 nm; column temperature: 30°C; flow rate: 1.0 ml / min.

[0096] Determination method: accurately pipette 10 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, and you get the result.

[0097] Determination of extract yield

[0098] Take 500 ml of the above extract and place it in an evaporating dish. First, concentrate it into a thick paste on a water bath, then transfer it to a vacuum drying oven for drying. Weigh the dried extract, calculate the extract yield, and you get the result.

[0099] 4. The test results are shown in Table 3.

[0100]

[0101] From the above table, it can be seen that the content of icaritin in the sample is the highest when water is used as the solvent for extraction. With increasing ethanol concentration, the content of icaritin tends to decrease. The extract yields are not significantly different when different solvents are used for extraction. The extract yield is slightly lower when the solvent has a higher ethanol concentration. Using ethanol as the solvent increases the cost and causes environmental pollution. Therefore, water is selected as the extraction solvent for Epimedium.

[0102] III. Process optimization of extraction solvent for salted Psoralea corylifolia

[0103] 1. Test design

[0104] Single-factor test was used to investigate the extraction effects of water, 50% ethanol, 60% ethanol, 70% ethanol, and 80% ethanol, with the contents of psoralen and isopsoralen as the indicators.

[0105] 2. Test method

[0106] Weigh five portions of salted Psoralea corylifolia, each 20 g. Extract twice by heating reflux, with 10 times the amount of solvent each time, for 1.5 hours. Filter the extract, combine, transfer the filtrate to a 500 ml volumetric flask, add the corresponding solvent to the mark, shake well, and prepare for use.

[0107] 3. Test method

[0108] (1) Determination of psoralen and isopsoralen

[0109] Preparation of the test solution: Accurately measure 5 ml of the above extract, place it in a 50 ml volumetric flask, add the appropriate solvent to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0110] Preparation of reference solutions: Accurately weigh appropriate amounts of psoralen reference standard and isopsoralen reference standard, and add methanol to prepare solutions containing 0.023 mg and 0.023 mg per ml, respectively.

[0111] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; mobile phase: methanol-water (55:45); detection wavelength: 246 nm; column temperature: 30 ℃; flow rate: 1.0 ml / min.

[0112] Assay: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0113] (2) Determination of extract yield

[0114] Take 200 ml of the above extract and place it in an evaporating dish. First, concentrate it into a thick paste in a water bath, then transfer it to a vacuum drying oven to dry. Weigh the sample and calculate the result.

[0115] 4. The test results are shown in Table 4.

[0116]

[0117] The experimental results show that when using 50%-70% ethanol as a solvent, the contents of psoralen and isopsoralen in the samples are relatively high; the yield of extract is not significantly different. Only when using 80% ethanol as a solvent is the yield of extract lower.

[0118] Based on the results of the solvent optimization experiments, it can be seen that extracting Cuscuta chinensis and Psoralea corylifolia with 50%-70% ethanol can better extract their effective components. Therefore, it is proposed to combine the two and use ethanol as the extraction solvent. At the same time, in order to ensure complete extraction of effective components and reduce the amount of ethanol used, the ethanol concentration is determined to be 60%. Epimedium is extracted with water as the solvent, which has a high transfer rate of effective components and saves energy. Therefore, Epimedium, Rehmannia glutinosa, and Salvia miltiorrhiza are combined for water extraction.

[0119] IV. Experimental Study on Optimization of Water Extraction Process

[0120] 1. Water absorption rate investigation

[0121] Weigh out 50g each of Epimedium, Rehmannia glutinosa, and Salvia miltiorrhiza, and then weigh out 30g each of Epimedium, Rehmannia glutinosa, and Salvia miltiorrhiza, for a total of 84g. Mix them together, add 10 times the amount of water to each, soak them, measure the water absorption, and calculate the water absorption rate. The results are shown in Table 5.

[0122]

[0123] The above experimental results show that: because Epimedium has a high water absorption rate, the water absorption rate is also high after the three herbs are mixed; Epimedium is light and large in volume, and is not easy to soak, so the amount of water added for water extraction is set at about 12 times to ensure the extraction effect.

[0124] 2. Orthogonal experimental design

[0125] Factors that significantly affect the extraction effect were selected as the following: (A) number of extractions, (B) extraction time, (C) water usage, and (D) blank. The contents of icariin and tanshinone sodium were used as indicators, and the results were analyzed at the following levels: L9(3) 4 The experiment was conducted using the table. The factor level table is shown in Table 6.

[0126]

[0127] 3. Test methods

[0128] Weigh out 15g of Epimedium, 15g of Rehmannia glutinosa, and 12g of Salvia miltiorrhiza, making a total of 9 portions. Under parallel operation conditions, follow L9 (3 4 The orthogonal design table is prepared by boiling in water, filtering the extract, combining the solutions, concentrating appropriately, transferring to a 1000ml volumetric flask, adding water to the mark, shaking well, and set aside.

[0129] (1) Determination of icariin content

[0130] Preparation of the test solution: Take the orthogonal test sample solution, filter it, and take the filtrate as the test solution.

[0131] Preparation of the reference solution and chromatographic conditions were performed in accordance with the optimal extraction solvent test.

[0132] Assay: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0133] (2) Determination of sodium tanshinone content

[0134] Preparation of the test solution: Take the orthogonal test sample solution, filter it, and take the filtrate as the test solution.

[0135] Preparation of reference solution: Accurately weigh an appropriate amount of tanshinone sodium reference standard, add 50% methanol to prepare a solution containing 0.04 mg per ml.

[0136] Chromatography conditions: octadecylsilane bonded silica gel as the filler; mobile phase: methanol-1% glacial acetic acid (15:85); detection wavelength: 280 nm; column temperature: 30°C; flow rate: 1.0 ml / min.

[0137] Determination method: 20 μl of the control solution and the sample solution was respectively injected into the liquid chromatograph, and then the determination was performed.

[0138] (3) Determination of the extract yield

[0139] The orthogonal test sample solution was taken, 200 ml of which was respectively placed in the evaporating pan, concentrated into thick paste on the water bath, and then transferred to the vacuum drying oven for drying. The weight was determined, and the calculation was performed to obtain the result.

[0140] 4. The test results of icariin, sodium danshensu and extract yield are shown in Table 7.

[0141]

[0142] 5. Analysis of icariin content determination and sodium danshensu content determination results

[0143] The test data was subjected to variance analysis, and the significance of the influence of each factor was tested. The results are shown in Tables 8 and 9.

[0144]

[0145] From the above table, it can be seen that, taking icariin content as the index, the influence of each factor on icariin content is in the order of extraction times > water amount > extraction time, and the best process is A3B3C3. Among them, the extraction times has a greater influence on icariin content, and has a significant difference.

[0146]

[0147] From the above table, it can be seen that, taking sodium danshensu content as the index, the influence of each factor on sodium danshensu content is in the order of extraction times > extraction time > water amount, and the best process is A3B3C3. Among them, the extraction times has the greatest influence on sodium danshensu content, and has a very significant difference, and the extraction time and water amount have a smaller influence on sodium danshensu content, and have no significant difference. Since in the A factor, the content of the III level is increased more than that of the II level, it is proposed to extract the fourth time after the extraction of the best process and the optimized process is completed, add 10 times the amount of water, extract for 1.5 hours, and collect separately to investigate the content change of sodium danshensu.

[0148] Based on the results of variance analysis of icariin and sodium danshensu, in order to ensure sufficient extraction of effective components and save labor and energy, the extraction is selected for three times, 2 hours for the first extraction, 12 times of water, 1 hour for the second and third extractions, 10 times of water. The optimized extraction process is A3B (3，1，1） C (2，1，1） .

[0149] V. Test study on optimization of alcohol extraction process

[0150] 1. Orthogonal test design

[0151] Select the factors that have greater influence on extraction effect: (A) extraction times, (B) extraction time, (C) alcohol amount, (D) blank as the observation factors, and take the content of hyperoside, psoralen and isopsoralen as the indexes, and perform the test according to the following levels in L9(34 4 ) table. See Table 10.

[0152]

[0153] 2. Test method

[0154] According to the prescription ratio, take 30 g of crushed salted semen abrus, 12 g of salted psoralen, and 9 portions of each, and take 60% ethanol as the extraction solvent. Under the condition of parallel operation, perform reflux extraction according to the L9(34 4 ) orthogonal design table, filter the extraction liquid, combine, appropriately concentrate, transfer to a 500 ml volumetric flask, add 60% ethanol to the calibration mark, shake well, and reserve.

[0155] (1) Determination of the content of hyperoside

[0156] Preparation of test sample solution: take the orthogonal test sample solution, filter, and take the filtrate as the test sample solution.

[0157] Preparation of reference solution and chromatographic conditions are the same as those in the "extraction solvent optimization test".

[0158] Determination method: precisely take 10 μl of the reference solution and the test sample solution respectively, inject into the liquid chromatograph, and determine, and then obtain.

[0159] (2) Determination of the content of psoralen and isopsoralen

[0160] Preparation of test sample solution: precisely take 5 ml of the orthogonal test sample solution, respectively, into a 50 ml volumetric flask, add 60% ethanol to the calibration mark, shake well, filter, and take the filtrate as the test sample solution.

[0161] Preparation of reference solution and chromatographic conditions are the same as those in the "extraction solvent optimization test".

[0162] Determination method: 10 μl of the control solution and the sample solution were precisely pipetted into the liquid chromatograph, and determination was carried out, and the content was obtained.

[0163] 3. The test results are shown in Table 11.

[0164]

[0165] 4. Analysis of the determination results of the hyperoside content, the psoralen content and the isopsoralen content

[0166] The test data were subjected to variance analysis, and the significance of the influence of each factor was tested, and the results are shown in Table 12 and Table 13.

[0167]

[0168] From the above table, it can be seen that, taking the hyperoside content as the index, the influence of each factor on the hyperoside content is in the order of extraction times > alcohol addition amount > extraction time, and the optimal process is A2B2C3, wherein the extraction times A has a significant influence on the hyperoside content.

[0169]

[0170] From the above table, it can be seen that, taking the psoralen and isopsoralen contents as the index, the influence of each factor on the psoralen and isopsoralen contents is in the order of extraction times > alcohol addition amount > extraction time, and the optimal process is A3B2C2; wherein the extraction times A and the alcohol addition amount C have a greater influence on the psoralen and isopsoralen contents, and have significant differences.

[0171] Combining the content analysis results of the hyperoside, the psoralen and the isopsoralen, in order to ensure the sufficient extraction of the effective components and save labor and energy, the extraction is selected twice, each time for 1.5 hours, 10 times of alcohol is added in the first time, and 8 times of alcohol is added in the second time. The process after optimization is A2B2C (3，2） .

[0172] Six, the crushing process of calcined oyster shell

[0173] 1. The powder yield investigation

[0174] Three portions of the calcined oyster shell were taken, each portion was 5 kg, and was crushed by a crusher, and was passed through a 120-mesh sieve, and the powder yield was calculated, and the results are shown in Table 14.

