CN118557703A - Application of bone polypeptide in the preparation of oral medicine for preventing and treating osteoporosis - Google Patents

Abstract

The invention discloses application of bone polypeptide in preparation of an oral medicine for preventing and treating osteoporosis, and belongs to the field of medicines. The invention builds a rat retinoic acid osteoporosis model, and compared with a model group, after the treatment of bone polypeptide with different dosages for 4 weeks, the bone polypeptide can reduce spleen and liver enlargement caused by retinoic acid; relieving testis and uterus atrophy caused by gonadal injury, and improving anorexia and weight loss caused by retinoic acid; can improve the level of microelements such as osteoporosis rat bone Ca, mg, P, fe, zn, raise the content of calcium and phosphorus in blood, and reduce the serum ALP level, which indicates that the bone polypeptide can improve bone metabolism, increase bone deposition, enhance bone density and improve bone quality and bone strength; can raise the activity of antioxidant enzyme SOD and GSH-Px in bone marrow of osteoporosis rat, reduce MDA production and reduce the oxidative damage of bone tissue. The invention provides a new method and a new direction for preventing and treating osteoporosis.

Description

Application of bone polypeptide in the preparation of oral medicine for preventing and treating osteoporosis

Technical Field

The present invention relates to the field of medicine, and in particular to the application of bone polypeptide in the preparation of oral medicine for preventing and treating osteoporosis.

Background Art

Osteoporosis is a degenerative disease of the skeletal system characterized by decreased bone mass, decreased bone density, damaged bone microstructure, increased fragility, and increased risk of fractures. It is common in middle-aged and elderly people, especially postmenopausal women, and seriously endangers their quality of life and health. The main clinical manifestations are bone pain, hunchback, easy fractures, limited movement, and even inability to take care of oneself. The disease is not easy to detect in the early stage, and the course of the disease is long. The incidence and fracture rates are as high as 50% and 33% respectively, and the disability rate and mortality rate are high. At present, there are more than 200 million osteoporosis patients in the world. With the intensification of global aging, osteoporosis has become a major public health problem faced by all mankind. Research on the prevention and treatment of osteoporosis is extremely urgent. At present, the first choice for the prevention and treatment of osteoporosis and fractures in the world is still estrogen replacement therapy (ERT), but estrogen is an anti-bone resorption drug. Long-term use will lead to decreased bone turnover and poor bone renewal. It will also increase the potential risk of breast cancer by 25% and the risk of endometrial cancer by 4-8 times. Therefore, finding osteoporosis prevention and treatment drugs that have definite efficacy, few toxic side effects, and can be taken for a long time is an urgent task in the field of osteoporosis drug research and development.

Food-derived active peptides are safe, highly bioactive, and have broad market development prospects. Osteogenic peptides derived from the bone marrow of different animals are an endogenous osteogenic growth factor with a conservative amino acid sequence and high homology. They participate in the communication between bone cells and have a strong effect on promoting the activity of osteoblasts and fibroblasts, thereby strongly promoting the mineralization of bone matrix, the increase of bone mass, and the enhancement of alkaline phosphatase activity. At present, osteogenic peptide products are mostly used in bone scaffolds, repair, and delivery. Literature has reported that many bone peptide products from different animal bones have the effects of regulating bone metabolism, promoting osteoblast growth, and increasing bone deposition. At present, the market for bone peptide products is dominated by injectable bone peptide products, and related research is mostly clinical observation, with relatively few basic studies, and basic research on osteoporosis using oral bone peptides from pig bones is even rarer.

Summary of the invention

The purpose of the present invention is to provide an application of bone polypeptide in the preparation of an oral drug for preventing and treating osteoporosis, so as to solve the problems existing in the above-mentioned prior art. Bone polypeptide can prevent and treat osteoporosis by reducing the damage of retinoic acid to rat gonads and the oxidative damage to bone tissue, increasing bone mineralization, and enhancing bone density.

To achieve the above object, the present invention provides the following solutions:

The present invention provides an application of a bone polypeptide in the preparation of an oral medicine for preventing and treating osteoporosis, wherein the osteoporosis is osteoporosis caused by retinoic acid.

Preferably, the bone polypeptide is derived from pig bones, and peptides with a relative molecular weight lower than 10,000 Da account for 100%.

Preferably, the bone polypeptide reduces gonadal damage and bone tissue oxidative damage caused by retinoic acid, increases bone mineralization, and enhances bone density, thereby preventing and treating osteoporosis.

The present invention also provides the use of the bone polypeptide in preparing a medicine for alleviating gonad damage and/or bone tissue oxidative damage caused by retinoic acid.

The present invention also provides the use of the bone polypeptide in preparing medicine for increasing bone mineralization and enhancing bone density.

Preferably, the drug has bone polypeptide as the only active ingredient and is an oral drug.

The present invention also provides an oral medicine for preventing and treating osteoporosis, wherein the oral medicine has bone polypeptide as the only effective component, the bone polypeptide is a bone polypeptide derived from pig bones, peptides with a relative molecular weight lower than 10,000 Da account for 100%, and the total nitrogen content of each gram of the bone polypeptide is 0.157 grams.

