WO2022247640A1

WO2022247640A1 - Pharmaceutical composition for preventing or treating primary osteoporosis and preparation method therefor

Info

Publication number: WO2022247640A1
Application number: PCT/CN2022/092321
Authority: WO
Inventors: 贾振华
Original assignee: 河北以岭医药研究院有限公司
Priority date: 2021-05-26
Filing date: 2022-05-11
Publication date: 2022-12-01
Also published as: CN115400177A

Abstract

A pharmaceutical composition for treating primary osteoporosis, which pharmaceutical composition is prepared from Cuscutae semen, Epimedii folium, Psoraleae fructus, Rehmanniae radix, Salviae miltiorrhizae radix et rhizoma and Ostreae concha, and has the effects of tonifying the kidneys, replenishing essence, nourishing yin and strengthening yang, and strengthening the tendons and bones. A method for preparing the composition comprises: preparing preparations such as capsules and tablets from the following materials: a clear paste extracted from Cuscutae semen and Psoraleae fructus with ethanol, a clear paste extracted from Epimedii folium, Rehmanniae radix and Salviae miltiorrhizae radix et rhizoma with water, and the finest powder obtained by means of crushing Ostreae concha.

Description

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

Cross References to Related Applications

This application claims the priority of the Chinese patent application with the application number 202110574568.4 and the invention title "a pharmaceutical composition for preventing or treating primary osteoporosis and its preparation method" submitted to the Chinese Patent Office on May 26, 2021 , the entire contents of these applications are incorporated by reference in this application.

technical field

The invention relates to a traditional Chinese medicine composition for preventing or treating primary osteoporosis and a preparation method thereof, which belongs to the field of application of Chinese herbal medicines and aims at primary osteoporosis, mostly postmenopausal osteoporosis patients with kidney essence deficiency It is the basic pathogenesis, which can effectively increase bone density, improve clinical symptoms and quality of life of patients, and at the same time, treat low back pain, soreness of waist and knees, difficulty in holding weight, cramps of lower limbs, dizziness, tinnitus, fatigue, night Systemic symptoms such as frequent urination also have significant curative effect, and the medicine is simple and effective, showing the characteristics and innovation of this prescription.

Background technique

Osteoporosis (OP) is a systemic bone disease characterized by low bone mass and damage to bone microarchitecture, resulting in increased bone fragility and susceptibility to fracture. In 2001, the National Institutes of Health (NIH) of the United States proposed that osteoporosis is a disease of the skeletal system characterized by decreased bone strength and increased risk of fracture. Bone strength reflects two main aspects of bones, namely bone mineral density and bone quality. Osteoporosis can occur in different sexes and ages, but it is more common in postmenopausal women and older men. Osteoporosis is divided into two categories: primary and secondary. Primary osteoporosis is divided into three types: postmenopausal osteoporosis (type I), senile osteoporosis (type II) and idiopathic osteoporosis (including juvenile type). Postmenopausal osteoporosis generally occurs in women within 5-10 years after menopause, and is the most common bone disease in menopause. Its onset is related to estrogen deficiency, genetic factors, menstrual cycle disorders, and early menopause; Osteoporosis generally refers to osteoporosis that occurs after the age of 70; secondary osteoporosis refers to osteoporosis caused by any disease and/or drug that affects bone metabolism, mainly occurs in adolescents, and the etiology is still unknown.

Osteoporosis is a degenerative disease whose risk increases with age. At present, there are about 200 million people in the world suffering from osteoporosis, and its incidence rate has jumped to the seventh among all kinds of common diseases in the world. With the extension of human life span and the arrival of aging society, osteoporosis has become the important health problems.

Osteoporotic fractures are one of the most serious consequences of osteoporosis. In osteoporosis, bone density and bone quality decrease, bone strength decreases, and fractures can occur after slight violence, so it is a fragility fracture. It is estimated that an osteoporotic fracture occurs every 3 seconds in the world, and the elderly population will have a higher risk of fracture and mortality due to factors such as poorer bone quality, more serious calcium and vitamin D deficiency, and easy falls . It is reported that the incidence rate of osteoporotic fractures in women in my country is 40%, which is much higher than the sum of the incidence rates of these major cancers compared with breast cancer, endometrial cancer and ovarian cancer. It can be seen that fracture is a serious consequence of osteoporosis, which can significantly increase the morbidity and mortality of patients, and the treatment and care of osteoporosis and osteoporotic fractures require huge manpower and material resources, and the cost is high. , causing heavy family, social and economic burdens. The incidence of osteoporosis is related to endocrine factors, nutritional factors, living habits and exercise load, immune factors and cytokines, and genetic factors.

At present, the prevention and treatment of osteoporosis covers three aspects: basic measures, drug intervention and rehabilitation treatment. The basic measures include lifestyle adjustments and basic supplements for bone health, namely calcium and vitamin D. At present, anti-osteoporosis drugs can be roughly divided into the following categories: (1) Bisphosphonates: bisphosphonates bind to bone hydroxyapatite with high affinity, and specifically bind to the bone surface where bone turnover is active On, inhibit the function of osteoclasts, thereby inhibiting 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) Estrogens: These drugs can inhibit bone turnover and prevent bone loss, including estrogen replacement therapy (ET) and estrogen and progesterone replacement therapy (EPT), which can prevent bone loss and reduce osteoporotic vertebral body, Risk of nonvertebral fractures. (4) Parathyroid hormone: It has the effect of promoting bone formation. (5) Selective estrogen receptor modulators: It is characterized by selectively acting on the target organs of estrogen, and after combining with different forms of estrogen receptors, different biological effects occur. For example, raloxifene binds to estrogen receptors on bones, exhibits estrogen-like activity, and inhibits bone resorption. (6) Strontium salt: Strontium is one of the essential trace elements for the human body and participates 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 tissues, blood, bones and teeth. (7) Active vitamin D and its analogues: including 1,25-dihydroxyvitamin D3 (calcitriol) and 1α-hydroxyvitamin D3 (α-calciferol). (8) Vitamin K2: Vitamin K2 is the coenzyme of γ-carboxylase and plays an important role in the formation of γ-carboxyglutamic acid. γ-Carboxyglutamic acid is necessary for the normal physiological function of osteocalcin, can promote bone formation, and has a certain effect on inhibiting bone resorption. Although the above-mentioned drugs can be applied to the prevention and treatment of osteoporosis, they also face corresponding barriers and application bottlenecks. For example, estrogen preparations can bind to receptors on osteoblasts, which can promote bone cells to secrete collagenase and release growth Factors and cytokines, etc., promote bone organic matter synthesis and bone reconstruction, and can also directly inhibit osteoclast resorption, but the application of estrogen is prone to side effects, mainly including postmenopausal vaginal bleeding, breast cancer, endometrial cancer, and cardiovascular accidents. And thromboembolism, etc., so it is rarely used alone. Combination of estrogen and progesterone, the addition of progesterone can counteract the endometrial proliferation effect of estrogen, and progesterone 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 progesterone on many other tissues including breast, uterus, cardiovascular system, and rare adverse reactions such as severe deep vein thrombosis. Oral bisphosphonates have upper gastrointestinal complications, in addition to toxic side effects including kidney, blood, liver and immunosuppression. Calcitonin is currently one of the first-choice drugs for the treatment of high-transition osteoporosis. It is suitable for those who forbid estrogen or who cannot tolerate estrogen or bone pain osteoporosis, but long-term use may cause "escape phenomenon""For patients using bone formation accelerators, some studies have shown that the application of fluoride can increase the BMD of the lumbar spine (bone density), but it cannot reduce vertebral fractures. Many clinical studies of the application of parathyroid hormone have shown that intermittent small-dose application It can promote bone formation and increase bone mass, while continuous high-dose application can promote bone resorption and cause bone loss. Vitamin K ₂ is an essential vitamin for carboxylation of bone calcium, but there are adverse reactions such as anemia, liver function damage, and erythrocytosis. To sum up, all the western medicines for anti-osteoporosis have different degrees of side effects, which limits the long-term use of these medicines for osteoporosis patients.

Traditional Chinese medicine has accumulated a wealth of theoretical and practical experience in the prevention and treatment of osteoporosis. According to syndrome differentiation and treatment, the overall regulation of viscera is used to dispatch prescriptions and medicines, and specific treatment methods are selected according to different clinical characteristics and conditions of patients, which can significantly promote the development of patients. Improvement of systemic symptoms, reduction of bone pain, improvement of patient's quality of life, and less side effects, easy to be accepted by patients, has obvious advantages in the treatment of osteoporosis.

Most of the Chinese patent medicines currently on the market for the treatment of osteoporosis are targeted at osteoporosis caused by blood stasis blocking collaterals, deficiency of kidney yang, and deficiency of liver and kidney. Treatment methods such as liver and kidney, most of which tend to warm and nourish the kidney yang, or tend to nourish yin and nourish the kidney, will inevitably cause the disadvantages of tonifying the kidney yang and tending to transform heat. Few applications.

Contents of the invention

The pharmaceutical composition of the present invention is a compound traditional Chinese medicine developed by applying the theory of traditional Chinese medicine to explore the pathogenesis and treatment of osteoporosis in combination with many years of clinical practice. It fully grasps the syndrome characteristics of osteoporosis with deficiency of kidney essence, and pays attention to nourishing the kidney in treatment. Replenishing essence, nourishing yin and supporting yang at the same time, the pharmacy, pharmacology and toxicology experiments carried out in the early stage have confirmed that the drug has the effects of treating osteoporosis, analgesia, anti-inflammation, and regulating immunity. The quality also shows good efficacy and is safe to take. It is planned to further evaluate the clinical effectiveness and safety of the drug on the basis of the above research, and provide safe and effective traditional Chinese medicine preparations for osteoporosis patients.

The pharmaceutical composition of the present invention belongs to the traditional Chinese medicine preparation, which is composed of salt dodder, epimedium, rehmannia glutinosa, salvia miltiorrhiza, calcined oyster, salt psoralen, etc., has the effect of "tonifying kidney and essence, nourishing yin and supporting yang", and is suitable for primary hair Osteoporosis (kidney essence deficiency type). Compared with the same kind of Chinese patent medicine products, the pharmaceutical composition of the present invention is innovative in the principle of cube and the composition of prescriptions. It is different from previous studies that mostly used drugs for warming and tonifying kidney yang to treat osteoporosis, but it is aimed at the disease of kidney essence deficiency. The foundation, Xie regulates yin and yang, avoiding the disadvantages of yin and yang. In the prescription, dodder nourishes kidney and essence, strengthens tendons and bones, epimedium warms kidney yang, strengthens muscles and bones, and rehmannia glutinosa nourishes kidney yin, so as to achieve both yin and yang and nourish the kidney Essence filling effect, at the same time choose oysters to nourish kidney and essence, nourish yin and subdue yang, strengthen joints, add psoralen to warm kidney and support yang, strengthen kidney and replenish essence, salvia miltiorrhiza promotes blood circulation, dredges collaterals, and relieves pain. This guideline is mainly for postmenopausal osteoporosis patients with primary osteoporosis. Kidney essence deficiency is the basic pathogenesis. It can effectively increase bone density, improve patients' clinical symptoms and quality of life, and at the same time treat the symptoms of kidney essence deficiency. It also has a significant curative effect on systemic symptoms such as low back pain, soreness of the waist and knees, difficulty in maintaining weight, cramps of lower limbs, dizziness and tinnitus, fatigue, frequent urination at night, etc. The simple medicine and great effect show the characteristics and innovation of this prescription.

The pharmaceutical composition for preventing or treating primary osteoporosis according to the present invention is characterized in that the composition includes the following components in parts by weight: 200-600 parts of dodder, 100-300 parts of epimedium, 100 parts of rehmannia glutinosa -300 parts, 80-240 parts of salvia miltiorrhiza, 25-80 parts of oysters, 80-240 parts of psoralen.