[0175]

[0176] The results show that the powder yield is more than 95%, which meets the requirements. BRIEF DESCRIPTION OF DRAWINGS

[0177] Figure 1: XDGM significantly improves the bone morphological characteristics of ovariectomy-induced osteoporotic rats

[0178] Figure 2 : XDGM significantly improves the bone morphological characteristics of ovariectomy-induced osteoporotic rats

[0179] Figure 3 : XDGM significantly improves the bone morphological characteristics of ovariectomy-induced osteoporotic rats

[0180] Functional test:

[0181] In order to illustrate the efficacy of the pharmaceutical composition of the present application, functional tests were performed on the samples of Example 1, Example 2 and Example 6 prepared by the examples.

[0182] First, the effect of the active ingredient of the pharmaceutical composition of the present application on ovariectomy-induced osteoporosis in rats

[0183] [Objective] Using a rat ovariectomy osteoporosis model, the effect of the active ingredient (XDGM1, XDGM2, XDGM6) of the pharmaceutical composition of the present application on ovariectomy-induced osteoporosis in rats was observed, and the therapeutic effect on osteoporosis was evaluated.

[0184] [Methods] After 3 months of ovariectomy surgery modeling, the rats were randomly divided into normal group, sham operation group, model group, positive drug (estradiol valerate) group, XDGM 1, 2, and 6 groups according to body weight, and intragastrically administered for 3 months. During the period, all rats were intraperitoneally injected with tetracycline hydrochloride for bone fluorescence labeling. After the administration was completed, blood was taken to detect serum calcium Ca, phosphorus P, alkaline phosphatase ALP, tartrate-resistant acid phosphatase TRACP, type I procollagen amino-terminal peptide PINP, osteocalcin BGP, parathyroid hormone PTH, and calcitonin CT. The left tibia, femur, right femur, and spine were taken to detect bone morphological, bone biomechanical, and bone density indicators.

[0185] Results 1. Bone morphological indexes showed that compared with the model group, XDGM 1 and 2 significantly increased the percentage of tibial trabecular bone volume TBV% (P<0.05 or P<0.01), reduced the percentage of trabecular bone resorption surface TRS%, the percentage of trabecular bone formation surface TFS%, the trabecular bone mineralization rate MAR and the bone cortical mineralization rate mAR (P<0.01 or P<0.05), and the high conversion type bone metabolism state of the ovariectomized rats could be reduced, and the bone morphological characteristics of the osteoporotic rats could be improved. 2. Bone biomechanical indexes showed that compared with the model group, XDGM 1 and 2 significantly increased the maximum load, bending strength and elastic modulus of the femur (P<0.05 or P<0.01), and the bone biomechanical characteristics of the osteoporotic rats were improved. 3. Bone density indexes showed that XDGM 1 significantly increased the bone density of the osteoporotic rats (P<0.05), and the bone density of XDGM 2 and 6 groups was also increased. 4. Bone component indexes showed that XDGM 1, 2 and 6 significantly increased the contents of serum Ca and P (P<0.01), which provided rich inorganic raw materials for bone formation; the high-dose XDGM significantly increased the content of bone formation marker PINP (P<0.01), which provided rich organic raw materials for bone formation. 5. Bone transformation marker indexes showed that XDGM 1, 2 and 6 significantly reduced the content of bone formation marker BGP (P<0.01 or P<0.05), and the contents of bone resorption markers ALP and TRACP were also reduced, and the high conversion type bone metabolism state could be reduced. 6. Hormone indexes showed that XDGM 1 significantly increased the contents of CT and PTH (P<0.01 or P<0.05), and the contents of CT and PTH in XDGM 2 and 6 groups were also increased, and the XDGM could improve the osteoporosis state, so that the bone formation was greater than the bone resorption.

[0186] Conclusion The experimental results show that the XDGM can significantly improve the bone morphological and biomechanical characteristics of the rats, increase the bone density, and the XDGM has a significant improvement effect on the osteoporotic bone of the ovariectomized rats. The improvement effect is related to the increase of the contents of blood Ca, P and PINP, the reduction of the high conversion type bone metabolism state and the stimulation of the secretion of CT and PTH.

[0187] Objective: To observe the effect of the active component of the pharmaceutical composition of the application on the osteoporosis of rats caused by ovariectomy by using the osteoporosis model of ovariectomized rats, and to evaluate the therapeutic effect on osteoporosis.

[0188] 1. Experimental materials

[0189] 1.1 Test sample

[0190] 1.1.1 Name: Active ingredient of the pharmaceutical composition of the invention, abbreviation: XDGM, test article number: TN-1318.

[0191] 1.1.2 Property: Brownish powder.

[0192] 1.1.3 Function and indication: Menopausal osteoporosis.

[0193] 1.1.4 Dose for clinical simulation: Oral, 14.1 g crude drug per day.

[0194] 1.1.5 Content and specification: 2.96 g crude drug / g dry paste powder.

[0195] 1.1.6 Source and batch number: Provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd., batch number: 20130301.

[0196] 1.1.7 Storage condition: Store in a sealed container.

[0197] 1.1.8 Validity period: 2 years.

[0198] 1.2 Positive drug and main reagent

[0199] 1.2.1 Estradiol valerate tablets: DELPHARM Lille S.A.S, batch number: 094A2.

[0200] 1.2.2 Tetracycline hydrochloride: SIGMA-ALDRICH.Co., batch number: SLBH4536V.

[0201] 1.2.3 Picric acid: Taishan City Chemical Factory Co., Ltd., batch number: 20071201.

[0202] 1.2.4 Sodium carboxymethylcellulose (CMC-Na): Tianjin Yongda Chemical Reagent Development Center, batch number: 20080702.

[0203] 1.2.5 Chloral hydrate: Tianjin Guangfu Fine Chemical Research Institute, batch number: 20131114.

[0204] 1.2.6 Anti-tartaric acid phosphatase TRACP: Biyun Tian Biotechnology Co., Ltd., batch number: 1202191412.

[0205] 1.2.7 Alkaline phosphatase ALP: Beijing Jiujiang Biotechnology Co., Ltd., batch number: 14-0612P.

[0206] 1.2.8 I type procollagen amino-terminal peptide PINP: Cloud-Clone Corp., batch number: L150120505.

[0207] 1.2.9 Phosphorus P: Beijing Jiujiang Biotechnology Co., Ltd., batch number: 14-0923.

[0208] 1.2.10 Calcium Ca: Beijing Jiujiang Biotechnology Co., Ltd., batch number: 14-0811.

[0209] 1.2.11 Bone Gla protein BGP: Beijing North Institute of Biotechnology, batch number: 20150220.

[0210] 1.2.12 Calcitonin CT: Beijing North Institute of Biotechnology, batch number: 20150220.

[0211] 1.2.13 Parathyroid hormone PTH: Beijing North Institute of Biotechnology, batch number: 20150220.

[0212] 1.3 Experimental system:

[0213] 1.3.1 Animal pedigree: SD rats.

[0214] 1.3.2 Animal level: SPF level.

[0215] 1.3.3 Animal gender and quantity: female, a total of 108.

[0216] 1.3.4 Animal age: 63~69 days.

[0217] 1.3.5 Animal weight: 200~220g.

[0218] 1.3.6 Animal source: purchased from Beijing Vantolliva Experimental Animal Technology Co., Ltd., qualified certificate number: 11400700047562, license number: SCXK (Jing) 2012-0001, received date May 21, 2014.

[0219] 1.3.7 Raising conditions: The animals were raised in the New Drug Evaluation Center of Hebei Province of Integrative Medicine Research Institute. The rats were cage-raised, with 12 hours of light per day, temperature 20~26℃, relative humidity 40~70%.

[0220] 1.3.8 Quarantine process: New animal quarantine period 3 days, due to the inability to buy suitable animals, the newly purchased animals were fed to about 6 months old before starting the experiment, during which the animals had normal drinking and eating, good health, no disease and death signs.

[0221] 1.3.9 Feed: Experimental animal full-value granular feed, provided by the Experimental Animal Center of the Chinese People's Liberation Army Military Medical Sciences Academy, qualified certificate number: 0025539.

[0222] 1.3.10 Drinking water: fill the drinking water bottle with normal water for animals to drink freely, and rinse the bottle and change the water once a day.

[0223] 1.3.11 Bedding: normal grade bedding for laboratory animals, provided by Hebei Province Laboratory Animal Center, production license: SCXK (Hebei) 2013-2-001.

[0224] 1.3.12 Identification: marked with 5% picric acid.

[0225] 2 Experimental method

[0226] 2.1 Basis for experimental design

[0227] 2.1.1 Standards used: Chinese Ministry of Health Drug Administration Bureau issued "Guidelines for the Study of New Drugs of Traditional Chinese Medicine (Pharmacy, Pharmacology, Toxicology)", "Preclinical Research Guidelines for New Drugs (Western Medicine) (Pharmacy, Pharmacology, Toxicology)", "Methodology of Traditional Chinese Medicine Pharmacological Research", "Methodology of Pharmacological Experiments" published by People's Medical Publishing House and related literature.

[0228] 2.1.2 Information provided by the client: after castration, rats show decreased bone mineral content, rapid increase in bone resorption, and decreased bone strength, which are similar to humans in many ways. Compared with other animal models, it has the advantages of fast reproduction, low cost, easy feeding and management, and clear genetic background. It has been used as the most commonly used animal model and is widely used in the evaluation and development of new drugs for postmenopausal osteoporosis treatment.

[0229] 2.2 Dose and grouping

[0230] 108 rats were randomly divided into 3 groups according to body weight, 10 in the normal group, 12 in the sham operation group (Sham), and 86 in the modeling group. The rats in the modeling group were subjected to ovariectomy surgery, and the bilateral ovaries were removed. The sham operation group had the same surgical procedure as the modeling group, but the ovaries were not removed, only a small amount of adipose tissue was removed. After modeling for about 3 months, the rats in the modeling group were randomly divided into 5 groups according to body weight, namely the model group (OVX), the estradiol valerate group, the XDGM 1, 2, and 6 groups. The approximate clinical dose of XDGM is 14.1g crude drug per day, calculated based on 60kg per person, the rat dose is 8 times the human clinical dose, which is 1.88 crude drug / kg, and the content is 2.96g crude drug / g dry extract powder, which is 0.635g dry extract powder / kg. According to the instructions of the positive drug and reference literature, the dose of estradiol valerate is 100μg / kg, see Appendix 15.

[0231]

[0232] 2.3 Administration method:

[0233] Intragastric administration, 10 ml / kg body weight, consistent with the clinically recommended oral route.

[0234] 2.4 Preparation and storage of test articles

[0235] The test drugs were prepared into experimental concentrations (see Table 15) with 0.5% CMC-Na for medication, and stored at 2-8°C after preparation for standby use. The positive drug was prepared on the spot.