Preferably, the composition includes the following components in parts by weight: 200 parts of dodder, 300 parts of epimedium, 100 parts of rehmannia glutinosa, 240 parts of salvia miltiorrhiza, 25 parts of oyster and 240 parts of psoralen.

The composition preferably includes the following components in parts by weight: 600 parts of dodder, 100 parts of epimedium, 300 parts of rehmannia glutinosa, 80 parts of salvia miltiorrhiza, 80 parts of oyster and 80 parts of psoralen.

The composition may also preferably include the following components in parts by weight: 400 parts of dodder, 200 parts of epimedium, 200 parts of rehmannia glutinosa, 160 parts of salvia miltiorrhiza, 53 parts of oyster, and 160 parts of psoralen.

More preferably, the composition comprises the following components in parts by weight: 380 parts of dodder, 220 parts of epimedium, 200 parts of rehmannia glutinosa, 155 parts of salvia miltiorrhiza, 55 parts of oyster, and 155 parts of psoralen.

In the composition, the dodder is preferably salt dodder, the oyster is preferably calcined oyster, and the psoralen is preferably salt psoralen.

The dosage forms that can be prepared from the composition include capsules, tablets, pills, oral liquids, granules, injections or powders.

The active ingredient of the composition is made by the following steps:

A. Weigh the dodder and psoralen according to the prescription amount, extract twice with 50-70% ethanol, each time for 1-3 hours, add ethanol in an amount 6-10 times of the medicinal material, filter the extract, combine, and decompress Concentrated to 60 ℃ heat measurement relative density of 1.10 ± 0.05 clear paste, set aside;

B. Weigh Epimedium, Rehmannia glutinosa, and Salvia miltiorrhiza according to the prescription amount, add 8-12 times the amount of water to extract three times, the extraction time is 1-3 hours, filter the extract, concentrate under reduced pressure to 60 ° C, and the relative density of thermal measurement is 1.10 ± 0.05 clear paste, combine the concentrated solution three times, and combine with the alcohol extraction clear paste obtained in step A, concentrate to a thick paste with a relative density of 1.20±0.05 by thermal measurement at 60°C, dry and pulverize to obtain a fine powder;

C. Oysters are crushed into 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 invention.

The preparation technology of this composition tablet is:

A. Weigh the dodder and psoralen according to the prescription amount, extract twice with 50-70% ethanol, each time for 1-3 hours, add ethanol in an amount 6-10 times of the medicinal material, filter the extract, combine, and decompress Concentrate to a clear paste with a thermally measured relative density of 1.10±0.05 at 60°C, set aside;

D. Combine the oyster powder obtained in step C and the fine powder obtained in step B, granulate according to the conventional process, granulate, and tablet.

The preparation technology of this composition tablet is preferably:

A, take dodder and psoralen by prescription quantity, dodder is broken first, extract twice with 60% ethanol, each time 1.5 hours, add 10 times of alcohol for the first time, add 8 times of alcohol for the second time, extract Filtrate the liquid, combine, concentrate under reduced pressure to a clear paste with a thermal measurement relative density of 1.10±0.05 at 60°C, and set aside;

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

The application of the pharmaceutical composition is preferably the application in the preparation of postmenopausal osteoporosis medicine.

The application of the pharmaceutical composition is preferably the application of the pharmaceutical composition in the preparation of a drug for reducing the degradation product β-CTX of β glue.

The application of the pharmaceutical composition is preferably the application of the pharmaceutical composition in the preparation of medicines for the treatment of deficiency of kidney essence.

The application of the pharmaceutical composition is preferably that the pharmaceutical composition increases serum Ca, P content, increases the content of bone formation marker PINP, reduces the content of bone formation marker BGP, reduces the content of bone resorption marker ALP and TRACP, or increases the content of CT , The application of PTH content medicine.

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

The pharmaceutical composition of the present invention is a prescription for treating postmenopausal osteoporosis established through years of clinical application to explore the pathogenesis and treatment of osteoporosis from the perspective of "deficiency of kidney essence" based on the theory of traditional Chinese medicine. It has obvious curative effect. For postmenopausal osteoporosis patients, this prescription can significantly relieve back pain, soreness of waist and knees, relieve lower limb cramps, dizziness, tinnitus, fatigue, frequent urination at night and other symptoms, and has no obvious adverse reactions.

According to the clinical manifestations of osteoporosis, it should belong to the categories of "bone impotence", "gubi", "bone dryness", "bone extreme", "bone shrinkage" and other categories in traditional Chinese medicine. Question·Atrophy Theory says: "When the kidney qi is hot, the waist and spine will not move, the bones will wither and the marrow will decrease, and the hair will become bone atrophy." Get up from the bed." "Su Wen Bi Lun" contains: "Bone numbness is endless, and the feeling is caused by evil again, and the interior is housed in the kidney. It has been clearly recorded in "Suwen Changshi Jielun": "The disease is in the bones, the bones are too heavy to lift, the bone marrow is sore, and the cold air is coming. It is called bone numbness." When the yin qi is exhausted, the bones wither." The above-mentioned records in ancient Chinese medical books are similar to the symptoms of back pain, limited limb function, and turtle back in modern osteoporosis, which provide a basis for exploring the etiology and pathogenesis of osteoporosis.

Traditional Chinese medicine believes that the kidney is the innate foundation, the root of life, and the kidney stores essence. "Su Wen · Ancient Innocence Theory" says: "The kidney governs water, and it is stored by the essence of the five internal organs and six fu organs." , Sealed, and closed features. Essence is the basic substance that constitutes the human body, and it is also the material basis for human growth and development and various functional activities. The essence stored in the kidney is not static, but changes regularly with the different stages of the human body's growth, growth, strength, and aging. Just as it is said in "Suwen·Ancient Innocence Theory": "A woman's seven-year-old kidney qi Prosperous, the teeth grow longer... At the fifth and seventh, the Yangming channel weakens, the face becomes scorched, and the hair begins to fall. At the sixth and seventh, the three yang channels weaken, the face becomes scorched, and the hair begins to turn white. At the seventh and seventh, the Ren channel becomes weak, and the Tai Chongmai declines, Tiangui exhausts, and the tunnel is blocked, so the shape is bad and childless." The argument of "Qiqi... Tiangui exhausted" pointed out in the article is very consistent with the age of women's menopause in modern medical research. Kidney, bone, marrow The close physiological connection between them is fully reflected in the whole process of bone growth and development. As we age, the growth of kidney essence changes, and the growth of bones also changes. Loss of nourishment, so back pain, shin knees soreness, fragile and easy to break the bones generally manifested as a high incidence of osteoporosis in the middle-aged and elderly population. For women, the kidney essence determines the strength of bones through the action of "Tiangui". With the increase of age, the kidney essence becomes weaker and weaker. During menopause, the acquired essence gradually becomes exhausted, and the essence energy in the kidney gradually declines. The source of bone marrow is insufficient, and the bone marrow cannot nourish the bone marrow, resulting in bone marrow emptiness. As a result, osteoporosis such as low back pain is prone to occur. happened.

To sum up, it can be seen that the location of the disease is the bone, the main organ of the disease is the kidney, and the main pathogenesis is the deficiency of kidney essence and bone marrow dystrophy. The etiology of this disease is mainly due to old age, physical weakness, chronic illness, infirmity, overwork, acquired deprivation, improper treatment, etc., resulting in deprivation of the bone marrow, resulting in bone flaccidity and weakness, failure to produce marrow and fill the bone, and bone marrow is empty. Based on the disease. In view of the main pathogenesis of kidney essence deficiency and bone marrow dystrophy in this disease, the treatment method should be to replenish the kidney essence and nourish the marrow to fill the bone. Too much, kidney essence must be replenished, yin and yang must be balanced, kidney qi must be transformed, and bone marrow must be nourished. Therefore, the treatment principle of this disease is to invigorate the kidney, replenish essence and marrow, and treat this type of disease from the perspective of kidney essence. Good curative effect will be achieved.

Aiming at the pathogenesis of postmenopausal osteoporosis with deficiency of kidney essence, the pharmaceutical composition of the present invention establishes a treatment method of replenishing kidney essence, nourishing yin and strengthening yang, and aims at tonifying kidney and replenishing essence to consolidate its essence, and regulating yin and yang to help It works. The kidney stores the essence, which transforms into kidney qi, kidney yin, and kidney yang. Both kidney yin and kidney yang are rooted in the kidney essence. Therefore, for osteoporosis, which is the root of kidney essence deficiency, the treatment needs to be based on nourishing the kidney and replenishing essence. At the same time, it takes into account the balance of yin and yang, and supports yang while nourishing yin. It is produced by kidney yin, and the steaming of yang is continuous. On the contrary, the solitary yin does not grow, and the product of replenishing essence and nourishing yin, there is yang in yin, and yin is assisted by yang, and the kidney yin is metabolized, so it is combined with nourishing kidney yin drug.

Focusing on the treatment method of "replenishing kidney essence, nourishing yin and supporting yang", the inventor established the prescription of the pharmaceutical composition of the present invention:

Monarch medicine: Dodder, pungent, sweet, slightly warm in nature, its function is to benefit the essence, strengthen the muscles and bones, take it as the king, and take it as the effect of warming and nourishing the essence, nourishing the marrow and strengthening the bones.

Ministerial drug: Epimedium, pungent, sweet, warm, has the effect of invigorating kidney and essence, warming and tonifying kidney yang, with Epimedium as minister, it has the effect of helping the emperor to revitalize kidney yang and strengthen bones and muscles.

Ministerial drug: Rehmannia glutinosa, sweet in the mouth, bitter, cold in nature, GUIXIN, liver, kidney meridian. Taking Rehmannia glutinosa as a minister, nourishing kidney yin, and sharing it with epimedium, tonifying the yin and yang of the kidney, and helping the monarch medicine to fill the kidney essence, so as to achieve the effect of seeking yin in yang and growing yin from yang.

Adjuvant medicine: oyster, salty, slightly cold, returns to the liver, gallbladder, and kidney meridian, oyster nourishes the kidney and strengthens essence, softens hard masses, nourishes yin and nourishes yang, and strengthens bones.

Adjuvant: Psoralen: pungent, bitter, warm in nature. Return kidney, spleen channel. It has the functions of warming the kidney and supporting yang, strengthening the kidney and filling the essence. Psoraleae has the functions of invigorating the kidney and warming the yang, astringing and consolidating the essence, and it is used together with oysters as an adjuvant drug to invigorate the kidney and strengthen the essence, strengthen the joints, and it is also a medicine for monarchs and ministers to invigorate the kidney and replenish essence.

Herbal medicine: Danshen, bitter in the mouth, slightly cold, enters the heart, liver meridian, functions in promoting blood circulation, dredging collaterals, relieving pain. Possesses the function of "Tongli Guanmai", using Salvia Miltiorrhiza as an envoy, can not only introduce medicine into the blood, but also enhance the effect of dredging collaterals and relieving pain.

Looking at the whole prescription, the medicinal dodder nourishes and nourishes the essence, nourishes the marrow and strengthens the bones, epimedium and rehmannia tonify the yin and yang in the kidney, one yin and one yang, tonify the kidney qi, oyster and psoralen strengthen the kidney and replenish essence, strengthen the yin and yang, and astringent It has the effect of strengthening astringency and sharpening the joints. It is combined with Danshen as an envoy to introduce the medicine into the blood to enhance the efficacy of the medicine.

To sum up, the pharmaceutical composition of the present invention aims at the pathogenesis of postmenopausal osteoporosis with deficiency of kidney essence. On the one hand, it focuses on replenishing kidney essence, while simultaneously nourishing yin and supporting yang, and regulating kidney yin and kidney yang. The prescription medicine is simple and effective, which helps to improve the symptoms of patients with low back pain caused by kidney essence deficiency, soreness of the waist and knees, difficulty in maintaining weight, cramps of lower limbs, dizziness and tinnitus, fatigue, frequent urination at night and other symptoms, and improves the quality of life.