[0236] 2.5 Administration of test articles

[0237] After 3 months of modeling, intragastric administration of test drugs was started according to experimental grouping. The normal, sham and model groups were given 0.5% CMC-Na, 1 time / day, for 3 consecutive months.

[0238] 2.6 Experimental procedures

[0239] The newly received SD rats were quarantined for 3 days, and experimental grouping and modeling were performed according to the above procedures. After 3 months of modeling, the groups were administered, and the normal, Sham and OVX groups were given 0.5% CMC-Na, as described previously. Body weight was recorded once a week. All rats were injected intraperitoneally with tetracycline hydrochloride 30 mg / kg for bone fluorescence labeling on the 16th and 4th days before sacrifice. After the end of the administration period, the rats were anesthetized with 10% chloral hydrate on the 2nd day, with a volume of 0.35 ml / 100 g body weight. Blood was collected from the abdominal aorta, and the rats were sacrificed. The samples were taken and the indicators were detected.

[0240] Model preparation method: The rats were anesthetized with 10% chloral hydrate, with a volume of 0.35 ml / 100 g body weight. The center was at the waist 1 cm below the rib on the middle axillary line, the hair was cut to a diameter of 2 cm, and the skin was tightened. A 0.8-1 cm incision was made along the longitudinal axis, and the subcutaneous superficial fascial tissue layer was separated with blunt forceps. The muscle layer was cut open longitudinally, and the abdominal cavity was exposed. The ovaries were carefully separated (mature ovaries were light red, oval in shape, with irregular nodular follicles on the surface, and the pink tubular uterine terminal was connected, surrounded by a lot of white fatty ligaments). The uterine terminal was ligated with silk, and the entire ovary was removed. The remaining tissue was returned to the abdominal cavity, and the muscle layer and skin were sutured in turn. The incision was disinfected. The Sham group was not ligated with the uterine terminal and the ovary was not removed, and the rest of the operation was the same as above. After the operation, the rats were placed in a warm environment, and each rat was given penicillin sodium salt 50,000 units per day for 3 consecutive days.

[0241] 2.7 Indicators for detection

[0242] 2.7.1 Detection of blood indicators

[0243] After the drug administration operation according to the experimental design, the rats were killed by abdominal aortic blood sampling after abdominal cavity anesthesia with 10% chloral hydrate. The whole blood was left to stand, and the serum was separated by centrifugation. The serum calcium Ca, phosphorus P, alkaline phosphatase ALP, tartrate-resistant acid phosphatase TRACP, type I procollagen amino-terminal peptide PINP, osteocalcin BGP, parathyroid hormone PTH, and calcitonin CT were determined.

[0244] 2.7.2 Bone mineral density (BMD) detection

[0245] Before detection, the frozen rat vertebrae were taken out of the refrigerator, warmed at room temperature, and the BMD of the L4-6 lumbar vertebrae of each group of rats was detected by Osteocore3 Digital 2D bone mineral density instrument.

[0246] Osteocore3 Digital 2D bone mineral density instrument, detection of BMD of L4-6 lumbar vertebrae of each group of rats.

[0247] 2.7.3 Detection of bone histomorphological indexes

[0248] The proximal 1 / 3 of the left tibia of the rat was taken and fixed in 4% paraformaldehyde solution (PH 7.4) for 24 h, then dehydrated, the procedure was 80% ethanol for 2d, 95% ethanol for 2d, 100% ethanol for 2d, and xylene for 2d. Then, the specimen was immersed in plastic polymerization liquid I, II, and III for 3d respectively, and the above fixation, dehydration and immersion process was carried out at 4°C. Finally, 400 μL of N, N-dimethyl-p-toluidine was added to 100 ml of pre-cooled (4°C) III liquid, stirred with a magnetic stirrer for 10 min, then about 7 ml of III liquid was injected into a penicillin vial, the bone specimen was placed in the same direction at the bottom of the vial, the air in the embedding bottle was evacuated with a syringe, and then placed in a-20°C freezer for polymerization for about 1 w, then it became a colorless transparent hard embedding block. After trimming, 5 μm longitudinal non-decalcified bone sections were cut from each bone tissue on a Reicheit-Jung 2040 microtome, 2 sections were obtained, one for toluidine blue staining and the other for fluorescence observation.

[0249] Bone histomorphometry method: Qwin Pro V3.5.0 image analysis system was used for morphometry of non-decalcified bone sections:

[0250] (1) Trabecular bone histomorphometry:

[0251] Trabecular bone volume percentage (TBV%): the percentage of trabecular bone volume to the total volume of the measured bone marrow cavity, which is the main marker for measuring bone mass level;

[0252] Trabecular bone resorption surface percentage (TRS%): the percentage of irregular, uneven trabecular surface to the trabecular surface, which can determine the activity of osteoclasts;

[0253] Trabecular bone formation surface percentage (TFS%): the percentage of osteoblast-covered osteoid surface to trabecular surface, which can determine the activity of osteoblasts;

[0254] Trabecular bone mineralization rate (MAR): the average distance of the fluorescence double-labeling band on the trabecular surface divided by the number of days between the two labels.

[0255] (2) Intracortical surface morphometry:

[0256] Osteoid average width (OSW): the average width of the osteoid covered by osteoblasts on the intracortical surface;

[0257] Cortical bone mineralization rate (mAR): the average distance of the fluorescence double-labeling band on the intracortical surface divided by the number of days between the two labels.

[0258] 2.7.4 Detection of bone biomechanics indicators

[0259] Before detection, the frozen right femur of the rat was taken out from the refrigerator and warmed at room temperature. The rat femur was subjected to three-point bending test: the femur was placed on the microcomputer-controlled electronic universal testing machine support, with a span of 16 mm, and pressed on the middle segment of the femur at a loading speed of 1 mm / min until the femur was broken, and the maximum load (N), bending strength (MPa) and elastic modulus (GPa) were detected.

[0260] 2.8 Notification of relevant staff

[0261] When the animal appears abnormal, notify the pathology room for treatment.

[0262] 2.9 Main instrument system

[0263] Hitachi 7080 automatic biochemical analyzer Hitachi Company, Japan

[0264] BT224S Sartorius precision analytical balance Sartorius Scientific Instruments Co., Ltd.

[0265] SIGMA-3K15 high-speed refrigerated centrifuge SIGMA Company, Germany

[0266] DT-2000 electronic balance Changshu Shuangjie Testing Instrument Factory

[0267] SpectraMax M2 enzyme label analyzer Molecular Devices

[0268] XH6080 Radioimmunoassay Analyzer, Xi'an Nuclear Instrument Factory

[0269] Microcomputer-controlled electronic universal testing machine Shenzhen Ruigeer Instrument Co., Ltd.

[0270] Qwin Pro V3.5.0 Image Analysis System, Leica GmbH (Germany)

[0271] 2040 slicer, Reicheit-Jung GmbH, Germany

[0272] Osteocore3 Digital 2D Bone Densitometer, Medilink (France)

[0273] (Dual-energy X-ray)

[0274] 2.10 Statistical Methods

[0275] Experimental data were analyzed using SPSS statistical software, and the statistical results are expressed as mean ± standard deviation. The results indicate that one-way ANOVA was used for comparing means, and the least significant difference (LSD) method was used for pairwise comparisons.

[0276] 3 Results

[0277] 3.1XDGM significantly improved the bone morphological characteristics of ovariectomized osteoporotic rats.

[0278] As shown in Appendix Table 16, compared with the sham-operated group, the tibial TBV% of rats in the model group was significantly decreased (P<0.01), while TRS%, TFS%, OSW, mAR, and MAR were significantly increased (P<0.01 or P<0.05), indicating that ovariectomized rats exhibited high-turnover bone metabolism. Compared with the model group, the positive control drug estradiol valerate significantly increased the tibial TBV% of rats (P<0.01) and decreased TRS%, TFS%, mAR, and MAR (P<0.01 or P<0.05). The XDGM1 and XDGM2 groups significantly increased the tibial TBV% of rats (P<0.05 or P<0.01) and decreased TRS%, TFS%, mAR, and MAR (P<0.01 or P<0.05). Figure 1 It is evident that the number and volume of trabeculae in the proximal tibia of XDGM rats were increased compared to the model group, indicating that XDGM can reduce the high-turnover bone metabolism state in ovariectomized rats and improve the bone morphological characteristics of osteoporotic rats.

[0279]

[0280] 3.2 XDGM significantly improved the bone biomechanical properties of ovariectomized osteoporotic rats.

[0281] As shown in Table 17, compared with the sham operation group, the maximum load, bending strength and elastic modulus of the femur of the model group were significantly reduced (P<0.01); compared with the model group, the maximum load, bending strength and elastic modulus of the femur of the positive drug estradiol valerate group were significantly increased (P<0.01), and the maximum load, bending strength and elastic modulus of the femur of the XDGM 1 and 2 groups were significantly increased (P<0.05 or P<0.01). It can be seen that XDGM can significantly improve the bone biomechanical properties of osteoporotic rats.

[0282]

[0283] 3.3 Effect of XDGM on bone mineral density of osteoporotic rats.

[0284] As shown in Table 18, compared with the sham operation group, the bone mineral density of the model group was significantly reduced (P<0.01). Compared with the model group, the bone mineral density of the positive drug estradiol valerate group was increased, but no significant difference was found (P>0.05); the bone mineral density of the XDGM 1 group was significantly increased (P<0.05), and the bone mineral density of the XDGM 2 and 6 groups was also increased. It can be seen that XDGM can increase the bone mineral density of ovariectomized osteoporotic rats.

[0285]

[0286] 3.4 Effect of XDGM on serum Ca and P contents of osteoporotic rats.

[0287] As shown in Table 19, compared with the sham operation group, the serum Ca content of the model group was significantly reduced (P<0.05), and the P content also showed a decreasing trend, but no significant difference was found (P>0.05). Compared with the model group, the serum Ca and P contents of the XDGM groups were significantly increased (P<0.01). It can be seen that XDGM can increase the contents of Ca and P, providing abundant inorganic material for bone formation.

[0288] 3.5 Effect of XDGM on serum bone markers of osteoporotic rats.

[0289] From the attached table 19, 20, compared with the sham operation group, the model group rats bone formation markers ALP, BGP and bone resorption markers TRACP increased significantly (P<0.01 or P<0.05), bone formation markers PINP content decreased, but no significant difference (P>0.05), can see the model group rats performance for a certain degree of high turnover bone metabolism. Compared with the model group, XDGM 1 group PINP increased significantly (P<0.01), XDGM 1, 2, 6 group BGP decreased significantly (P<0.01 or P<0.05), ALP and TRACP decreased, but no significant difference (P>0.05). Can see XDGM can increase PINP, provide rich organic raw materials for bone formation, while reducing the high turnover bone metabolism state.