Traditional decoctions are cumbersome to decoct, have poor taste, unstable quality, are not convenient for patients to carry and take, and are greatly limited in use. Therefore can choose to make capsule, tablet, granule, oral liquid or other dosage forms. Among them, compared with other dosage forms, tablets use less auxiliary materials, are easier to form, are small in size, accurate in dosage, and are more convenient to carry, transport and take.

In addition, the tablet is a dry solid with stable and controllable quality. Some drugs that are prone to oxidative deterioration and deliquescence can be protected by coating. Light, air, moisture, etc. have little effect on them, and can also cover up the bad smell of traditional Chinese medicine.

Furthermore, tablet production is mechanized and has a high degree of automation, which is suitable for industrialization promotion. In order to give full play to the social and economic value of this medicine, we combine the characteristics of the traditional Chinese medicine taste, and preferably use tablets as the dosage form of this prescription.

Selection of preparation process:

In the research process of the preparation process, we started from various aspects and completed the selection of the process.

1. Salt dodder

1. The impact of crushing on the extraction effect of Cuscuta chinensis

(1) Experimental design

Salt dodder is a kind of seed, the seed is small, and the texture is hard. In order to extract the active ingredients completely, the impact of crushing on the extraction effect was investigated. The salt dodder was crushed, extracted with 60% ethanol, and compared with the unbroken salt dodder, and the hyperin content was used as an index to investigate the extraction effect.

(2) Test method

Take an appropriate amount of Cuscuta salt, break it, weigh 30 g each of Cuscuta salt and the broken Cuscuta salt, extract twice with 60% ethanol under reflux, each time for 1.5 hours, add 10 times the amount of solvent, filter the extract, combine, and concentrate appropriately, Transfer to a 500ml measuring bottle, add 60% ethanol to the mark, shake well, and set aside.

(3) Inspection method

Determination of Hyperin

Preparation of the test solution: take the above extract, filter, and take the subsequent filtrate to obtain.

Preparation of reference substance solution: Accurately weigh an appropriate amount of hyperin reference substance, add methanol to make a solution containing 0.04968mg per 1ml, and obtain it.

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

Determination method: Precisely draw 10 μl each of the reference substance solution and the test solution, inject it into a liquid chromatograph, measure it, and obtain it.

(4) The test results are shown in Table 1.

Table 1 The results of investigation on the crushing of dodder salt

The test results show that whether the salt dodder is broken or not has a great influence on the extraction effect, and the salt dodder should be broken before extraction.

2. Process research on extraction solvent optimization

(1) Experimental design

The extraction effects of six solvents including water, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol and 90% ethanol were investigated by using the single factor investigation method, taking the content of hyperin and the yield of ointment as indicators.

(2) Test method

Weigh six parts of broken salt dodder, each part 30g, under the condition of parallel operation, heat and reflux with different solvents to extract twice, add 10 times the amount of solvent each time, extract for 1.5 hours, filter the extract, combine, and properly Concentrate, transfer to a 500ml measuring bottle, add the corresponding solvent to the mark, shake well, and set aside.

(3) Inspection method

Determination of Hyperin

The preparation of the reference solution and the chromatographic conditions are the same as the test of "Effect of Crushing on the Extraction Effect of Cuscuta Semen Salt".

Determination of paste yield

Take the above extracts, measure 200ml respectively, put them in evaporating dishes, first concentrate them on a water bath to form a thick paste, then transfer them to a vacuum drying oven for drying, weigh them, calculate, and you get it.

(4) The test results are shown in Table 2.

Table 2 salt dodder solvent optimization results

The test results show that 50%-70% ethanol is used as solvent for extraction, and the content of hyperin in the sample and the yield of paste are higher. Therefore, the extraction solvent of Cuscuta sativa is preliminarily determined to be 50%-70% ethanol.

2. Process research on optimal solvent extraction of Epimedium

1. Experimental design

The extraction effect of water, 40% ethanol, 50% ethanol, 60% ethanol, and 70% ethanol was investigated by single factor test method, with the content of icariin and the yield of ointment as indicators.

2. Test method

Weigh five parts of Epimedium, each 15g, under the condition of parallel operation, use different solvents to reflux extract twice, add 12 times of solvent each time, extract for 1.5 hours, filter, transfer to a 1000ml measuring bottle, add corresponding The solvent to the mark, shake well, set aside.

3. Inspection method

Determination of Icariin

Preparation of reference substance solution: Accurately weigh an appropriate amount of icariin reference substance, add methanol to make a solution containing 0.1mg per 1ml, and obtain it.

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

Determination of paste yield

Take the above extracts, measure 500ml respectively, put them in evaporating dishes, first concentrate them on a water bath to form a thick paste, then transfer them to a vacuum drying oven for drying, weigh them, calculate, and you get it.

4. The test results are shown in Table 3.

Table 3 Epimedium solvent optimization results

It can be seen from the results in the above table that when using water as a solvent to extract Epimedium, the content of icariin in the sample is the highest, and with the increase of ethanol concentration, the content of icariin tends to decrease; There is little difference in the ointment rate, and the solvent with high ethanol concentration has a slightly lower ointment yield; and using ethanol as a solvent will increase the cost and cause environmental pollution, so water is selected as the extraction solvent of Epimedium.

3. Process research on optimal extraction solvent of salt psoraleae

1. Experimental design

The extraction effect of water, 50% ethanol, 60% ethanol, 70% ethanol and 80% ethanol was investigated by using the single factor test and investigation method, taking the content of psoralen and isopsoralen as the index.

2. Test method

Weigh five portions of salt psoralen, 20g each, heat and reflux for extraction twice, add 10 times the amount of solvent each time, extract for 1.5 hours, filter the extract, combine, transfer the filtrate to a 500ml measuring bottle, and add the corresponding solvent To the mark, shake well, set aside.

3. Inspection method

(1) Determination of psoralen and isopsoralen

Preparation of the test solution: Accurately measure 5ml of the above-mentioned extract, put it in a 50ml measuring bottle, add the corresponding solvent to the mark, shake well, filter, and take the subsequent filtrate to obtain final product.

Preparation of reference substance solution: Accurately weigh the appropriate amount of psoralen reference substance and isopsoralen reference substance, and add methanol respectively to make solutions containing 0.023mg and 0.023mg per 1ml.

Chromatographic conditions: octadecylsilane bonded silica gel as filler; mobile phase: methanol-water (55:45); detection wavelength: 246nm; column temperature: 30°C; flow rate: 1.0ml/min.

(2) Determination of paste yield

4. The test results are shown in Table 4.

Table 4 salt psoralen solvent preferred result

The test results show that the content of psoralen and isopsoralen in the sample is higher when 50%-70% ethanol is used as solvent for extraction; , the paste rate is low.

Based on the above solvent optimization test results, it can be seen that extracting Cuscuta salt and psoralen salt with 50%-70% ethanol can better extract their active ingredients, so it is proposed to combine the two and use ethanol as the extraction solvent for extraction. At the same time, in order to ensure that the active ingredients can be extracted completely and reduce the amount of ethanol, the ethanol concentration is determined to be 60%; Epimedium is extracted with water as a solvent, the transfer rate of active ingredients is high, and energy is saved, so Epimedium and Rehmannia glutinosa , Salvia miltiorrhiza combined with water extraction.

4. Experimental research on water extraction process optimization

1. Investigation on water absorption rate of water extraction

Weigh 50g each of Epimedium, Dihuang and Salvia, and then weigh 30g of Epimedium, 30g of Rehmannia, and 24g of Salvia in total, mix them, add 10 times the amount of water each, soak, measure the water absorption, and calculate the water absorption. The results are shown in table 5.

Table 5 water absorption test results

From the above test results, it can be seen that because Epimedium has a high water absorption rate, the water absorption rate after mixing the three medicinal materials is also high; Epimedium is light, large in size, and difficult to infiltrate, so the amount of water added in the water extraction is set at about 12 times , to ensure the extraction effect.

2. Orthogonal experimental design

The factors that have a greater impact on the extraction effect (A) extraction times, (B) extraction time, (C) water consumption, (D) blank are selected as the investigation factors, and the contents of icariin and danshensu sodium are used as indicators. The test was performed with the L ₉ (3 ⁴ ) table at the following levels. See Table 6 for the factor level table.

Table 6 water extraction orthogonal test factor level table

3. Test method

Weigh 15g of epimedium, 15g of rehmannia glutinosa, and 12g of salvia miltiorrhiza, 9 parts in total. Under the condition of parallel operation, according to the L ₉ (3 ⁴ ) orthogonal design table, decoct with water, filter the extract, combine, and concentrate appropriately. Transfer to a 1000ml measuring bottle, add water to the mark, shake well, and set aside.

(1) Determination of content of icariin

Preparation of the testing solution: get the orthogonal test sample solution, filter, and get the subsequent filtrate as the testing solution.

The preparation of the reference solution and the chromatographic conditions were the same as the optimization test of the extraction solvent.

(2) Determination of the content of Danshensu Sodium

Preparation of reference substance solution: Accurately weigh an appropriate amount of Danshensu sodium reference substance, add 50% methanol to make a solution containing 0.04mg per 1ml, and obtain it.

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

Determination method: Precisely draw 20 μl each of the reference substance solution and the test solution, inject it into a liquid chromatograph, measure it, and obtain it.

(3) Determination of paste yield

Take the orthogonal test sample solution, measure 200ml respectively, put it in an evaporating dish, concentrate it on a water bath to form a thick paste, then transfer it to a vacuum drying oven for drying, weigh it, and calculate it.

4. See Table 7 for the test results of icariin, danshensu sodium and ointment yield.

Table 7 Water extraction orthogonal table L ₉ (3 ⁴ ) experimental design and results

5. Analysis of icariin content determination and danshensu sodium content determination results

The experimental data were analyzed by variance, and the significance of the influence of each factor was tested. The results are shown in Table 8 and Table 9.

Table 8 icariin content analysis of variance results

F _{0.01(2, 2)} = 99 F _{0.05(2, 2)} = 19

As can be seen from the above table, taking the content of icariin as the investigation index, the influence of various factors on the content of icariin is in the following order: extraction times>water addition>extraction time, and the best process is A ₃ B ₃ C ₃ ; The number of extractions had a great influence on the content of icariin, and there was a significant difference.

Table 9 danshensu sodium content analysis of variance results

F _{0.01(2, 2)} = 99 F _{0.05(2, 2)} = 19

It can be seen from the above table that taking the content of Danshensu sodium as the investigation index, the influence of various factors on the content of Danshensu sodium is in the following order: extraction times>extraction time>water addition, and the best process is A ₃ B ₃ C ₃ ; The content of danshensu sodium has the greatest impact, and there is a very significant difference. The extraction time and the amount of water have little effect on the content of danshensu sodium, and there is no significant difference. Because in factor A, the content of level Ⅲ has a larger increase than that of level Ⅱ, it is planned to extract the fourth time after the extraction of the optimal process and the optimal process, add 10 times the amount of water, extract for 1.5 hours, collect separately, and investigate Danshensu sodium content changes.

Combined with the results of variance analysis of icariin and danshensu sodium, in order to ensure the full extraction of active ingredients and save labor and energy, choose to extract three times, the first extraction is 2 hours, add water 12 times, the second and third extractions are 1 hour , add water 10 times. The extraction process determined after optimization is: A ₃ B _(3,1,1) C _(2,1,1) .

5. Experimental research on the optimization of alcohol extraction process

1. Orthogonal experimental design

Select the factors that greatly affect the extraction effect: (A) extraction times, (B) extraction time, (C) alcohol addition, (D) blank as the investigation factors, the content of hyperin, psoralen and iso The content of psoralen was used as an index, and the test was carried out in the form of L ₉ (3 ⁴ ) at the following levels. See Table 10.