[0290] 3.6 XDGM on the influence of serum hormone levels in osteoporotic rats.

[0291] From the attached table 20, compared with the sham operation group, the model group rats CT, PTH decreased significantly (P<0.01). Compared with the model group, XDGM 1 group CT, PTH content increased significantly (P<0.01 or P<0.05), 2, 6 groups CT, PTH content also increased. Can see XDGM improve the state of osteoporosis, so that bone formation is greater than bone resorption.

[0292]

[0293]

[0294] 4 Conclusion

[0295] The results of this experiment show that XDGM significantly improves the bone morphology and bone biomechanical properties of rats, increases bone density, and can see that XDGM has obvious improvement effect on osteoporotic bone caused by ovariectomy in rats. This improvement is related to the increase of blood Ca, P, PINP content, the decrease of high turnover bone metabolism state, and the stimulation of CT, PTH secretion.

[0296] 5 Abnormal situation

[0297] After ovariectomy modeling, some animals died, the reasons were infection, internal hemorrhage and intestinal distension. At the end of 3 months of modeling, the number of remaining animals was 8 in the sham operation group and 60 in the model group.

[0298] 6 Discussion

[0299] Osteoporosis (OP) is a systemic bone disease characterized by low bone mass, microarchitectural deterioration of bone tissue, and increased bone fragility and susceptibility to fracture (WHO). OP can be divided into primary and secondary. Primary OP includes postmenopausal osteoporosis (type I, high turnover) and senile osteoporosis (type II, low turnover). High turnover OP is a pathological state of increased bone turnover rate with increased bone resorption and bone formation. Low turnover OP is a pathological state of low turnover rate with decreased bone formation rate although bone resorption is increased or decreased. Secondary OP refers to OP caused by any disease or drug affecting bone metabolism. In the process of studying the pharmacology and mechanism of postmenopausal osteoporosis, animal models such as mice, rats, rabbits, dogs, pigs, and sheep have been used. The selection of experimental animals requires convenience, relevance, and suitability, and has the stability of repeatability, and OP should be consistent with human performance in histopathology. The decrease in bone mineral content, rapid increase in bone resorption, and decrease in bone strength after ovariectomy in rats are similar to those in humans. Compared with other animal models, rats have the advantages of fast breeding, low cost, easy feeding and management, and clear genetic background, and have been used as the most common animal model. They are widely used in the evaluation and development of new drugs for postmenopausal osteoporosis. Some studies have shown that using rats over six months of age can achieve better results. In this study, six-month-old female rats were used to study postmenopausal osteoporosis.

[0300] Bone histomorphometry is a technique that can quantitatively observe and study bone tissue morphology and structure. It has become one of the important research methods for quantitatively detecting the effects of drugs on animal osteoporosis models, exploring the mechanisms of drugs, and providing theoretical guidance for clinical practice. Bone histomorphometry has different classification methods. According to the different sampling sites, it can be divided into tibia, femur, lumbar spine, etc. According to the different observation sites, it can be divided into cortical bone, cancellous bone, femoral neck, etc. According to the different data, it can be divided into static parameters and dynamic parameters, etc. Cancellous bone is a small trabecular bone located on the inner surface of cortical bone. In the upper segment of the rat tibia, the epiphysis of three-month-old rats is not closed, and it is completely or partially closed until 12 months of age. Due to abundant blood supply and nutrition, active metabolism, strong bone regeneration ability, and high bone turnover rate, drugs can directly reach the local site. Therefore, the upper segment of the rat tibia is a sensitive site for inducing osteoporosis models and observing the effects of drugs. It is also the most ideal site for various osteoporosis models and drug prevention and treatment research. The results of this experiment show that XDGM significantly increases the TBV% of rat tibia, reduces the TRS%, TFS%, mAR, and MAR, inhibits the high turnover state of bone metabolism in ovariectomized rats, and improves the bone morphological characteristics of osteoporotic rats.

[0301] Bone biomechanics is based on the theory of engineering mechanics, to study the mechanical properties of bone tissue under the action of the outside world and the biological effects of bone after stress, which is a reliable method for bone quality assessment. The study of bone biomechanics can be generally investigated from two aspects: structural mechanics and material mechanics. Structural mechanics mainly includes maximum load, fracture load, elastic load and other indicators, while material mechanics mainly includes fracture strain, elastic modulus and other indicators. The performance of structural mechanics is mainly related to the size and geometry of the bone, while the material mechanics is mainly related to the strength and toughness of the bone, which is related to the microstructure of the bone, the mineral content in the bone and the bone density. The results of this experiment show that XDGM significantly increases the maximum load, bending strength and elastic modulus of the femur of rats, and significantly improves the bone biomechanical properties of osteoporotic rats.

[0302] Bone density, which is the abbreviation of bone mineral density, refers to the bone mass per unit volume of bone tissue. Bone density is commonly used to assess bone fragility and the risk of fracture. Bone density measurement is the best quantitative indicator for diagnosing osteoporosis, predicting the risk of osteoporotic fractures, monitoring the natural course of the disease, and evaluating the efficacy of drug intervention. The results of this experiment show that high-dose XDGM significantly increases the bone density of ovariectomized osteoporotic rats.

[0303] Bone turnover biochemical markers are the metabolic products of bone tissue itself, which are referred to as bone markers. Bone turnover markers are divided into bone formation markers and bone resorption markers. The former represents the metabolic products of osteoblast activity and bone formation, while the latter represents the metabolic products of osteoclast activity and bone resorption, especially bone matrix degradation products. Bone formation markers mainly include serum alkaline phosphatase, osteocalcin, and type I collagen N-terminal propeptide. Bone resorption markers mainly include serum tartrate-resistant acid phosphatase and serum type I collagen cross-linked C-terminal peptide. This experiment selected bone formation markers such as serum alkaline phosphatase, osteocalcin, and type I collagen N-terminal propeptide, and bone resorption markers such as serum tartrate-resistant acid phosphatase. The results show that XDGM significantly increases PINP, decreases BGP, ALP, and TRACP. It can be seen that XDGM can increase PINP, providing abundant organic raw materials for bone formation, while also reducing the high turnover state of bone metabolism.

[0304] At the molecular level, bone matrix mainly includes organic components (about 35%) and inorganic components (about 65%). The organic matrix is composed of collagen and glycoprotein, and the inorganic components mainly include hydroxyapatite, cations (calcium, magnesium, sodium, potassium and strontium) and anions (phosphorus and chloride). The calcium in the human body accounts for 1.5%-2% of the body weight, 99% of which is stored in bones and teeth, and the remaining 1% is in the blood, which is called blood calcium. When the ratio of blood calcium and phosphorus is 2:1, the absorption of calcium can be promoted, and when too much phosphorus-containing food is ingested, the ratio of calcium and phosphorus can be as high as 1:10 or even 1:20, at this time, phosphorus will drive calcium out of the body. When the blood calcium concentration is insufficient to meet normal physiological functions, the body mobilizes a number of corresponding measures. Bone is a calcium reservoir, and when the calcium concentration in the blood is high, calcium is stored in the bone, which is called osteogenesis; when the blood calcium concentration is low, calcium is dissolved from the bone to supplement the blood calcium deficiency, which is called osteolysis. These two effects are regulated by hormones. Therefore, when the blood calcium is too low, the body automatically secretes PTH, which is synthesized and secreted by the chief cells of the parathyroid gland. PTH can stimulate the synthesis of vitamin D, which is a promoter of osteolysis, and can dissolve calcium in the bone into the blood. Vitamin D can also promote the absorption of calcium in the intestine and the reabsorption of calcium in the urine, which all increase the concentration of blood calcium. PTH has two effects on bone, one is to enhance the activity of osteoclasts, promote bone resorption, and release bone calcium into the blood; the other is to increase the number of osteoblasts while enhancing the activity of osteoclasts, promote the release of bone growth factors by osteoblasts, and promote bone formation and increase bone mass. Intermittent PTH injection has been widely confirmed to stimulate bone formation. When the blood calcium concentration is too high, on the one hand, the calcium excreted in the urine through the kidneys increases, and on the other hand, calcium is guided to the bone to deposit, which is the aforementioned osteogenesis. CT is a peptide hormone synthesized and secreted by the parafollicular cells of the thyroid gland, which can reduce the concentration of calcium and phosphorus in the blood and inhibit the absorption of calcium and phosphorus. It is currently widely used in the treatment of osteoporosis, and its main mechanism is to significantly inhibit the activity of osteoclasts and reduce bone resorption. The experimental results show that XDGM can increase the content of blood Ca and P, provide abundant inorganic material for bone formation. At the same time, XDGM can stimulate the secretion of CT and PTH, promote bone formation, and inhibit bone resorption.

[0305] Second, the effect of the active component of the pharmaceutical composition of the present application on tretinoin-induced osteoporosis in rats

[0306] Experimental purpose

[0307] The effect of the active component of the pharmaceutical composition of the present application on tretinoin-induced osteoporosis in rats was observed, and the therapeutic effect on osteoporosis was determined.

[0308] 1. Experimental Materials

[0309] 1.1 Test sample

[0310] 1.1.1 Name: Active component of the pharmaceutical composition of the present invention, abbreviation: XDGM, test item number: TN-1318.

[0311] 1.1.2 Appearance: Brownish-brown powder.

[0312] 1.1.3 Indications: Osteoporosis in menopausal women.

[0313] 1.1.4 Proposed clinical dosage: Oral administration, 14.1g crude drug / day.

[0314] 1.1.5 Content and Specifications: 2.96g crude drug / g dry extract powder.

[0315] 1.1.6 Source and batch number: Provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd., batch number: 20130301.

[0316] 1.1.7 Storage conditions: Store in a sealed container.

[0317] 1.1.8 Validity period: 2 years.

[0318] 1.2 Positive testing agents, tool reagents, and main reagents

[0319] 1.2.1 Xianling Gubao Capsules: Guizhou Tongjitang Pharmaceutical Co., Ltd., batch number: 1310079.

[0320] 1.2.2 Retinoic acid: Meilun Biotechnology, batch number: A0304A.

[0321] 1.2.3 Tetracycline hydrochloride: SIGMA-ALDRICH .Co., batch number: SLBH4536V.

[0322] 1.2.4 Picric acid: Taishan Chemical Plant Co., Ltd., batch number: 20071201.

[0323] 1.2.5 Sodium carboxymethyl cellulose (CMC-Na): Tianjin Yongda Chemical Reagent Development Center, batch number: 20080702.

[0324] 1.2.6 Chloral hydrate: Tianjin Guangfu Fine Chemical Research Institute, batch number: 20131114.

[0325] 1.3 Experimental System

[0326] 1.3.1 Animal strain: SD rat.

[0327] 1.3.2 Animal grade: SPF grade.

[0328] 1.3.3 Animal gender and number: 72 female animals.

[0329] 1.3.4 Animal age: 63~69 days.