Table 10 Ethanol extraction factor level table

2. Test method

According to the ratio of the prescription, 30 g of broken salt dodder and 12 g of salt psoralen were weighed, 9 parts each, and 60% ethanol was used as the extraction solvent. Under the condition of parallel operation, follow the L ₉ (3 ⁴ ) orthogonal design table, reflux extraction, filter the extract, combine, concentrate in an appropriate amount, transfer to a 500ml measuring bottle, add 60% ethanol to the mark, shake well, and set aside .

(1) Determination of hyperin content

The preparation of the reference solution and the chromatographic conditions are the same as in the "extraction solvent optimization test".

(2) Determination of psoralen and isopsoralen content

The preparation of need testing solution: precision measures each 5ml of orthogonal test sample solution, puts in 50ml measuring bottle, adds 60% ethanol to scale respectively, shakes up, filters, and gets continued filtrate as need testing solution.

3. The test results are shown in Table 11.

Table 11 Alcohol extraction orthogonal test design and results

4. Analysis of the determination results of alcohol-extracted hyperoside content, psoralen and isopsoralen content

The experimental data were analyzed by variance, and the significance of the influence of each factor was tested. The results are shown in Table 12 and Table 13.

Table 12 Analysis of variance table for hyperoside content

F _{0.05(2, 4)} = 6.94 F _{0.01(2, 4)} = 18.00

It can be seen from the above table that taking the hyperin content as the investigation index, the influence of various factors on the hyperin content is in the following order: extraction times>alcohol addition>extraction time, the best process is A ₂ B ₂ C ₃ , Among them, the extraction times of factor A had a significant effect on the content of hyperin.

Table 13 Analysis of variance table of psoralen and isopsoralen content

F _{0.01(2, 2)} = 99 F _{0.05(2, 2)} = 19

As can be seen from the above table, taking the content of psoralen and isopsoralen as the investigation index, the order of influence of various factors on psoralen and isopsoralen is: extraction times>alcohol addition>extraction time, The optimal process is A ₃ B ₂ C ₂ ; the extraction times A and the amount of alcohol added C have a greater impact on psoralen and isopsoralen, with significant differences.

Combined with the content analysis results of hyperoside, psoralen and isopsoralen, in order to ensure the full extraction of active ingredients and save labor and energy, two extractions were selected, each extraction was 1.5 hours, and alcohol was added for the first time 10 times the amount, add alcohol 8 times the amount for the second time. After optimization, the process is determined to be A ₂ B ₂ C _(3,2) .

6. Research on crushing process of calcined oyster

1. Inspection of powder extraction rate

Take three portions of calcined oysters, 5 kg each, crush them with a pulverizer, pass through a 120-mesh sieve, and calculate the powder yield. The results are shown in Table 14.

Table 14 powder extraction rate investigation results

The results show that the powder extraction rate is above 95%, meeting the requirements.

Description of drawings:

Figure 1: XDGM significantly improved bone morphology in rats with osteoporosis induced by ovariectomy

Figure 2: XDGM significantly improved bone morphology in rats with osteoporosis induced by retinoic acid

Figure 3: XDGM significantly improved bone morphology in rats with osteoporosis induced by retinoic acid

Functional test:

In order to illustrate the efficacy of the pharmaceutical composition of the present invention, functional tests were carried out with the samples of Example 1, Example 2, and Example 6 prepared in the examples.

One, the influence of the active ingredient of the pharmaceutical composition of the present invention on rat osteoporosis caused by ovariectomy

【Objective】Ovariectomized rat osteoporosis model was used to observe the effect of the active ingredients (XDGM1, XDGM2, XDGM6) of the pharmaceutical composition of the present invention on the osteoporosis caused by ovariectomized rats, and to evaluate its treatment of osteoporosis effect.

[Method] After 3 months of ovariectomized surgery, the animals were randomly divided into three groups: normal group, sham operation group, model group, positive drug (estradiol valerate) group,

XDGM

1, 2, and 6 groups according to body weight, and fed After 3 months of gastric administration, all rats were intraperitoneally injected with tetracycline hydrochloride for bone fluorescence labeling. After the administration, blood was collected to detect serum calcium Ca, phosphorus P, alkaline phosphatase ALP, tartrate-resistant acid phosphatase TRACP, and pre-type I Collagen amino telopeptide PINP, osteocalcin BGP, parathyroid hormone PTH, calcitonin CT. The left tibial femur, right femur, and spine were taken to detect bone morphology, bone biomechanics, and bone density indicators.

[Results] 1. Bone morphology indicators showed that compared with the model group,

XDGM

1 and 2 significantly increased tibial trabecular bone volume percentage TBV% (P<0.05 or P<0.01), decreased trabecular bone resorption surface percentage TRS%, Bone trabecular formation surface percentage TFS%, trabecular bone mineralization rate MAR, bone cortical mineralization rate mAR (P<0.01 or P<0.05), it can be seen that XDGM can reduce the high-transition bone metabolism state of rats after ovariectomized, Improvement of bone morphological properties in osteoporotic rats. 2. Bone biomechanical indicators showed that compared with the model group, XDGM1 and 2 significantly increased the maximum load, bending strength and elastic modulus of the femur (P<0.05 or P<0.01), and improved the bone biomechanical properties of osteoporotic rats. 3. Bone density indicators showed that XDGM 1 significantly increased bone density in osteoporotic rats (P<0.05), and

XDGM

2 and 6 groups also increased bone density. 4. Bone composition indicators showed that XDGM1, 2, and 6 significantly increased serum Ca and P levels (P<0.01), providing abundant inorganic raw materials for bone formation; high-dose XDGM significantly increased the bone formation marker PINP content (P<0.01 ), providing rich organic raw materials for bone formation. 5. The indicators of bone turnover markers showed that

XDGM

1, 2, and 6 significantly reduced the content of bone formation marker BGP (P<0.01 or P<0.05), and bone resorption markers ALP and TRACP also decreased. It can be seen that XDGM reduces high conversion state of bone metabolism. 6. Hormone indicators showed that XDGM1 significantly increased CT and PTH content (P<0.01 or P<0.05), and XDGM2 and 6 groups also increased CT and PTH content. It can be seen that XDGM can improve the state of osteoporosis, making bone formation greater than bone absorb.

【Conclusion】The results of this experiment show that XDGM can significantly improve the bone morphology and biomechanical properties of rats, and increase bone density. It can be seen that XDGM can significantly improve the osteoporotic bone in rats caused by ovariectomy. This improving effect is related to the increase of blood Ca, P, PINP content, reduction of high-transformation bone metabolism state, and stimulation of CT and PTH secretion.

The purpose of the experiment: to observe the effect of the active components of the pharmaceutical composition of the present invention on the osteoporosis of rats caused by ovariectomy by adopting a rat model of ovariectomized osteoporosis, and to evaluate its therapeutic effect on osteoporosis.

1 Experimental materials

1.1 Test product

1.1.1 Name: active ingredient of the pharmaceutical composition of the present invention, abbreviation: XDGM, test product number: TN-1318.

1.1.2 Properties: brown powder.

1.1.3 Functions and indications: menopausal osteoporosis.

1.1.4 Intended clinical dosage: oral administration, 14.1g crude drug/day.

1.1.5 Content and specification: 2.96g crude drug/g dry cream powder.

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

1.1.7 Storage conditions: sealed storage.

1.1.8 Validity period: 2 years.

1.2 Positive drugs and main reagents

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

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

1.2.3 Picric acid: Taishan Chemical Factory Co., Ltd., batch number: 20071201.

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

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

1.2.6 Tartrate-resistant acid phosphatase TRACP: Biyuntian Biotechnology Co., Ltd., batch number: 1202191412.

1.2.7 Alkaline phosphatase ALP: Beijing Jiuqiang Biotechnology Co., Ltd., batch number: 14-0612P.

1.2.8 Type I procollagen amino terminal peptide PINP: Cloud-Clone Corp., batch number: L150120505.

1.2.9 Phosphorus P: Beijing Jiuqiang Biotechnology Co., Ltd., batch number: 14-0923.

1.2.10 Calcium Ca: Beijing Jiuqiang Biotechnology Co., Ltd., batch number: 14-0811.

1.2.11 Osteocalcin BGP: Beijing North Institute of Biotechnology, batch number: 20150220.

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

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

1.3 Experimental system:

1.3.1 Animal species: SD rats.

1.3.2 Animal grade: SPF grade.

1.3.3 Sex and number of animals: female, 108 in total.

1.3.4 Animal age: 63-69 days.

1.3.5 Animal weight: 200-220g.

1.3.6 Animal source: purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., certificate number: 11400700047562, license number: SCXK (Beijing) 2012-0001, date of receipt on May 21, 2014.

1.3.7 Raising conditions: Animals were raised in the New Drug Evaluation Center of Hebei Academy of Integrated Traditional Chinese and Western Medicine. Rats were housed in a cage with 12 hours of light per day, a temperature of 20-26°C, and a relative humidity of 40-70%.

1.3.8 Quarantine process: The quarantine period for new animals is 3 days. Since no suitable animals can be purchased, the newly purchased animals are fed until they are about 6 months old to start the experiment. During this period, the animals drink water and eat normally, are in good health, and have no signs of disease or death.

1.3.9 Feed: Full-price pellet feed for experimental animals, provided by the Experimental Animal Center of the Academy of Military Medical Sciences of the Chinese People's Liberation Army, certificate number: 0025539.

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

1.3.11 Bedding: Common grade bedding for experimental animals, provided by Hebei Experimental Animal Center, production license: SCXK (Ji) 2013-2-001.

1.3.12 Labeling: 5% picric acid is used for labeling.

2 Experimental methods

2.1 Experimental Design Basis

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

2.1.2 Information provided by the entrusting unit: After castration, the bone mineral content decreases, the bone resorption increases rapidly, and the bone strength decreases, all of which have many similarities with humans. Compared with other animal models, they have the advantages of fast reproduction and low cost Low cost, easy to raise and manage, and clear genetic background have been used as the most commonly used animal models, and are now widely used in the evaluation and development of new drugs for the treatment of postmenopausal osteoporosis.

2.2 Dosage and grouping

108 rats were randomly divided into 3 groups according to body weight, namely, 10 normal groups, 12 sham operation groups (Sham), and 86 model group. Ovariectomies were performed on rats in the model group, and bilateral ovaries were removed. The operation process of the sham operation group was the same as that of the model group, but the ovary was not removed, and only a small amount of fat tissue was removed. About 3 months after modeling, the rats in the modeling group were randomly divided into 5 groups according to body weight, namely the model group (OVX), estradiol valerate group,

XDGM

1, 2, and 6 groups, and the proposed clinical dosage of XDGM It is 14.1g crude drug/day, calculated on the basis of 60kg per person, the dose for rats is 8 times of the human clinical dosage, i.e. 1.88 crude drug/kg, and the content is 2.96g crude drug/g dry ointment powder, i.e. 0.635g dry ointment powder/kg. Referring to the positive drug instructions and references, the dose of estradiol valerate is set at 100 μg/kg, see Attached Table 15.

Table 15 The effect of the active components of the pharmaceutical composition of the present invention on the impact of ovariectomized rat osteoporosis experimental group control table

2.3 Administration method:

Oral administration, 10ml/kg body weight, is consistent with the clinically recommended oral route.

2.4 Preparation and storage of the test product

The test drug was prepared with 0.5% CMC-Na to the concentration used in the experiment (see attached table 15), and after preparation, it was stored at 2-8°C for future use, and the positive drug was prepared immediately after use.

2.5 Administration of the test article

After 3 months of modeling, the experimental group was given the test drug by gavage. The normal, sham and model groups were given 0.5% CMC-Na once a day for 3 consecutive months.