[0330] 1.3.5 Animal weight: 200~220g.

[0331] 1.3.6 Animal source: purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., certificate number: 11400700047563, license number: SCXK (Jing) 2012-0001, received on May 21, 2014.

[0332] 1.3.7 Feeding conditions: The animals were bred in the New Drug Evaluation Center of Hebei Province of Integrative Medicine Research Institute. The rats were kept in cages with 12 hours of light per day, a temperature of 20~26℃, and a relative humidity of 40~70%.

[0333] 1.3.8 Quarantine process: The new animals were quarantined for 3 days. Since suitable animals of the required age could not be purchased, the newly purchased animals were fed for 4 weeks before starting the experiment. During this period, the animals had normal drinking and eating, were in good health, and showed no signs of disease or death.

[0334] 1.3.9 Feed: The experimental animals were fed with full-value granular feed provided by the Experimental Animal Center of the Chinese People's Liberation Army Military Medical Sciences Academy, with a certificate number of 0025539.

[0335] 1.3.10 Drinking water: The animals were given free access to ordinary water, which was replaced once a day.

[0336] 1.3.11 Bedding: The experimental animals were provided with ordinary-grade bedding provided by the Hebei Province Experimental Animal Center, with a production license number of SCXK (Ji) 2013-2-001.

[0337] 1.3.12 Identification: 5% picric acid was used for marking.

[0338] 2 Experimental methods

[0339] 2.1 Experimental design basis

[0340] 2.1.1 Standards used: The Guidelines for New Drug Research of Traditional Chinese Medicine (Pharmacy, Pharmacology, Toxicology) issued by the Drug Administration Bureau of the Ministry of Public Health of the People's Republic of China, the Preclinical Research Guidelines for New Drugs (Western Medicine) Compilation (Pharmacy, Pharmacology, Toxicology), the Pharmacological Research Methodology of Traditional Chinese Medicine and the Pharmacological Experimental Methodology published by the People's Health Publishing House, and relevant literature.

[0341] 2.1.2 Experimental system selection: Retinoic acid is a synthetic derivative of vitamin A, mainly used for the treatment of tumors and skin diseases, but has a significant impact on bone quality, with the adverse reaction of osteoporosis. Because of the typicality of retinoic acid-induced osteoporosis model in the changes of bone tissue microstructure and the reversibility after drug action, the model has been listed by the Ministry of Health as one of the standard models for evaluating the efficacy of osteoporosis drugs.

[0342] 2.2 Dose and grouping

[0343] The animals were randomly divided into 6 groups according to body weight, 12 in each group, namely normal group, model group, positive drug (Xianling Gubao) group, XDGM 1, 2, 6 group. The quasi-clinical dose of XDGM is 14.1g of crude drug per day, according to 60kg per person, the rat is 8 times the human clinical dose, that is, 1.88g of crude drug / kg, the content is 2.96g of crude drug / g of dry extract powder, that is, 0.635 dry extract powder / kg. According to the instructions of Xianling Gubao, the rat was given a dose of 0.40g / kg, which was 8 times the human clinical dose. See Appendix 21.

[0344]

[0345] 2.3 Method of administration

[0346] Gavage, 10ml / kg body weight, consistent with the recommended oral route in clinical.

[0347] 2.4 Preparation and storage of test products

[0348] The test drug was prepared with 0.5% CMC-Na into the experimental concentration (see Appendix 21), and stored at 2~8℃ after preparation for standby use, and the positive drug was prepared immediately before use.

[0349] 2.5 Administration of test products

[0350] After 4 weeks of modeling, the test drug was given according to the experimental grouping, the normal group and the model group were given 0.5% CMC-Na, 1 time / day, for 15 days.

[0351] 2.6 Experimental procedures

[0352] Newly received SD rat number, quarantine for 3 days, feed for 4 weeks, and then randomly group according to body weight, as described previously. After grouping, give retinoic acid 70 mg / kg modeling, volume is 10 ml / kg body weight, gavage for 4 weeks, 6 days a week, the normal group is given solvent 0.5% CMC-Na, after modeling, the next day, gavage the test drug, once a day, for 15 days, the normal group and the model group are given 0.5% CMC-Na, record body weight once a week during modeling and giving the test drug, all rats are injected with tetracycline hydrochloride 30 mg / kg intraperitoneally for bone fluorescence labeling on the 15th day and the 3rd day before being sacrificed, after the end of the drug administration period, the 2nd day, use 10% chloral hydrate for intraperitoneal anesthesia, volume is 0.35 ml / 100 g body weight, abdominal aortic blood sampling for sacrifice, and detect the indexes.

[0353] 2.7 Detection index

[0354] 2.7.1 Detection of bone tissue morphological index

[0355] Take the proximal 1 / 3 of the left tibia of the rat, fix it in 4% paraformaldehyde solution (PH 7.4) for 24 h, and then dehydrate, the procedure is 80% ethanol for 2 days, 95% ethanol for 2 days, 100% ethanol for 2 days, and xylene for 2 days. Then, the specimen is sequentially immersed in plastic polymerization liquid I, II, and III for 3 days each, and the above fixation, dehydration, and immersion processes are all carried out at 4°C. Finally, add 400 μL of N, N-dimethyl-p-toluidine to 100 ml of pre-cooled (4°C) III liquid, stir with a magnetic stirrer for 10 min, then inject about 7 ml of III liquid into a penicillin vial, place the bone specimen in the same direction at the bottom of the vial, use a syringe to evacuate the air in the embedding vial, and then place it in a-20°C freezer for polymerization for about 1 week, which can then be changed into a colorless transparent hard embedding block. After trimming, use a tungsten steel knife on a Reicheit-Jung 2040 microtome to cut 5 μm longitudinal non-decalcified bone sections from each bone tissue, 2 sections each, one for toluidine blue staining and the other for fluorescence observation.

[0356] Bone histomorphometry method: use Qwin Pro V3.5.0 image analysis system to perform morphometry on non-decalcified bone sections:

[0357] (1) Bone trabecular tissue morphometry:

[0358] Trabecular bone volume percentage (TBV%): the percentage of trabecular bone volume to the total volume of the measured bone marrow cavity, which is the main indicator for measuring bone mass level;

[0359] Trabecular bone resorption surface percentage (TRS%): the percentage of irregular, uneven trabecular surface to the trabecular surface, which can determine the activity of osteoclasts;

[0360] Trabecular bone formation surface percentage (TFS%): the percentage of osteoblast-covered osteoid surface to the trabecular surface, which can determine the activity of osteoblasts;

[0361] Trabecular bone mineralization rate (MAR): the average distance of the fluorescence double-labeling band on the trabecular surface divided by the number of days between the two labels.

[0362] (2) Intracortical surface morphometry:

[0363] Osteoid average width (OSW): the average width of the osteoid covered by osteoblasts on the intracortical surface;

[0364] Cortical bone mineralization rate (mAR): the average distance of the fluorescence double-labeling band on the intracortical surface divided by the number of days between the two labels.

[0365] 2.7.2 Detection of bone biomechanics indicators

[0366] Before detection, the frozen right femur of the rat was taken out from the refrigerator and warmed at room temperature. The three-point bending test was performed on the rat femur: the femur was placed on the microcomputer-controlled electronic universal testing machine support with a span of 16 mm, and the middle segment of the femur was pressed at a loading speed of 1 mm / min until the femur was broken, and the maximum load (N), bending strength (MPa) and elastic modulus (GPa) were detected.

[0367] 2.8 Notification of relevant staff

[0368] When the animal appears abnormal, notify the pathology room for processing.

[0369] 2.9 Main instrument system

[0370] BT224S Sartorius precision analytical balance Sartorius Scientific Instrument Co., Ltd.

[0371] DT-2000 electronic balance Changshu Shuangjie Testing Instrument Factory

[0372] Microcomputer-controlled electronic universal testing machine Shenzhen Regal Instrument Co., Ltd.

[0373] Qwin Pro V3.5.0 image analysis system Leica Company, Germany

[0374] 2040 microtome Reicheit-Jung Company, Germany

[0375] 2.10 Statistical method

[0376] The experimental data were analyzed by SPSS statistical software, and the statistical results were expressed as mean ± standard deviation (x±s). The mean comparison was performed by One-Way ANOVA, and the pairwise comparison was performed by the least significant difference (LSD).

[0377] 3 Results

[0378] 3.1 XDGM significantly improved the bone morphological characteristics of rats with retinoic acid-induced osteoporosis.

[0379] As shown in Table 22, the bone morphological index showed that compared with the normal group, the TBV% of the tibia of the model group rats was significantly reduced (P<0.01), the TRS%, TFS%, and MAR were increased, but no significant difference was found (P>0.05), indicating that the model group animals showed a certain degree of high transformation, and the mAR was significantly reduced (P<0.05), and the OSW was reduced, indicating that the model had an impact on bone cortical mineralization; compared with the model group, the positive drug Xianlinggubao significantly increased the TBV% of the tibia of the rats (P<0.05), and the XDGM 1 and 2 groups significantly increased the TBV% of the tibia of the rats (P<0.05 or P<0.01), and the TRS%, TFS%, and MAR were reduced, but no significant difference was found (P>0.05). As shown in Table 22, the bone morphological index showed that compared with the normal group, the TBV% of the tibia of the model group rats was significantly reduced (P<0.01), the TRS%, TFS%, and MAR were increased, but no significant difference was found (P>0.05), indicating that the model group animals showed a certain degree of high transformation, and the mAR was significantly reduced (P<0.05), and the OSW was reduced, indicating that the model had an impact on bone cortical mineralization; compared with the model group, the positive drug Xianlinggubao significantly increased the TBV% of the tibia of the rats (P<0.05), and the XDGM 1 and 2 groups significantly increased the TBV% of the tibia of the rats (P<0.05 or P<0.01), and the TRS%, TFS%, and MAR were reduced, but no significant difference was found (P>0.05). As shown in Table 22, the bone morphological index showed that compared with the normal group, the TBV% of the tibia of the model group rats was significantly reduced (P<0.01), the TRS%, TFS%, and MAR were increased, but no significant difference was found (P>0.05), indicating that the model group animals showed a certain degree of high transformation, and the mAR was significantly reduced (P<0.05), and the OSW was reduced, indicating that the model had an impact on bone cortical mineralization; compared with the model group, the positive drug Xianlinggubao significantly increased the TBV% of the tibia of the rats (P<0.05), and the XDGM 1 and 2 groups significantly increased the TBV% of the tibia of the rats (P<0.05 or P<0.01), and the TRS%, TFS%, and MAR were reduced, but no significant difference was found (P>0.05). Figure 2 As shown in Table 22, the bone morphological index showed that compared with the normal group, the TBV% of the tibia of the model group rats was significantly reduced (P<0.01), the TRS%, TFS%, and MAR were increased, but no significant difference was found (P>0.05), indicating that the model group animals showed a certain degree of high transformation, and the mAR was significantly reduced (P<0.05), and the OSW was reduced, indicating that the model had an impact on bone cortical mineralization; compared with the model group, the positive drug Xianlinggubao significantly increased the TBV% of the tibia of the rats (P<0.05), and the XDGM 1 and 2 groups significantly increased the TBV% of the tibia of the rats (P<0.05 or P<0.01), and the TRS%, TFS%, and MAR were reduced, but no significant difference was found (P>0.05).

[0380]

[0381] 3.2 XDGM significantly improved the bone biomechanical properties of rats with retinoic acid-induced osteoporosis.

[0382] As shown in Table 23, the bone biomechanical index showed that compared with the normal group, the maximum load, bending strength, and elastic modulus of the femur of the model group rats were significantly reduced (P<0.01); compared with the model group, the positive drug Xianlinggubao significantly increased the maximum load, bending strength, and elastic modulus of the femur of the rats (P<0.01 or P<0.05), the XDGM 1 and 2 groups significantly increased the maximum load and bending strength of the femur of the rats (P<0.05 or P<0.01), and the XDGM 1 group also significantly increased the elastic modulus of the femur of the rats (P<0.01), indicating that XDGM could significantly improve the bone biomechanical properties of rats with osteoporosis.

[0383]

[0384] 4 Conclusion

[0385] The results of the experiment show that the XDGM significantly increases the TBV% of the tibia of the rats, significantly increases the maximum load, bending strength and elastic modulus of the femur, and the XDGM has obvious improvement effects on the bone morphology and bone biomechanical characteristics of the rats with the osteoporosis caused by retinoic acid.

[0386] 5Abnormal situation

[0387] When the rats are intragastrically administered with the retinoic acid for 4 weeks, one animal in each of the XDGM 1 and XDGM 2 groups has a fracture, which affects the feeding, and is thus killed, and it is found through dissection that the tibia is fractured.

[0388] Third, the influence of the active component of the pharmaceutical composition on the prevention and treatment of the osteoporosis of the rats caused by retinoic acid

[0389] Experimental purpose

[0390] The osteoporosis model of the rats is caused by retinoic acid, the influence of the active component of the pharmaceutical composition on the osteoporosis of the rats caused by retinoic acid is observed, and the preventive effect of the active component on the osteoporosis is determined.

[0391] 1Experimental materials

[0392] 1.1 Test product

[0393] 1.1.1 Name: the active component of the pharmaceutical composition of the application, abbreviation: XDGM, test product number: TN-1318.

[0394] 1.1.2 Appearance: brownish powder.

[0395] 1.1.3 Function and indication: menopausal osteoporosis.

[0396] 1.1.4 Approximate clinical dosage: oral administration, 14.1 g of crude drug per day.

[0397] 1.1.5 Content and specification: 2.96 g of crude drug per g of dry paste powder.

[0398] 1.1.6 Source and batch number: provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd., batch number: 20130301.

[0399] 1.1.7 Storage condition: stored in a sealed manner.

[0400] 1.1.8 Validity period: 2 years.

[0401] 1.2 Positive drug, tool drug and main reagent

[0402] 1.2.1 Xianling Gubao capsules: Guizhou Tongjitang Pharmaceutical Co., Ltd., batch number: 1406020.

[0403] 1.2.2 Retinoic acid: Meilun Biological Technology Co., Ltd., batch number: A0304A.

[0404] 1.2.3 Tetracycline hydrochloride: SIGMA-ALDRICH Co., batch number: SLBH4536V.

[0405] 1.2.4 Picric acid: Taishan Chemical Factory Co., Ltd., batch number: 20071201.

[0406] 1.2.5 Sodium carboxymethylcellulose (CMC-Na): Tianjin Yongda Chemical Reagent Development Center, batch number: 20080702.

[0407] 1.2.6 Chloral hydrate: Tianjin Guangfu Fine Chemical Institute, batch number: 20131114.

[0408] 1.3 Experimental system

[0409] 1.3.1 Animal strain: SD rats.

[0410] 1.3.2 Animal level: clean level.

[0411] 1.3.3 Animal gender and number: female, of which 60 were used for formal experiment and 5 were for practice.

[0412] 1.3.4 Animal age: 3 months old.

[0413] 1.3.5 Animal weight: 220~260g.

[0414] 1.3.6 Animal source: purchased from Hebei Province Experimental Animal Center, certificate number: 1501078, license number: SCXK (Ji) 2013-1-003, received on January 23, 2015.

[0415] 1.3.7 Feeding conditions: the animals were bred in the New Drug Evaluation Center of Hebei Province of Integrative Medicine Research Institute. The rats were kept in cages, with 12 hours of light per day, temperature of 20~26℃, and relative humidity of 40~70%.

[0416] 1.3.8 Quarantine process: the quarantine period for new animals was 4 days, during which the animals had normal drinking and eating, good health, and no signs of disease and death.

[0417] 1.3.9 Feed: full-value granular feed for experimental animals, provided by the Experimental Animal Center of the Chinese People's Liberation Army Military Medical Sciences Academy, certificate number: 0025539.

[0418] 1.3.10 Drinking water: ordinary water was filled for the animals to drink freely, and the drinking bottles were washed and changed once a day.

[0419] 1.3.11 Bedding: general grade bedding for laboratory animals, provided by Hebei Province Laboratory Animal Center, production license: SCXK (Hebei) 2013-2-001.

[0420] 1.3.12 Identification: marked with 5% picric acid.

[0421] 2 Experimental Methods

[0422] 2.1 Basis for Experimental Design

[0423] 2.1.1 Standards Adopted: The Research Guidelines for New Drugs of Traditional Chinese Medicine (Pharmacy, Pharmacology, Toxicology) issued by the Drug Administration Bureau of the Ministry of Public Health of the People's Republic of China, The Preclinical Research Guidelines for New Drugs (Western Medicine) Compilation (Pharmacy, Pharmacology, Toxicology), The Research Methodology of Traditional Chinese Medicine Pharmacology, The Methodology of Pharmacological Experiments, and related literature.

[0424] 2.1.2 Explanation of Experimental System Selection: Retinoic acid is a synthetic derivative of vitamin A, mainly used for the treatment of tumors and skin diseases, but has a significant impact on bone mass, with the adverse reaction of causing osteoporosis. Due to the typicality of retinoic acid-induced osteoporosis model in the changes of bone tissue microstructure and the reversibility after drug action, this model has been listed by the Ministry of Public Health as one of the standard models for evaluating the efficacy of osteoporosis drugs.

[0425] 2.2 Dose and Grouping

[0426] Animals were randomly divided into 6 groups according to body weight, 10 in each group, namely normal group, model group, positive drug (Xianling Gubao) group, XDGM1, 2, 6 groups. The quasi-clinical dose of XDGM was 14.1g of crude drug per day, calculated according to 60kg per person, the dose for rats was 8 times the human clinical dose, i.e. 1.88g of crude drug per kg, the content was 2.96g of crude drug per g of dry extract powder, i.e. 0.635g of dry extract powder per kg. Referring to the instructions of Xianling Gubao, the dose for rats was set to 0.40g / kg, which was 8 times the human clinical dose. See Appendix 24.

[0427]

[0428] 2.3 Method of Administration

[0429] Gavage, 10ml / kg body weight, consistent with the recommended oral route in clinical practice.

[0430] 2.4 Preparation and Storage of Test Products

[0431] The test drug was prepared with 0.5% CMC-Na to the experimental concentration (see Appendix 24), and stored at 2-8°C after preparation for future use. The positive drug was prepared immediately before use.

[0432] 2.5 Administration of test article

[0433] The test drug was administered by oral gavage according to the experimental grouping. The normal group and the model group were administered 0.5% CMC-Na, 1 time / day, for 2 weeks.

[0434] 2.6 Experimental procedure

[0435] The newly received SD rats were randomly grouped by weight after quarantine for 4 days, as previously described. In addition to the normal group, all groups were administered 70 mg / kg of tretinoin in the morning, with a volume of 10 ml / kg of body weight, and the normal group was administered 0.5% CMC-Na. In the afternoon, the drug administration group was administered the corresponding test drug, and the normal group and the model group were administered 0.5% CMC-Na. Oral gavage was performed continuously for 2 weeks, and body weight was recorded once a week. All rats were intraperitoneally injected with tetracycline hydrochloride at 30 mg / kg on the 14th day and the 3rd day before sacrifice for bone fluorescence labeling. After the end of the administration period, on the 2nd day, the rats were anesthetized with 10% chloral hydrate intraperitoneally, with a volume of 0.35 ml / 100 g of body weight, and blood was collected from the abdominal aorta for sacrifice. The detection indicators were measured.

[0436] 2.7 Detection indicators

[0437] 2.7.1 Detection of bone histomorphometric indicators

[0438] The proximal 1 / 3 of the left tibia of the rats was taken and fixed in 4% paraformaldehyde solution (PH 7.4) for 24 h, followed by dehydration, with the following procedure: 80% ethanol for 2 days, 95% ethanol for 2 days, 100% ethanol for 2 days, and xylene for 2 days. Then, the specimens were sequentially immersed in plastic polymerization solution I, II, and III for 3 days each. The above fixation, dehydration, and immersion processes were all performed at 4°C. Finally, 400 μL of N, N-dimethyl-p-toluidine was added to 100 ml of pre-cooled (4°C) III solution, and stirred with a magnetic stirrer for 10 min. About 7 ml of III solution was injected into a penicillin vial, and the bone specimens were placed in the vial with the same orientation. Air was removed from the embedding vial using a syringe, and then the vial was placed in a -20°C freezer for polymerization for about 1 week, after which it became a colorless, transparent, and hard embedding block. After trimming, 5 μm longitudinal undecalcified bone sections were cut from each bone tissue using a tungsten steel knife on a Reicheit-Jung 2040 microtome, with 2 sections per bone tissue. One section was used for toluidine blue staining, and the other was used for fluorescence observation.

[0439] Bone histomorphometry method: Qwin Pro V3.5.0 image analysis system was used for morphometric analysis of undecalcified bone sections:

[0440] (1) Bone trabecular histomorphometry:

[0441] Percentage of trabecular bone volume (TBV%): the percentage of trabecular bone volume in the total volume of the measured bone marrow cavity, which is the main indicator of bone mass level;

[0442] Percentage of trabecular bone resorption surface (TRS%): the percentage of irregular and uneven trabecular surface in the trabecular surface, which can judge the activity of osteoclasts;

[0443] Percentage of trabecular bone formation surface (TFS%): the percentage of osteoid surface covered by osteoblasts in the trabecular surface, which can judge the activity of osteoblasts;

[0444] Trabecular bone mineralization rate (MAR): the average distance of the fluorescence double-labeling band on the trabecular surface divided by the number of days between the two labels.