2.6 Experimental steps

Newly received SD rats, quarantined for 3 days, carried out experimental grouping and modeling according to the above operations, and divided into groups for administration after 3 months of modeling. As mentioned above, normal, Sham and OVX groups were given 0.5% CMC-Na, weekly Record the body weight once, all rats were intraperitoneally injected with tetracycline hydrochloride 30 mg/kg on the 16th and 4th days before execution for bone fluorescence labeling, and after the administration period, on the 2nd day, they were intraperitoneally anesthetized with 10% chloral hydrate with a volume of 0.35ml /100g body weight, sacrificed after abdominal aortic blood collection, collected materials, and detected indicators.

Model preparation method: Rats were anesthetized intraperitoneally with 10% chloral hydrate, the volume was 0.35ml/100g body weight, centered on the midaxillary line 1cm below the ribs, the diameter of the sheared hair was 2cm, disinfected with iodine and alcohol in sequence, and the surgical incision was located under the ribs Tighten the skin between one finger’s width to the upper edge of the thigh, make a 0.8-1cm incision along the longitudinal axis, bluntly separate the subcutaneous superficial fascial tissue layer with hemostatic forceps, incise the muscle layer longitudinally, expose the abdominal cavity, and carefully separate the ovary ( The mature ovary is light red, oval, with irregular nodular follicles on the surface, connected to the terminal end of the pink tubular uterus, surrounded by more white fat frenulum). The end of the uterus was ligated with silk thread, all ovaries were removed, and the remaining tissues were returned to the abdominal cavity. The muscle layer and skin were sutured in turn, and the incision was disinfected. In the Sham group, the operations were the same as above except that the uterus was not ligated and the ovaries were not removed. After the operation, they were placed in a warm environment, and each mouse was given 50,000 units of penicillin sodium salt every day for 3 consecutive days.

2.7 Detection indicators

2.7.1 Blood index detection

After the drug administration was completed according to the experimental design, intraperitoneal anesthesia was performed with 10% chloral hydrate, blood was collected from the abdominal aorta and sacrificed, the whole blood was allowed to stand still, and the serum was separated by centrifugation to determine serum calcium Ca, phosphorus P, alkaline phosphatase ALP, anti-tartaric acid Acid phosphatase TRACP, type I procollagen amino terminal peptide PINP, osteocalcin BGP, parathyroid hormone PTH, calcitonin CT.

2.7.2 Bone Density (BMD) Detection

Before the test, the frozen rat vertebrae were taken out from the refrigerator and rewarmed at room temperature, and the BMD of lumbar vertebrae 4-6 (L4-6) of rats in each group was detected by Osteocore3Digital 2D bone densitometer.

2.7.3 Detection of bone histomorphological indicators

Take the proximal 1/3 of the left tibia of the rat, place it in 4% paraformaldehyde solution (PH7.4) for 24 hours, and then carry out dehydration. Xylene 2d. Next, the specimens were soaked in plastic polymer solution I, II and III for 3 days in turn, and the above fixation, dehydration and soaking processes were all carried out at 4°C. Finally, add 400 μL of N,N-dimethyl-p-toluidine (N,N-Dimethyl-p-toluidine) into 100ml of pre-cooled (4°C) III solution, stir with a magnetic stirrer for 10 minutes, and then inject it into the penicillin vial About 7ml of liquid III, put the bone specimen into the bottom of the bottle in the same direction, use a syringe to evacuate the air in the embedding bottle, and then put it in a -20°C refrigerator to polymerize for about 1w, and it will become a colorless and transparent hard embedding block . After trimming, on a Reicheit-Jung2040 microtome, use a tungsten steel knife to cut out two 5 μm longitudinal non-decalcified bone slices for each bone tissue, one of which is used for toluidine blue staining, and the other is used for fluorescence observation .

Bone histomorphometric method: use Qwin Pro V3.5.0 image analysis system to perform morphometric measurement on non-decalcified bone slices:

(1) Histomorphometry of trabecular bone:

Trabecular volume percentage (TBV%): the percentage of trabecular bone volume in the total volume of the measured bone marrow cavity, which is the main indicator to measure the level of bone mass;

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

Trabecular forming surface percentage (TFS%): the percentage of osteoid surface covered by osteoblasts to the surface of trabecular bone, which can determine the activity of osteoblasts;

Mineralization rate of trabecular bone (MAR): The average distance of fluorescent double-labeled bands on the surface of trabecular bone divided by the number of days between two labelings.

(2) Morphometrics of the inner surface of the cortex:

Osteoid mean width (OSW): mean width of osteoid with osteoblast-coated inner surface of cortex;

Cortical mineralization rate (mAR): the average distance of fluorescent double-labeled bands on the inner surface of the cortex divided by the number of days between two labelings.

2.7.4 Detection of bone biomechanical indicators

Before testing, the frozen rat right femurs were taken out from the refrigerator and rewarmed at room temperature. Three-point bending test of rat femur: place the femur on the support of a microcomputer-controlled electronic universal testing machine with a span of 16 mm, press down on the middle part of the femur at a loading speed of 1 mm/min until the femur breaks, and detect its maximum load (N), flexural strength (MPa) and elastic modulus (GPa).

2.8 Relevant staff notification

Notify the animal room when purchasing animals, and notify the pathology room for processing when animals appear abnormal.

2.9 Main instrument systems

(dual energy X-ray)

2.10 Statistical Methods

- The experimental data is analyzed and processed by SPSS statistical software, and the statistical results are average ± standard deviation

One-way analysis of variance (One-Way ANOVA) was used for comparison of means, and least significant difference method (LSD) was used for pairwise comparison.

3 results

3.1XDGM significantly improved bone morphological characteristics in rats with osteoporosis caused by ovariectomy.

It can be seen from the attached table 16 that compared with the sham operation group, the tibia TBV% of rats in the model group was significantly reduced (P<0.01), and TRS%, TFS%, OSW, mAR, and MAR were significantly increased (P<0.01 or P<0.05) , it can be seen that the rats after ovariectomized showed high bone metabolism; compared with the model group, the positive drug estradiol valerate significantly increased rat tibial TBV% (P<0.01), decreased TRS%, TFS%, mAR, MAR (P<0.01 or P<0.05); XDGM1 and 2 groups significantly increased rat tibial TBV% (P<0.05 or P<0.01), decreased TRS%, TFS%, mAR, MAR (P<0.01 or P<0.05) . It can be seen from Figure 1 that the number and volume of bone trabeculae in the proximal tibia of XDGM rats were increased compared with the model group. It can be seen that XDGM can reduce the high-transformation bone metabolism state of rats after ovariectomy and improve bone loss in osteoporotic rats. Morphological properties.

Table 16 The influence of the active components of the pharmaceutical composition of the present invention on bone morphological indicators in rats with osteoporosis caused by ovariectomy

Note: Compared with the sham operation group: ^Δ P<0.05, ^ΔΔ P<0.01; compared with the model group: ^* P<0.05, ^** P<0.01.

3.2 XDGM significantly improved bone biomechanical properties in rats with osteoporosis induced by ovariectomy.

It can be seen from the attached table 17 that compared with the sham operation group, the maximum load, bending strength and elastic modulus of the rat femur in the model group were significantly decreased (P<0.01); compared with the model group, the positive drug estradiol valerate significantly increased the The maximum load, bending strength and elastic modulus of the femur (P<0.01), XDGM1, 2 significantly increased the maximum load, bending strength and elastic modulus of the rat femur (P<0.05 or P<0.01), it can be seen that XDGM can significantly improve bone quality Biomechanical properties of osteoporotic rat bone.

Table 14 Effects of Active Components of the Pharmaceutical Composition of the Present Invention on Bone Biomechanical Indexes in Rats with Ovariectomized Osteoporosis

Note: Compared with the sham operation group: ^Δ P<0.05, ^ΔΔ P<0.01; compared with the model group: ^* P<0.05, ^** P<0.01

3.3 Effect of XDGM on bone mineral density in osteoporotic rats.

It can be seen from the attached table 18 that compared with the sham operation group, the bone mineral density of the rats in the model group was significantly lower (P<0.01). Compared with the model group, the bone density of the positive drug estradiol valerate group increased, but there was no significant difference (P>0.05); the bone density of the XDGM 1 group increased significantly (P<0.05), and the bone density of the

XDGM

2 and 6 groups also increased. It can be seen that XDGM can increase the bone density of ovariectomized osteoporotic rats.

Table 18 The effect of the active components of the pharmaceutical composition of the present invention on the bone density of osteoporosis rats caused by ovariectomy

3.4 Effect of XDGM on serum Ca and P contents in osteoporotic rats.

It can be seen from the attached table 19 that compared with the sham operation group, the serum Ca content of the rats in the model group decreased significantly (P<0.05), and the P content also tended to decrease, but no significant difference was seen (P>0.05). Compared with the model group, the contents of serum Ca and P in each dose group of XDGM increased significantly (P<0.01). It can be seen that XDGM can increase blood Ca and P content, and provide abundant inorganic raw materials for bone formation.

3.5 Effect of XDGM on serum bone markers in osteoporotic rats.

It can be seen from the attached tables 19 and 20 that compared with the sham operation group, the bone formation markers ALP, BGP and bone resorption marker TRACP in the model group increased significantly (P<0.01 or P<0.05), and the bone formation marker PINP content decreased , but no significant difference (P>0.05), it can be seen that the rats in the model group showed a certain degree of high conversion bone metabolism. Compared with the model group, PINP in XDGM1 group increased significantly (P<0.01), BGP in

XDGM

1, 2, and 6 groups decreased significantly (P<0.01 or P<0.05), and ALP and TRACP tended to decrease, but there was no significant difference ( P>0.05). It can be seen that XDGM can increase PINP, provide rich organic raw materials for bone formation, and at the same time reduce the state of high-transformation bone metabolism.

3.6 Effect of XDGM on serum hormone levels in osteoporotic rats.

It can be seen from the attached table 20 that compared with the sham operation group, the CT and PTH of rats in the model group were significantly lower (P<0.01). Compared with the model group, the contents of CT and PTH in XDGM 1 group increased significantly (P<0.01 or P<0.05), and the contents of CT and PTH in

groups

2 and 6 also increased. It can be seen that XDGM improves the state of osteoporosis, making bone formation greater than bone resorption.

Table 19 The influence of the active components of the pharmaceutical composition of the present invention on the biochemical indicators of osteoporosis rats caused by ovariectomy

Table 20 The influence of the active components of the pharmaceutical composition of the present invention on hormone indexes in rats with osteoporosis caused by ovariectomy

4 Conclusion

The results of this experiment showed that XDGM significantly improved the bone morphology and biomechanical properties of rats, and increased bone density. It can be seen that XDGM has a significant effect on improving the osteoporotic bone in rats caused by ovariectomy. This improving effect is related to the increase of blood Ca, P, PINP content, reduction of high-transformation bone metabolism state, and stimulation of CT and PTH secretion.

5 Abnormal situations

Animals died after ovariectomized surgery for modeling, due to infection, internal bleeding and intestinal flatulence. At the end of 3 months after modeling, the remaining animals were 8 sham-operated animals and 60 models.

6 discussion

Osteoporosis (OP) is a systemic bone disease characterized by low bone mass and damage to bone microarchitecture, resulting in increased bone fragility and susceptibility to fracture (World Health Organization, WHO). Osteoporosis can be divided into primary and secondary categories. Primary osteoporosis includes postmenopausal osteoporosis (type Ⅰ, high conversion type) and senile osteoporosis (type Ⅱ, low conversion type). High turnover osteoporosis is a pathological state in which the rate of bone turnover is increased with increased bone resorption and bone formation. Low turnover osteoporosis is a pathological state in which bone resorption increases or decreases, but bone formation rate decreases, thus showing a low turnover rate. Secondary osteoporosis refers to osteoporosis caused by any disease or drug that affects bone metabolism. In the process of studying the pharmacology and mechanism related to postmenopausal osteoporosis, animal models such as mice, rats, rabbits, dogs, pigs, and sheep have been used. Reasonable selection of experimental animals requires compliance with the three principles of convenience, relevance, and suitability, and has reproducible stability. Try to make the OP histopathologically consistent with human performance. After castration, the bone mineral content decreases, the bone resorption increases rapidly, and the bone strength decreases, etc., which have many similarities with humans. Compared with other animal models, they have the advantages of fast reproduction, low cost, easy feeding and management, With the advantages of clear genetic background, it has been used as the most commonly used animal model, and is now widely used in the evaluation and development of new drugs for the treatment of postmenopausal osteoporosis. There are literatures showing that rats aged over six months are used for experiments, and the model effect is better. In this experiment, female rats aged about six months were used for bilateral ovariectomized surgery to create models for the study of menopausal osteoporosis.