[0445] (2) Cortical inner surface morphometry:

[0446] Average width of osteoid (OSW): the average width of osteoid covered by osteoblasts on the inner surface of the cortex;

[0447] Mineralization rate of bone cortex (mAR): the average distance of the fluorescence double-labeling band on the inner surface of the cortex divided by the number of days between the two labels.

[0448] 2.7.2 Detection of bone biomechanics indicators

[0449] Before detection, take the frozen right femur of the rat from the refrigerator and warm it at room temperature. Perform three-point bending test on the rat femur: place the femur on the microcomputer-controlled electronic universal testing machine support, with a span of 16mm, and press the middle section of the femur at a loading speed of 1mm / min until the femur is broken, and detect the maximum load (N), bending strength (MPa) and elastic modulus (GPa).

[0450] 2.8 Notification of relevant staff

[0451] Notify the animal room when purchasing animals, and notify the pathology room for processing when the animals show abnormal conditions.

[0452] 2.9 Main instrument system

[0453] BT224S Sartorius precision analytical balance Sartorius Scientific Instruments Co., Ltd.

[0454] DT-2000 electronic balance Changshu Shuangjie Testing Instrument Factory

[0455] Microcomputer-controlled electronic universal testing machine Shenzhen Regal Instruments Co., Ltd.

[0456] Qwin Pro V3.5.0 Image Analysis System, Leica GmbH (Germany)

[0457] 2040 slicer, Reicheit-Jung GmbH, Germany

[0458] 2.10 Statistical Methods

[0459] Experimental data were analyzed using SPSS statistical software, and the statistical results are expressed as mean ± standard deviation. The results indicate that one-way ANOVA was used for comparing means, and the least significant difference (LSD) method was used for pairwise comparisons.

[0460] 3 Results

[0461] 3.1XDGM significantly improved the bone morphological characteristics of retinoic acid-induced osteoporosis rats.

[0462] As shown in Appendix Table 25, bone morphology parameters revealed that, compared with the normal group, the tibial TBV% of rats in the model group was significantly decreased (P<0.01), TRS% was significantly increased (P<0.01), and TFS%, OSW, and MAR were slightly increased, but without significant differences (P>0.05). This indicates that the model group animals exhibited a certain degree of high bone turnover and stronger bone resorption. Compared with the model group, the positive control drug Xianling Gubao significantly increased the tibial TBV% of rats (P<0.05) and decreased TRS% (P<0.01). XDGM1 and XDGM2 groups significantly increased the tibial TBV% of rats (P<0.05 or P<0.01) and decreased TRS% (P<0.05 or P<0.01). XDGM1 group significantly decreased MAR (P<0.05), while other parameters showed no significant differences (P>0.05). Figure 3 It is evident that the number and volume of trabeculae in the proximal tibia of rats in the XDGM group were increased compared with those in the model group. This indicates that XDGM significantly reduced the intensity of bone resorption in osteoporotic rats, improved the high-transformation state to a certain extent, and improved the bone morphological characteristics of osteoporotic rats.

[0463]

[0464] 3.2 XDGM significantly improved the bone biomechanical properties of retinoic acid-induced osteoporotic rats.

[0465] As shown in Table 26, the bone biomechanics index showed that compared with the normal group, the maximum load, bending strength and elastic modulus of the femur of the model group rats were significantly reduced (P<0.01); compared with the model group, the positive drug Xianling Gubao significantly increased the maximum load, bending strength and elastic modulus of the femur of the rats (P<0.05), and the medium and high doses of XDGM significantly increased the maximum load, bending strength and elastic modulus of the femur of the rats (P<0.05 or P<0.01), which showed that XDGM could significantly improve the bone biomechanics characteristics of osteoporotic rats.

[0466]

[0467] 4 Conclusion

[0468] The experimental results showed that XDGM significantly increased the TBV% of the tibia of rats, and increased the maximum load, bending strength and elastic modulus of the femur, which showed that XDGM had obvious improvement effect on the bone morphology and bone biomechanics characteristics of rats with osteoporosis caused by retinoic acid.

[0469] 5 Discussion

[0470] Retinoic acid is a synthetic derivative of vitamin A, mainly used for the treatment of tumors and skin diseases, but has obvious effect on bone quality and has the adverse reaction of causing osteoporosis. The animal model of retinoic acid-induced osteoporosis in rats was first created by Chinese scholar Shao Jinying in 1989, and this model has been listed by the Ministry of Health as one of the standard models for evaluating the therapeutic effect of osteoporosis drugs. In this experiment, retinoic acid was used to induce osteoporosis in rats for 2 weeks, aiming to simulate a high turnover type of osteoporosis animal model.

[0471] Bone histomorphometry is a technique that can quantitatively observe and study the morphology and structure of bone tissue. At present, this technique has become one of the important scientific research methods for quantitatively detecting the effects of drugs on animal osteoporosis models, exploring the mechanisms of drugs, and providing theoretical guidance for clinical practice. Bone histomorphometry has different classification methods. According to the different sampling sites, it can be divided into tibia, femur, lumbar spine, etc. According to the different observation sites, it can be divided into cortical bone, cancellous bone, femoral neck, etc. According to the different data, it can be divided into static parameters and dynamic parameters, etc. Cancellous bone is a small trabecular bone located on the inner surface of cortical bone. In the upper segment of the rat tibia, the epiphysis of three-month-old rats is not closed until 12 months of age. Due to rich blood supply and nutrition, active metabolic function, strong bone regeneration ability, and high bone turnover rate, drugs can directly reach the local site. Therefore, the upper segment of the rat tibia is a sensitive site for inducing osteoporosis models and observing the effects of drugs, and it is also the most ideal site for various osteoporosis models and drug prevention and treatment research. In this experiment, three-month-old female animals were used for osteoporosis research. The results showed that the TBV% of the model group rats was significantly decreased, the TRS% was significantly increased, and the XDGM was significantly increased. The TBV% of the rat tibia was decreased, the TRS% and MAR were decreased. It can be seen that XDGM significantly reduces bone resorption intensity in osteoporotic rats, improves high transformation to a certain extent, and improves the bone morphological characteristics of osteoporotic rats. Bone biomechanics is based on the theory of engineering mechanics to study the mechanical properties of bone tissue under external action and the biological effects of bone after being stressed. It is a reliable method for evaluating bone quality. The study of bone biomechanics can be generally investigated from two aspects: structural mechanics and material mechanics. Structural mechanics mainly includes maximum load, fracture load, elastic load, and other indicators. Material mechanics mainly includes fracture strain, elastic modulus, etc. The performance of structural mechanics is mainly related to the size and geometry of the bone, while the material mechanics is mainly related to the strength and toughness of the bone, which is related to the microstructure of the bone, the mineral content in the bone, and the bone density. The results of this experiment showed that the maximum load, bending strength, and elastic modulus of the femur of the model group rats were significantly decreased, and the above three indicators of the XDGM group were significantly increased. It can be seen that XDGM can significantly improve the bone biomechanical properties of osteoporotic rats.

[0472] Conclusion

[0473] The results of the ovariectomy-induced osteoporosis model in rats show that the XDGM 1 and 2 groups significantly increase the percentage of tibial trabecular bone volume, reduce the high turnover bone metabolism state of the ovariectomized rats, improve the bone morphological characteristics of the osteoporotic rats, increase the maximum load, bending strength and elastic modulus of the femur, improve the bone biomechanical characteristics of the osteoporotic rats, increase the bone mineral density of the osteoporotic rats, and increase the serum Ca, P, PINP content and CT, PTH level, provide rich raw materials and hormone stimulation for bone formation, improve the osteoporosis state, and make the bone formation greater than the bone resorption. In addition, the results of the XDGM in preventing and treating the retinoic acid-induced osteoporosis model show that the XDGM 1 and 2 groups significantly increase the percentage of tibial trabecular bone volume, significantly increase the maximum load, bending strength and elastic modulus of the femur, and have a significant improvement effect on the bone morphological and bone biomechanical characteristics of the osteoporotic rats.

[0474] In combination with the above results, the XDGM reduces the high turnover bone metabolism state, improves the bone morphological and bone biomechanical characteristics, and increases the bone mineral density, which indicates that the XDGM can be used for the treatment of menopausal osteoporosis. DETAILED DESCRIPTION

[0475] Example 1

[0476] The crude drug formula is: Semen Eurycoma 380 g, Herba Epimedii 220 g, Radix Rehmanniae 200 g, Radix Salviae Miltiorrhizae 155 g, Oyster Shell Calcined 55 g, and Salted Fructus Psoraleae 155 g.

[0477] A. The Semen Eurycoma and Fructus Psoraleae are weighed according to the prescription amount, extracted twice with 70% ethanol, each time for 2 hours, and the amount of added ethanol is 10 times the amount of the medicinal materials. The extraction liquid is filtered, combined, and concentrated under reduced pressure to a clear extract with a relative density of 1.10±0.05 at 60°C, which is ready for use;

[0478] B. The Herba Epimedii, Radix Rehmanniae and Radix Salviae Miltiorrhizae are weighed according to the prescription amount, extracted three times with 12 times the amount of water, and the extraction time is 1 hour. The extraction liquid is filtered, concentrated under reduced pressure to a clear extract with a relative density of 1.10±0.05 at 60°C, and the three concentrated liquids are combined. The combined concentrated liquid is combined with the alcohol extract clear extract obtained in step A, concentrated to a thick extract with a relative density of 1.20±0.05 at 60°C, dried, and pulverized to obtain a fine powder;

[0479] C. The Oyster Shell is pulverized into the finest powder and sterilized by 60Co irradiation;

[0480] The fine powder obtained by mixing step B and step C together constitutes the active component of the pharmaceutical composition of the present application.

[0481] Example 2

[0482] Raw material formula: Semen cuscutae 400g, Herba epimedii 200g, Radix rehmanniae 200g, Salvia miltiorrhiza 160g, calcined concha margaritiferae 53g, salted Psoralea corylifolia 160g.

[0483] A. Weigh Semen cuscutae and Psoralea corylifolia according to the prescription amount, crush Semen cuscutae first, extract twice with 60% ethanol, 1.5 hours each time, add 10 times of alcohol in the first time and 8 times of alcohol in the second time, filter the extract, combine, and concentrate under reduced pressure to the clear extract with the relative density of 1.10±0.05 at 60°C, for standby;

[0484] B. Weigh Herba epimedii, Radix rehmanniae, and Salvia miltiorrhiza according to the prescription amount, extract three times with water, add 12 times of water in the first time, extract for 2 hours; add 10 times of water in the second and third times, extract for 1 hour each time, filter the extract, concentrate under reduced pressure to the clear extract with the relative density of 1.10±0.05 at 60°C, combine the three concentrated extracts, combine with the alcohol extract obtained in step A, concentrate to the thick extract with the relative density of 1.20±0.05 at 60°C, dry and crush to obtain fine powder;

[0485] C. Crush concha margaritiferae into the finest powder, sterilize with 60Co;

[0486] D. Combine the concha margaritiferae powder obtained in step C and the fine powder obtained in step B, granulate according to the conventional process, integrate the granules, and press into tablets.

[0487] Example 3:

[0488] Semen cuscutae 600g, Herba epimedii 100g, Radix rehmanniae 300g, Salvia miltiorrhiza 80g, concha margaritiferae 80g, Psoralea corylifolia 80g.

[0489] A. Weigh Semen cuscutae and Psoralea corylifolia according to the prescription amount, crush Semen cuscutae first, extract twice with 50% ethanol, 1.5 hours each time, add 10 times of alcohol in the first time and 8 times of alcohol in the second time, filter the extract, combine, and concentrate under reduced pressure to the clear extract with the relative density of 1.10±0.05 at 60°C, for standby;

[0490] B. Weigh Herba epimedii, Radix rehmanniae, and Salvia miltiorrhiza according to the prescription amount, extract three times with 10 times of water, extract for 2 hours each time, filter the extract, concentrate under reduced pressure to the clear extract with the relative density of 1.10±0.05 at 60°C, combine the three concentrated extracts, combine with the alcohol extract obtained in step A, concentrate to the thick extract with the relative density of 1.20±0.05 at 60°C, dry and crush to obtain fine powder;

[0491] C. Crush concha margaritiferae into the finest powder, sterilize with 60Co;

[0492] D. Combine the concha margaritiferae powder obtained in step C and the fine powder obtained in step B, granulate according to the conventional process, integrate the granules, and fill into capsules.

[0493] Example 4:

[0494] Cuscuta 200g, Herba Epimedii 300g, Rehmanniae 100g, Salviae Miltiorhizae 240g, Concha Margaritiferae 25g, Psoraleae 240g.

[0495] A. Take Cuscuta and Psoraleae by prescription amount, Cuscuta is broken first, extract twice with 60% ethanol, 1.5 hours each time, 10 times of alcohol is added in the first time, 8 times of alcohol is added in the second time, filter the extract, combine, reduce pressure to concentrate to the clear extract of 1.10±0.05 of relative density at 60℃, reserve;

[0496] B. Take Herba Epimedii, Rehmanniae and Salviae Miltiorhizae by prescription amount, extract three times with 10-12 times of water, extract time is 1-2 hours, filter the extract, reduce pressure to concentrate to the clear extract of 1.10±0.05 of relative density at 60℃, combine the three times of concentrated liquid and the alcohol extract of step A, concentrate to the thick extract of 1.20±0.05 of relative density at 60℃, dry, crush to get fine powder;

[0497] C. Concha Margaritiferae is crushed to the finest powder, 60Co irradiation sterilization;

[0498] D. Combine the Concha Margaritiferae of step C and the fine powder of step B, granulate according to the conventional process, whole granulation, prepare to get granules.

[0499] Example 5:

[0500] Cuscuta 500g, Herba Epimedii 220g, Rehmanniae 220g, Salviae Miltiorhizae 180g, Concha Margaritiferae 60g, Psoraleae 180g.

[0501] A. Take Cuscuta and Psoraleae by prescription amount, Cuscuta is broken first, extract twice with 60% ethanol, 1.5 hours each time, 10 times of alcohol is added in the first time, 8 times of alcohol is added in the second time, filter the extract, combine, reduce pressure to concentrate to the clear extract of 1.10±0.05 of relative density at 60℃, reserve;

[0502] B. Take Herba Epimedii, Rehmanniae and Salviae Miltiorhizae by prescription amount, extract three times with 10-12 times of water, extract time is 1-2 hours, filter the extract, reduce pressure to concentrate to the clear extract of 1.10±0.05 of relative density at 60℃, combine the three times of concentrated liquid and the alcohol extract of step A, concentrate to the thick extract of 1.20±0.05 of relative density at 60℃;

[0503] C. Concha Margaritiferae is crushed to the finest powder, 60Co irradiation sterilization;

[0504] D. Combine the Concha Margaritiferae of step C and the thick extract of step B, prepare to get oral liquid according to the conventional process.

[0505] Example 6:

[0506] Cuscuta 330g, Herba Epimedii 180g, Rehmanniae 180g, Salviae Miltiorhizae 125g, Concha Margaritiferae 60g, Fructus Psoraleae 150g.

[0507] A, Cuscuta and Fructus Psoraleae were weighed according to the prescription, Cuscuta was first broken, and extracted twice with 60% ethanol, 1.5 hours each time, 10 times of alcohol was added in the first time, and 8 times of alcohol was added in the second time, the extract was filtered, combined, and concentrated under reduced pressure to the clear extract with the relative density of 1.10±0.05 at 60°C, for standby;

[0508] B, Herba Epimedii, Rehmanniae, and Salviae Miltiorhizae were weighed according to the prescription, and extracted with 10-12 times of water for three times, 1-2 hours each time, the extract was filtered, concentrated under reduced pressure to the clear extract with the relative density of 1.10±0.05 at 60°C, the three times of concentrated liquid was combined, and combined with the alcohol extract obtained in step A, and concentrated to the thick extract with the relative density of 1.20±0.05 at 60°C;

[0509] C, Concha Margaritiferae was crushed into the finest powder, and sterilized by 60Co irradiation;

[0510] D, the Concha Margaritiferae obtained in step C and the thick extract obtained in step B were prepared into injections according to the conventional process.

Claims

1. A pharmaceutical composition for preventing or treating primary osteoporosis, characterized by comprising a parathyroid hormone and a pharmaceutically acceptable carrier. The composition is made from the following components by weight: Semen cuscutae 200-600 parts, Herba epimedii 100-300 parts, Radix rehmanniae 100-300 parts, Salvia miltiorrhiza 80-240 parts, Concha mallei 25-80 parts, Fructus psoraleae 80-240 parts, wherein the Semen cuscutae is salted Semen cuscutae, the Concha mallei is calcined Concha mallei, and the Fructus psoraleae is salted Fructus psoraleae.

2. The composition of claim 1, wherein The composition is made from the following components by weight: Semen cuscutae 200 parts, Herba epimedii 300 parts, Radix rehmanniae 100 parts, Salvia miltiorrhiza 240 parts, Concha mallei 25 parts, Fructus psoraleae 240 parts.

3. The composition of claim 1, wherein The composition is made from the following components by weight: Semen cuscutae 600 parts, Herba epimedii 100 parts, Radix rehmanniae 300 parts, Salvia miltiorrhiza 80 parts, Concha mallei 80 parts, Fructus psoraleae 80 parts.

4. The composition of claim 1, wherein The composition is made from the following components by weight: Semen cuscutae 400 parts, Herba epimedii 200 parts, Radix rehmanniae 200 parts, Salvia miltiorrhiza 160 parts, Concha mallei 53 parts, Fructus psoraleae 160 parts.

5. The composition of claim 1, wherein The composition is made from the following components by weight: Semen cuscutae 380 parts, Herba epimedii 220 parts, Radix rehmanniae 200 parts, Salvia miltiorrhiza 155 parts, Concha mallei 55 parts, Fructus psoraleae 155 parts.

6. The composition according to any one of claims 1 to 5, characterized in that The preparation form of the composition is capsule, tablet, pill, oral liquid, granule, injection or powder.

7. The composition according to any one of claims 1 to 5, characterized in that The active component of the composition is made from the following steps: A. Semen cuscutae and Fructus psoraleae are weighed according to the prescription, and extracted twice with 50-70% ethanol for 1-3 hours each time, with the amount of ethanol being 6-10 times the amount of the medicinal materials; the extract is filtered, combined, and concentrated under reduced pressure to a clear extract with a relative density of 1.10±0.05 at 60°C, for standby use; B. Herba epimedii, Radix rehmanniae and Salvia miltiorrhiza are weighed according to the prescription, and extracted three times with 8-12 times the amount of water for 1-3 hours each time; the extract is filtered, concentrated under reduced pressure to a clear extract with a relative density of 1.10±0.05 at 60°C, and combined with the alcohol extract obtained in step A; the combined extract is concentrated to a thick extract with a relative density of 1.20±0.05 at 60°C, dried, and pulverized to obtain fine powder; C. Concha mallei is pulverized to the finest powder, and sterilized by 60Co irradiation; The fine powder obtained by mixing step B and step C together constitutes the active component of the pharmaceutical composition of the present application.

8. The composition of claim 7, wherein The preparation process of the tablet is as follows: A. Semen cuscutae and Fructus psoraleae are weighed according to the prescription, and extracted twice with 50-70% ethanol for 1-3 hours each time, with the amount of ethanol being 6-10 times the amount of the medicinal materials; the extract is filtered, combined, and concentrated under reduced pressure to a clear extract with a relative density of 1.10±0.05 at 60°C, for standby use; B. Herba epimedii, Radix rehmanniae and Salvia miltiorrhiza are weighed according to the prescription, and extracted three times with 8-12 times the amount of water for 1-3 hours each time; the extract is filtered, concentrated under reduced pressure to a clear extract with a relative density of 1.10±0.05 at 60°C, and combined with the alcohol extract obtained in step A; the combined extract is concentrated to a thick extract with a relative density of 1.20±0.05 at 60°C, dried, and pulverized to obtain fine powder; C. Concha mallei is pulverized to the finest powder, and sterilized by 60Co irradiation; D. The Concha mallei powder obtained in step C and the fine powder obtained in step B are combined, granulated according to the conventional process, and tabletted to obtain the tablet.

9. The composition of claim 8, wherein The preparation process of the tablet is as follows: A. Weigh the Cuscuta and Psoralea according to the prescription, crush the Cuscuta first, extract twice with 60% ethanol, 1.5 hours each time, add 10 times the amount of alcohol for the first time and 8 times the amount of alcohol for the second time, filter the extract, combine, and concentrate under reduced pressure to a clear extract with a relative density of 1.10±0.05 at 60°C, and reserve; B. Weigh the Epimedium, Rehmannia, and Salvia miltiorrhiza according to the prescription, extract three times with 10-12 times the amount of water, 1-2 hours each time, filter the extract, concentrate under reduced pressure to a clear extract with a relative density of 1.10±0.05 at 60°C, combine the three concentrated extracts, combine with the alcohol extract from step A, concentrate to a thick extract with a relative density of 1.20±0.05 at 60°C, dry and pulverize to obtain a fine powder; C. Pulverize the Ostrea into the finest powder, sterilize with 60Co irradiation; D. Combine the Ostrea powder from step C and the fine powder from step B, granulate according to the conventional process, sieve, and compress into tablets.

10. The use of the composition according to any one of claims 1-5 in the preparation of a drug for treating postmenopausal osteoporosis.

11. Use according to claim 10, characterized in that The drug has the effect of increasing the percentage of tibial trabecular bone volume, increasing the maximum load, bending strength, and elastic modulus of the femur, and improving the bone morphology and bone biomechanical properties.

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