Bone histomorphometry is a technology that can quantitatively observe and study the morphology and structure of bone tissue. At present, this technology has become a quantitative detection of the effect of drugs on animal models of osteoporosis, exploring the mechanism of drug action, and providing clinical information. One of the important scientific research methods guided by theory. There are different classification methods for bone histomorphometry, which can be divided into tibia, femur, lumbar spine and other parts according to the different parts of the sample; it can be divided into cortical bone, cancellous bone and femoral neck according to the different parts of observation; according to different data, it can be divided into Divided into static parameters and dynamic parameters. Cancellous bone is a compact trabecular bone located on the inner surface of the cortical bone. In the upper part of the tibia of the rat, the epiphysis of this part of the three-month-old rat is not closed, and it is not completely or partially closed until the 12-month-old. Due to blood supply and nutrition Rich, active metabolic function, strong bone regeneration ability, relatively high bone turnover rate, and drugs can directly reach the local area, so the upper part of the rat tibia is a sensitive part for inducing osteoporosis models and observing the effects of drugs, as well as various osteoporosis models And the most ideal site for drug prevention and control research. The results of this experiment showed that XDGM significantly increased rat tibial TBV%, decreased TRS%, TFS%, mAR, MAR, inhibited the high-transition bone metabolism state of ovariectomized rats, and improved bone morphological characteristics of osteoporotic rats.

Bone biomechanics is based on the theory of engineering mechanics. It studies the mechanical properties of bone tissue under external influence and the biological effect of bone after stress. It is a reliable method for evaluating bone quality. The study of bone biomechanics can generally be investigated from two aspects: structural mechanical properties and material mechanical properties. Structural mechanical properties mainly include indicators such as maximum load, fracture load, and elastic load. Material mechanical properties mainly include fracture strain, elastic modulus, etc.; The performance of structural mechanics is mainly related to the size and geometry of bone, while the mechanical properties of materials mainly reflect the strength and toughness of bone itself, and are related to the fine structure of bone, mineral content in bone and bone density. The results of this experiment showed that XDGM significantly increased the maximum load, bending strength and elastic modulus of rat femur, and significantly improved the bone biomechanical properties of osteoporotic rats.

Bone mineral density is the abbreviation of bone mineral density, and its meaning refers to the bone mass per unit bone tissue volume. Bone density is often used to evaluate bone fragility and the risk of fracture. Clinically, bone density measurement is used to diagnose osteoporosis and predict bone density. It is the best quantitative indicator for the risk of osteoporotic fractures, monitoring the natural history, and evaluating the efficacy of drug interventions. The results of this experiment showed that high-dose XDGM significantly increased the bone mineral density of ovariectomized osteoporotic rats.

Bone turnover biochemical markers are metabolites of bone tissue itself, referred to as bone markers. Bone turnover markers are divided into bone formation markers and bone resorption markers, the former represents the metabolites of osteoblast activity and bone formation, and the latter represents the metabolites of osteoclast activity and bone resorption, especially the degradation of bone matrix product. Bone formation markers mainly include serum alkaline phosphatase, osteocalcin, type Ⅰ procollagen N-terminal propeptide and so on. Bone resorption markers mainly include serum anti-tartrate acid phosphatase, serum type Ⅰ collagen cross-linked C-terminal peptide, etc. In this experiment, bone formation markers serum alkaline phosphatase, osteocalcin, type Ⅰ procollagen N-terminal propeptide and bone resorption marker serum tartrate-resistant acid phosphatase were selected. The results showed that XDGM significantly increased PINP, decreased BGP, ALP, TRACP. It can be seen that XDGM can increase PINP, provide rich organic raw materials for bone formation, and at the same time reduce the state of high-transformation bone metabolism.

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 glycoproteins, and the inorganic components are mainly hydroxyapatite, cations (calcium, magnesium, sodium, potassium, and strontium) and anions (phosphorus and chloride). Calcium in the human body accounts for about 1.5%-2% of the body weight, 99% 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 to phosphorus is 2:1, it can promote the absorption of calcium. When eating too much phosphorus-containing food, the ratio of calcium to phosphorus may 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 drops below enough to cope with normal physiological functions, many corresponding measures will be mobilized in the body at this time. Bone is a storehouse of calcium. When the concentration of calcium in the blood is high, calcium will be sent to the bone for storage, which is called osteogenesis; when the blood calcium concentration is low, calcium will be dissolved from the bone to supplement the lack of blood calcium. , called osteolysis. Both effects are regulated by hormones. Therefore, when the blood calcium is too low, the body automatically secretes PTH. PTH is synthesized and secreted by the parathyroid chief cells. PTH can stimulate the synthesis of vitamin D. Vitamin D is an accelerator of osteolysis, which can dissolve calcium in the bones into the blood Vitamin D can also promote intestinal absorption of calcium and reabsorption of calcium in urine, all of which can increase blood calcium concentration. 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 and promote osteogenesis while the activity of osteoclasts is enhanced. Cells release bone growth factors to promote bone formation and increase bone mass. Intermittent PTH injections have been widely demonstrated to stimulate bone formation. When the blood calcium concentration is too high, on the one hand, the excretion of calcium from the urine through the kidneys increases; Peptide hormones synthesized and secreted by parableb cells can reduce the concentration of calcium and phosphorus in plasma and inhibit the absorption of calcium and phosphorus. It has been widely used in the treatment of osteoporosis. The main mechanism is that it can significantly inhibit the activity of osteoclasts. Reduce bone resorption. The results of this experiment show that XDGM can increase the content of Ca and P in blood, and provide abundant inorganic raw materials for bone formation. At the same time, XDGM can stimulate the secretion of CT and PTH, promote bone formation and inhibit bone resorption.

Two, the active component of pharmaceutical composition of the present invention is to the influence of rat osteoporosis caused by retinoic acid

Purpose

The rat osteoporosis model induced by retinoic acid is used to observe the effect of the active components of the pharmaceutical composition of the present invention on the rat osteoporosis induced by retinoic acid, and judge its therapeutic effect on osteoporosis.

1 Experimental materials

1.1 Test product

1.1.2 Properties: brown powder.

1.1.3 Functions and indications: menopausal osteoporosis.

1.1.4 Intended clinical dosage: oral administration, 14.1g crude drug/day.

1.1.5 Content and specification: 2.96g crude drug/g dry cream powder.

1.1.7 Storage conditions: sealed storage.

1.1.8 Validity period: 2 years.

1.2 Positive drugs, tool drugs and main reagents

1.2.1 Xianling Gubao Capsule: Guizhou Tongjitang Pharmaceutical Co., Ltd., batch number: 1310079.

1.2.2 Retinoic acid: Meilun Biology, batch number: A0304A.

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

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

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

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

1.3 Experimental system

1.3.1 Animal species: SD rats.

1.3.2 Animal grade: SPF grade.

1.3.3 Sex and number of animals: female, 72 in total.

1.3.4 Animal age: 63-69 days.

1.3.5 Animal weight: 200-220g.

1.3.6 Animal source: purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., certificate number: 11400700047563, license number: SCXK (Beijing) 2012-0001, receiving date on May 21, 2014.

1.3.8 Quarantine process: The quarantine period for new animals is 3 days. Since no animals of suitable age for the experiment can be purchased, the experiment starts after 4 weeks of feeding the newly purchased animals. During this period, the animals drink water and eat normally, and are in good health without any disease or death. sign.

1.3.12 Labeling: 5% picric acid is used for labeling.

2 Experimental methods

2.1 Experimental Design Basis

2.1.2 Description of experimental system selection: Retinoic acid is a synthetic derivative of vitamin A, which is mainly used for the treatment of tumors and skin diseases, but it has obvious effects on bone quality and has adverse reactions of osteoporosis. Due to the typicality of the retinoic acid-induced osteoporosis model in the microstructural changes of bone tissue and the reversibility after drug action, this model has been listed by the Ministry of Health as one of the standard models for evaluating the efficacy of osteoporosis drugs.

2.2 Dosage and grouping

The animals were randomly divided into 6 groups according to body weight, 12 in each group, which were normal group, model group, positive drug (Xianlinggubao) group,

XDGM

1, 2, 6 groups, and the proposed clinical dosage of XDGM was 14.1g crude drug/ day, calculated on the basis of 60kg per person, the clinical dosage for rats is 8 times that of humans, that is, 1.88g crude drug/kg, and the content is 2.96g crude drug/g dry ointment powder, that is, 0.635 dry ointment powder/kg. Referring to the instructions of Xianling Gubao, the dosage for rats is set at 0.40g/kg, which is 8 times the clinical dosage for humans. See Schedule 21.

Table 21 The effect of active components of the pharmaceutical composition of the present invention on rat osteoporosis caused by retinoic acid experimental grouping and dosage

2.3 Administration method

2.4 Preparation and storage of the test product

The test drug was prepared with 0.5% CMC-Na to the concentration used in the experiment (see attached table 21), and after preparation, it was stored at 2-8°C for future use, and the positive drug was prepared immediately after use.

2.5 Administration of the test article

After 4 weeks of modeling, the test drug was given according to the experimental group, and the normal group and the model group were given 0.5% CMC-Na, once a day for 15 consecutive days.

2.6 Experimental steps

Newly received SD rats were quarantined for 3 days and fed for 4 weeks, and randomly grouped according to body weight, as described above. After grouping, retinoic acid 70mg/kg was given to build models, with a volume of 10ml/kg body weight, and was administered by gavage for 4 weeks, 6 days a week. The normal group was given solvent 0.5% CMC-Na. , once a day, for 15 consecutive days, the normal group and the model group were given 0.5% CMC-Na, the body weight was recorded once a week during the period of modeling and administration of the test drug, and all rats were intraperitoneally injected with hydrochloric acid on the 15th and 3rd days before being sacrificed Tetracycline 30mg/kg was used for bone fluorescence labeling. After the administration period, on the 2nd day, 10% chloral hydrate was used for intraperitoneal anesthesia with a volume of 0.35ml/100g body weight. Blood was collected from the abdominal aorta and sacrificed to detect indicators.

2.7 Detection indicators

2.7.1 Detection of bone histomorphological indicators

Take the proximal 1/3 of the left tibia of the rat, place it in 4% paraformaldehyde solution (PH7.4) for 24 hours, and then carry out dehydration. Xylene 2d. Next, the specimens were soaked in plastic polymer solution I, II and III for 3 days in sequence, and the above fixation, dehydration and soaking processes were all carried out at 4°C. Finally, add 400 μL of N,N-dimethyl-p-toluidine (N,N-Dimethyl-p-toluidine) into 100ml of pre-cooled (4°C) III solution, stir with a magnetic stirrer for 10 minutes, and then inject it into the penicillin vial About 7ml of liquid III, put the bone specimen into the bottom of the bottle in the same direction, use a syringe to evacuate the air in the embedding bottle, and then put it in a -20°C refrigerator to polymerize for about 1w, and it will become a colorless and transparent hard embedding block . After trimming, on a Reicheit-Jung2040 microtome, use a tungsten steel knife to cut out two longitudinal non-decalcified bone slices of 5 μm each, one of which is used for toluidine blue staining, and the other is used for fluorescence observation .

(1) Histomorphometry of trabecular bone:

(2) Morphometrics of the inner surface of the cortex:

2.7.2 Detection of bone biomechanical indicators

Before testing, the frozen right femurs of rats were taken out from the refrigerator and rewarmed at room temperature. Three-point bending test of rat femur: place the femur on the support of a microcomputer-controlled electronic universal testing machine with a span of 16 mm, press down on the middle part of the femur at a loading speed of 1 mm/min until the femur breaks, and detect its maximum load (N), flexural strength (MPa) and elastic modulus (GPa).

2.8 Relevant staff notification

2.9 Main instrument systems

2.10 Statistical Methods

The experimental data was analyzed and processed by SPSS statistical software, and the statistical results were expressed as mean ± standard deviation

3 results

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

It can be seen from the attached table 22 that the bone morphology index shows that compared with the normal group, the rat tibia TBV% of the model group is significantly reduced (P<0.01), and the TRS%, TFS%, and MAR are increased, but there is no significant difference ( P>0.05), it can be seen that the animals in the model group showed a certain degree of high transformation state, while the mAR was significantly reduced (P<0.05), and the OSW was reduced. It can be seen that this model has an impact on the mineralization of the bone cortex; compared with the model group, the positive Yaoxian Linggubao significantly increased rat tibial TBV% (P<0.05), XDGM1 and 2 groups significantly increased rat tibial TBV% (P<0.05 or P<0.01), TRS%, TFS%, MAR decreased, But no significant difference was found (P>0.05). It can be seen from Figure 2 that the number and volume of bone trabeculae in the proximal tibia of rats in the XDGM group were increased compared with those in the model group. It can be seen that XDGM can significantly improve the bone morphological characteristics of osteoporotic rats.

Table 22 The influence of the active components of the pharmaceutical composition of the present invention on the bone morphological indexes of rats with osteoporosis caused by retinoic acid

Note: Compared with the normal group: ^Δ P<0.05, ^ΔΔ P<0.01; compared with the model group: ^* P<0.05, ^** P<0.01.

3.2 XDGM significantly improved bone biomechanical properties of retinoic acid-induced osteoporosis rats.

It can be seen from the attached table 23 that the bone biomechanical indicators show that compared with the normal group, the maximum load, bending strength and elastic modulus of the rat femur in the model group 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 rat femur (P<0.01 or P<0.05),

XDGM

1 and 2 groups significantly increased the maximum load and bending strength of rat femur (P<0.05 or P<0.01), XDGM1 XDGM also significantly increased the elastic modulus of rat femur (P<0.01), showing that XDGM can significantly improve the bone biomechanical properties of osteoporotic rats.

Table 23 The influence of the active components of the pharmaceutical composition of the present invention on the bone biomechanical indexes of rats with osteoporosis caused by retinoic acid

4 Conclusion

The results of this experiment showed that XDGM significantly increased the TBV% of the rat tibia, and significantly increased the maximum load, bending strength and elastic modulus of the femur. It can be seen that XDGM significantly improved the bone morphology and bone biomechanical properties of rats with osteoporosis induced by retinoic acid. effect.

5 Abnormal situations

Four weeks after intragastric administration of retinoic acid, one animal in

XDGM

1 and 2 groups had a fracture, which prevented them from eating, so they were killed. Anatomy revealed a tibial fracture.

3. Effects of the active components of the pharmaceutical composition of the present invention on the prevention and treatment of osteoporosis in rats caused by retinoic acid

Purpose

The rat osteoporosis model induced by retinoic acid is used to observe the effect of the active components of the pharmaceutical composition of the present invention on the rat osteoporosis induced by retinoic acid, and judge its preventive effect on osteoporosis.

1 Experimental materials

1.1 Test product

1.1.2 Properties: brown powder.

1.1.3 Functions and indications: menopausal osteoporosis.

1.1.4 Intended clinical dosage: oral administration, 14.1g crude drug/day.

1.1.5 Content and specification: 2.96g crude drug/g dry cream powder.

1.1.7 Storage conditions: sealed storage.

1.1.8 Validity period: 2 years.

1.2 Positive drugs, tool drugs and main reagents

1.2.1 Xianling Gubao Capsule: Guizhou Tongjitang Pharmaceutical Co., Ltd., batch number: 1406020.

1.2.2 Retinoic acid: Meilun Biology, batch number: A0304A.

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

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

1.3 Experimental system

1.3.1 Animal species: SD rats.

1.3.2 Animal level: clean level.

1.3.3 Sex and number of animals: female, 60 of which are used for formal experiments and 5 for practice.

1.3.4 Animal age: 3 months old.

1.3.5 Animal weight: 220-260g.

1.3.6 Source of animals: purchased from Hebei Experimental Animal Center, certificate number: 1501078, license number: SCXK (Ji) 2013-1-003, date of receipt on January 23, 2015.

1.3.8 Quarantine process: The quarantine period for new animals is 4 days. During this period, the animals drink water and eat normally, are in good health, and have no signs of disease or death.

1.3.12 Labeling: 5% picric acid is used for labeling.

2 Experimental methods

2.1 Experimental Design Basis

2.2 Dosage and grouping

The animals were randomly divided into 6 groups according to body weight, 10 animals in each group, which were normal group, model group, positive drug (Xianling Gubao) group, XDGM1, 2, and 6 groups, and the proposed clinical dosage of XDGM was 14.1g crude drug/day , calculated on the basis of 60kg per person, the dose for rats is 8 times the clinical dosage for humans, namely 1.88 crude drug/kg, and the content is 2.96g crude drug/g dry ointment powder, i.e. 0.635 dry ointment powder/kg. Referring to the instructions of Xianling Gubao, the dosage for rats is set at 0.40g/kg, which is 8 times the clinical dosage for humans. See Schedule 24.

Table 124 Experimental grouping of the effect of the active components of the inventive pharmaceutical composition on the prevention and treatment of osteoporosis in rats caused by retinoic acid

2.3 Administration method

2.4 Preparation and storage of the test product

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

2.5 Administration of the test article

The test drug was given by intragastric administration according to the experimental group, and the normal group and the model group were given 0.5% CMC-Na, once a day for 2 consecutive weeks.

2.6 Experimental steps

Newly received SD rats, quarantined for 4 days, were randomized by body weight, as previously described. Except the normal group, each group was given retinoic acid 70mg/kg in the morning, with a volume of 10ml/kg body weight, the normal group was given 0.5% CMC-Na, the afternoon administration group was given the corresponding test drug, and the normal group and the model group were given 0.5% CMC-Na. Na, gavage continuously for 2w, and record body weight once a week. All rats were intraperitoneally injected with tetracycline hydrochloride 30mg/kg on the 14th and 3rd days before execution for bone fluorescence labeling. Aldehyde intraperitoneal anesthesia, the volume is 0.35ml/100g body weight, abdominal aortic blood sampling and sacrifice, testing indicators.

2.7 Detection indicators

2.7.1 Detection of bone histomorphological indicators

(1) Histomorphometry of trabecular bone:

(2) Morphometrics of the inner surface of the cortex:

2.7.2 Detection of bone biomechanical indicators

2.8 Relevant staff notification

2.9 Main instrument systems

2.10 Statistical Methods

3 results

It can be seen from the attached table 25 that the bone morphology index shows that compared with the normal group, the rat tibia TBV% in the model group is significantly reduced (P<0.01), TRS% is significantly increased (P<0.01), TFS%, OSW, MAR have increased, but no significant difference (P>0.05), it can be seen that the animals in the model group showed a certain degree of high transformation state, and the degree of bone resorption was stronger; compared with the model group, the positive drug Xianling Gubao significantly increased the Tibia TBV% (P<0.05), decreased TRS% (P<0.01), XDGM1, 2 groups significantly increased rat tibial TBV% (P<0.05 or P<0.01), decreased TRS% (P<0.05 or P<0.01 ), the XDGM 1 group significantly reduced MAR (P<0.05), and there was no significant difference in other indicators (P>0.05). It can be seen from Figure 3 that the number and volume of bone trabeculae in the proximal tibia of rats in the XDGM group were increased compared with those in the model group. It can be seen that XDGM significantly reduced the bone resorption intensity of osteoporotic rats, improved the high transformation state to a certain extent, and improved bone loss. Morphological characteristics of bone in osteoporotic rats.

Table 25 The influence of the active components of the pharmaceutical composition of the present invention on the bone morphological indexes of rats with osteoporosis caused by refractory acid

It can be seen from the attached table 26 that the bone biomechanical indicators show that compared with the normal group, the maximum load, bending strength and elastic modulus of the rat femur in the model group 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 rat femur (P<0.05), middle and high doses of XDGM significantly increased the maximum load, bending strength and elastic modulus of rat femur (P<0.05 or P<0.01), it can be seen XDGM can significantly improve the bone biomechanical properties of osteoporotic rats.

Table 26 The influence of the active components of the pharmaceutical composition of the present invention on bone biomechanical indicators in rats with osteoporosis caused by retinoic acid

4 Conclusion

The results of this experiment showed that XDGM significantly increased the TBV% of the rat tibia, increased the maximum load, bending strength and elastic modulus of the femur, and it can be seen that XDGM can significantly improve the bone morphology and bone biomechanical properties of rats with osteoporosis induced by retinoic acid .

5 discussions

Retinoic acid is a synthetic derivative of vitamin A. It is mainly used for the treatment of tumors and skin diseases, but it has obvious effects on bone quality and has adverse reactions of osteoporosis. The animal model of rat osteoporosis induced by retinoic acid was first created by Chinese scholar Shao Jinying in 1989. This model has been listed by the Ministry of Health as one of the standard models for evaluating the efficacy of osteoporosis drugs. In this experiment, rats were induced with osteoporosis by intragastric administration of retinoic acid for 2 weeks, and it was intended to simulate a high-transition osteoporosis animal model.

Bone histomorphometry is a technology that can quantitatively observe and study the morphology and structure of bone tissue. At present, this technology has become a quantitative detection of the effect of drugs on animal models of osteoporosis, exploring the mechanism of drug action, and providing clinical information. One of the important scientific research methods guided by theory. There are different classification methods for bone histomorphometry, which can be divided into tibia, femur, lumbar spine and other parts according to the different parts of the sample; it can be divided into cortical bone, cancellous bone and femoral neck according to the different parts of observation; according to different data, it can be divided into Divided into static parameters and dynamic parameters. Cancellous bone is a compact trabecular bone located on the inner surface of the cortical bone. In the upper part of the tibia of the rat, the epiphysis of this part of the three-month-old rat is not closed, and it is not completely or partially closed until the 12-month-old. Due to blood supply and nutrition Rich, active metabolic function, strong bone regeneration ability, relatively high bone turnover rate, and drugs can directly reach the local area, so the upper part of the rat tibia is a sensitive part for inducing osteoporosis models and observing the effects of drugs, as well as various osteoporosis models And the most ideal site for drug prevention and control research. In this experiment, three-month-old female animals were used to study osteoporosis. The results showed that the TBV% of rats in the model group was significantly reduced, and the TRS% was significantly increased. XDGM significantly increased the rat tibia TBV%, decreased TRS%, and MAR. It can be seen that XDGM significantly reduces the bone resorption intensity of osteoporotic rats, improves the high transformation state to a certain extent, and improves the bone morphological characteristics of osteoporotic rats. Bone biomechanics is based on the theory of engineering mechanics. It studies the mechanical properties of bone tissue under external influence and the biological effect of bone after stress. It is a reliable method for evaluating bone quality. The study of bone biomechanics can generally be investigated from two aspects: structural mechanical properties and material mechanical properties. Structural mechanical properties mainly include indicators such as maximum load, fracture load, and elastic load. Material mechanical properties mainly include fracture strain, elastic modulus, etc.; The performance of structural mechanics is mainly related to the size and geometry of bone, while the mechanical properties of materials mainly reflect the strength and toughness of bone itself, and are related to the fine structure of bone, mineral content in bone and bone density. The results of this experiment showed that the maximum load, bending strength and elastic modulus of the rat femur in the model group were significantly reduced, and the above three indexes were significantly increased in the XDGM group. It can be seen that XDGM can significantly improve the bone biomechanical properties of osteoporotic rats.

in conclusion

The experimental results of the rat osteoporosis model induced by ovariectomy showed that

XDGM

1 and 2 groups significantly increased the volume percentage of tibial trabecular bone, decreased the high-transition bone metabolism state of the rats after ovariectomy, and improved the bone morphology of osteoporotic rats. Properties; increase femoral maximum load, bending strength and elastic modulus, improve bone biomechanical properties of osteoporotic rats; increase bone density of osteoporotic rats; at the same time XDGM can increase serum Ca, P, PINP content and CT, PTH levels , provide rich raw materials and hormone stimulation for bone formation, improve the state of osteoporosis, and make bone formation greater than bone resorption. In addition, the results of XDGM prevention and treatment of retinoic acid-induced osteoporosis model also showed that XDGM1 and 2 groups significantly increased the volume percentage of tibial trabecular bone, significantly increased the maximum load, bending strength and elastic modulus of the femur, and had a significant effect on the bone morphology of osteoporotic rats. Significantly improved bone biomechanical properties.

Based on the above results, XDGM can reduce the state of high-transition bone metabolism, improve bone morphology and bone biomechanical properties, and increase bone density, suggesting that XDGM can be used in the treatment of menopausal osteoporosis.

Detailed ways

Example 1:

The raw material medicine formula is: salt dodder 380g, epimedium 220g, rehmannia glutinosa 200g, salvia miltiorrhiza 155g, calcined oyster 55g, salt psoralen 155g.

A. Weigh the dodder and psoralen according to the prescription amount, extract twice with 70% ethanol, each time for 2 hours, add 10 times the amount of ethanol as the medicinal material, filter the extract, combine, concentrate under reduced pressure to 60°C and heat test Clear ointment with a relative density of 1.10±0.05, set aside;

B. Weigh Epimedium, Rehmannia glutinosa, and Salvia miltiorrhiza according to the prescription amount, add 12 times the amount of water to extract three times, the extraction time is 1 hour, filter the extract, concentrate under reduced pressure to a clear paste with a thermal measurement relative density of 1.10±0.05 at 60°C , combining the concentrated solutions three times, and merging with the alcohol extraction paste obtained in step A, concentrating to a thick paste with a thermal relative density of 1.20±0.05 at 60°C, drying and pulverizing to obtain a fine powder;

Example 2:

The raw material medicine formula is: salt dodder 400g, epimedium 200g, rehmannia glutinosa 200g, salvia miltiorrhiza 160g, calcined oyster 53g, salt psoralen 160g.

A, take dodder and psoralen by prescription quantity, dodder is broken first, extract twice with 60% ethanol, each time 1.5 hours, first time add alcohol 10 times amount, second time add alcohol 8 times amount, extract Filtrate, combine, concentrate under reduced pressure to a clear paste with a thermal measurement relative density of 1.10±0.05 at 60°C, and set aside;

B. Weigh Epimedium, Rehmannia glutinosa, and Salvia miltiorrhiza according to the prescription amount, add water to extract three times, add 12 times the amount of water for the first time, and extract for 2 hours; add 10 times the amount of water for the second and third times, and the extraction time is 1 hour , filter the extract, concentrate under reduced pressure to a clear paste with a thermal relative density of 1.10±0.05 at 60°C, combine three concentrates, and combine with the alcohol extraction clear paste obtained in step A, and concentrate to a thermal relative density of 1.20±0.05 at 60°C The thick paste is dried and pulverized to obtain fine powder;

Example 3:

Cuscuta 600g, Epimedium 100g, Rehmannia glutinosa 300g, Salvia miltiorrhiza 80g, oyster 80g, psoralen 80g.

A, take dodder and psoralen by prescription quantity, dodder is broken first, extract twice with 50% ethanol, each time 1.5 hours, add alcohol 10 times of amount for the first time, add alcohol 8 times of amount for the second time, extract Filtrate, combine, concentrate under reduced pressure to a clear paste with a thermal measurement relative density of 1.10±0.05 at 60°C, and set aside;

B. Weigh Epimedium, Rehmannia glutinosa, and Salvia miltiorrhiza according to the prescription amount, add 10 times the amount of water to extract three times, the extraction time is 2 hours, filter the extract, concentrate under reduced pressure to a clear paste with a thermal measurement relative density of 1.10±0.05 at 60°C , combining the concentrated solutions three times, and merging with the alcohol extraction paste obtained in step A, concentrating to a thick paste with a thermal relative density of 1.20±0.05 at 60°C, drying and pulverizing to obtain a fine powder;

D. Combine the oyster powder obtained in step C and the fine powder obtained in step B, granulate according to the conventional process, granulate, and pack into capsules.

Example 4:

Cuscuta 200g, Epimedium 300g, Rehmannia glutinosa 100g, Salvia miltiorrhiza 240g, oyster 25g, psoralen 240g.

D. Combining the oyster powder obtained in step C and the fine powder obtained in step B, granulating and sizing according to a conventional process to prepare granules.

Example 5:

Cuscuta 500g, Epimedium 220g, Rehmannia glutinosa 220g, Salvia miltiorrhiza 180g, oyster 60g, psoralen 180g.

B. Weigh Epimedium, Rehmannia glutinosa, and Salvia miltiorrhiza according to the prescription amount, add 10-12 times the amount of water to extract three times, the extraction time is 1-2 hours, filter the extract, concentrate under reduced pressure to 60°C, and the relative density of thermal measurement is 1.10± 0.05 clear cream, combine the concentrated solution three times, and combine with the alcohol extraction clear cream obtained in step A, and concentrate to a thick cream with a thermal relative density of 1.20±0.05 at 60°C;

D. Combine the oyster powder obtained in step C and the thick paste obtained in step B, and prepare oral liquid according to a conventional process.

Embodiment 6:

Cuscuta 330g, Epimedium 180g, Rehmannia glutinosa 180g, Salvia miltiorrhiza 125g, oyster 60g, psoralen 150g.

D. The oyster powder obtained in step C and the thick paste obtained in step B are prepared according to conventional techniques to obtain injections.

Claims

A pharmaceutical composition for preventing or treating primary osteoporosis, characterized in that the composition comprises the following components in parts by weight: 200-600 parts of Cuscuta, 100-300 parts of Epimedium, and 100-300 parts of Rehmannia glutinosa , Salvia 80-240 parts, oysters 25-80 parts, psoralen 80-240 parts.
The composition according to claim 1, characterized in that the composition comprises the following components by weight: 200 parts of dodder, 300 parts of epimedium, 100 parts of rehmannia glutinosa, 240 parts of salvia miltiorrhiza, 25 parts of oysters, 240 parts of psoralen share.
The composition according to claim 1, characterized in that the composition comprises the following components by weight: 600 parts of dodder, 100 parts of epimedium, 300 parts of rehmannia root, 80 parts of salvia miltiorrhiza, 80 parts of oyster, 80 parts of psoralen share.
The composition according to claim 1, characterized in that the composition comprises the following components by weight: 400 parts of dodder, 200 parts of epimedium, 200 parts of rehmannia glutinosa, 160 parts of salvia miltiorrhiza, 53 parts of oyster, 160 parts of psoralen share.
The composition according to claim 1, characterized in that the composition comprises the following components by weight: 380 parts of dodder, 220 parts of epimedium, 200 parts of rehmannia glutinosa, 155 parts of salvia miltiorrhiza, 55 parts of oyster, 155 parts of psoralen share.
The composition according to any one of claims 1-5, characterized in that among the components of the composition, dodder is preferably dodder salt, oyster is preferably calcined oyster, and psoralen is preferably psoralen salt.
The composition according to any one of claims 1-5, characterized in that the dosage form of the composition is capsule, tablet, pill, oral liquid, granule, injection or powder.
The composition according to any one of claims 1-5, characterized in that the active component of the composition is made by the following steps:

A. Weigh the dodder and psoralen according to the prescription amount, extract twice with 50-70% ethanol, each time for 1-3 hours, add ethanol in an amount 6-10 times of the medicinal material, filter the extract, combine, and decompress Concentrated to 60 ℃ heat measurement relative density of 1.10 ± 0.05 clear paste, set aside;

B. Weigh Epimedium, Rehmannia glutinosa, and Salvia miltiorrhiza according to the prescription amount, add 8-12 times the amount of water to extract three times, the extraction time is 1-3 hours, filter the extract, concentrate under reduced pressure to 60 ° C, and the relative density of thermal measurement is 1.10 ± 0.05 clear paste, combine the concentrated solution three times, and combine with the alcohol extraction clear paste obtained in step A, concentrate to a thick paste with a relative density of 1.20±0.05 by thermal measurement at 60°C, dry and pulverize to obtain a fine powder;

C. Oysters are crushed into 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 invention.
Composition according to claim 7, is characterized in that the preparation technology of tablet is:

A. Weigh the dodder and psoralen according to the prescription amount, extract twice with 50-70% ethanol, each time for 1-3 hours, add ethanol in an amount 6-10 times of the medicinal material, filter the extract, combine, and decompress Concentrated to 60 ℃ heat measurement relative density of 1.10 ± 0.05 clear paste, set aside;

B. Weigh Epimedium, Rehmannia glutinosa, and Salvia miltiorrhiza according to the prescription amount, add 8-12 times the amount of water to extract three times, the extraction time is 1-3 hours, filter the extract, concentrate under reduced pressure to 60 ° C, and the relative density of thermal measurement is 1.10 ± 0.05 clear paste, combine the concentrated solution three times, and combine with the alcohol extraction clear paste obtained in step A, concentrate to a thick paste with a relative density of 1.20±0.05 by thermal measurement at 60°C, dry and pulverize to obtain a fine powder;

C. Oysters are crushed into the finest powder and sterilized by 60Co irradiation;

D. Combine the oyster powder obtained in step C and the fine powder obtained in step B, granulate according to the conventional process, granulate, and tablet.
Composition according to claim 9, is characterized in that the preparation technology of tablet is preferably:

A, take dodder and psoralen by prescription quantity, dodder is broken first, extract twice with 60% ethanol, each time 1.5 hours, first time add alcohol 10 times amount, second time add alcohol 8 times amount, extract Filtrate, combine, concentrate under reduced pressure to a clear paste with a thermal measurement relative density of 1.10±0.05 at 60°C, and set aside;

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

C. Oysters are crushed into the finest powder and sterilized by 60Co irradiation;

D. Combine the oyster powder obtained in step C and the fine powder obtained in step B, granulate according to the conventional process, granulate, and tablet.
The composition according to any one of claims 1-5, characterized in that the pharmaceutical composition is used in the preparation of postmenopausal osteoporosis medicine.
The composition according to any one of claims 1-5, characterized in that the pharmaceutical composition is used in the preparation of drugs for reducing the degradation product β-CTX of β glue.
The composition according to any one of claims 1-5, characterized in that the pharmaceutical composition is used in the preparation of medicines for the treatment of deficiency of kidney essence.
According to the composition described in any one of claims 1-5, it is characterized in that the pharmaceutical composition increases serum Ca, P content, increases bone formation marker PINP content, reduces bone formation marker BGP content, and reduces bone resorption marker in preparation. ALP and TRACP content or increase the application of CT, PTH content of drugs.
According to the composition described in any one of claims 1-5, it is characterized in that the pharmaceutical composition increases tibial trabecular bone volume percentage in preparation, increases femur maximum load, bending strength and modulus of elasticity, improves bone morphology and bone biology Application of mechanical properties in medicine.