The present invention discloses the following technical effects:

The present invention uses 3-month-old SD rats as experimental subjects, and administers retinoic acid for 2 weeks by gavage, and successfully replicates an osteoporosis animal model. After 4 weeks of treatment with different doses of bone polypeptide, compared with the model group, bone polypeptide can reduce the enlargement of the spleen and liver caused by retinoic acid; reduce testicular and uterine atrophy caused by gonadal damage, and improve the loss of appetite and weight loss caused by retinoic acid; it can improve the levels of trace elements such as bone Ca, Mg, P, Fe, and Zn in osteoporotic rats, increase the calcium and phosphorus content in the blood, and reduce the serum ALP level, indicating that bone polypeptide can improve bone metabolism, increase bone deposition, enhance bone density and improve bone quality and bone strength; it can increase the activity of bone marrow antioxidant enzymes SOD and GSH-Px in osteoporotic rats, reduce MDA production, and reduce oxidative damage to bone tissue. The experimental results of the present invention show that bone polypeptide can play a role in preventing and treating osteoporosis at a dose of 50-200 mg/kg by counteracting the damage of retinoic acid to rat gonads and oxidative damage to bone tissue, increasing bone mineralization, and enhancing bone density.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

Figure 1 shows the effects of bone polypeptide on body weight (A), food intake (B) and water intake (C) of rats with osteoporosis induced by retinoic acid;

Figure 2 shows the effect of bone polypeptide on serum ALP (A), Ca (B) and P (C) levels in rats with osteoporosis induced by retinoic acid;

Figure 3 shows the effects of bone polypeptide on the contents of bone marrow SOD (A), GSH-Px (B) and MDA (C) in rats with osteoporosis model induced by retinoic acid.

DETAILED DESCRIPTION

Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

Example 1

1. Experimental Materials

Bone polypeptide (total nitrogen 0.157g/g protein powder, peptides with relative molecular weight less than 10000Da accounted for 100%), produced by Hebei Zhitong Biopharmaceutical Co., Ltd.; Fosamax (alendronate sodium tablets) produced by Hangzhou Merck Co., Ltd.; alkaline phosphatase (ALP), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA), calcium, phosphorus, magnesium and other test kits were purchased from Nanjing Jiancheng Biological Research Institute Co., Ltd.; hydrochloric acid, anhydrous ethanol, glacial acetic acid, methanol, analytical grade, provided by Tianjin Komiou Chemical Reagent Co., Ltd. Retinoic acid, produced by Chongqing Huabang Pharmaceutical Co., Ltd.

2. Experimental Animals

60 3-month-old SPF SD rats, half male and half female, with a body weight of 240-260 g, were provided by Sibeifu (Beijing) Biotechnology Co., Ltd., Animal License No.: SCXK (Beijing) 2019-0010. They were housed in a barrier environment IVC system with a temperature of 20-23°C, a relative humidity of 40-60%, a light of 12 hours/day, and free food.

3. Experimental methods

3.1 Grouping, modeling, and drug administration

As shown in Table 1, rats were randomly divided into 6 groups according to body weight: normal group, model group, Fosamax (positive control), low-, medium-, and high-dose groups of bone polypeptide. Except for the rats in the normal group, the remaining rats were gavaged with 70 mg/kg retinoic acid for 2 consecutive weeks to construct the OP model. The construction method was based on the study of the role of bovine bone peptide in preventing osteoporosis by Li Yaran et al. After the model was successfully established, rats in the normal group and model group were gavaged with 0.5% sodium carboxymethyl cellulose, the Fosamax group was gavaged with 1.33 mg/kg·day sodium alendronate, and the low-, medium-, and high-dose groups of bone polypeptide were gavaged with 50, 100, and 200 mg/kg·day bone polypeptide, respectively. The administration volume was 10 mL/kg, once a day, and the drug intervention was continued for 4 weeks.

Table 1 Effect of bone polypeptide on retinoic acid-induced osteoporosis model in rats Experimental groups and dosage

3.2 Source

After 4 weeks of drug administration according to the experimental design, the rats were fasted overnight but not water-deprived. The next day, they were anesthetized with sodium pentobarbital and killed after blood was drawn from the abdominal aorta. Serum was prepared to test calcium (Ca), phosphorus (P), and alkaline phosphatase (ALP). The main organs, heart, liver, spleen, kidney, adrenal gland, and gonads (uterus, prostate, testis) were taken to measure organ indexes. The left femur was taken to test bone wet weight, dry weight, bone length, and width; the right tibia was taken to test bone density; the right femur was taken to test bone trace elements, and the left tibia bone marrow was taken to test oxidation indicators.

3.3 Observation of general condition of rats

During the experiment, body weight and food intake were recorded once a week, and the general conditions of the rats, including their mental state, were observed.

3.4 Determination of major organ indexes

The heart, liver, spleen, kidney, adrenal gland, uterus, prostate and testicles were weighed and the organ index (the number of organs in grams per kilogram of body weight) was calculated.

3.5 Determination of serum ALP, Ca, and P levels

Serum was prepared according to the kit instructions and the Ca, P and ALP in serum were detected.

3.6 Bone weight, width, length, and bone density measurement

The bone tissue was cleaned of attached muscles and soft tissues, the wet weight and dry weight of the left femur were measured, and the femur length and bone width were measured with a vernier caliper. The right tibia was scanned with a Hologic QDR 2000 dual-energy X-ray absorptiometry to measure bone density.

3.7 Detection of bone metabolism related indicators

The bone specimens were dried to constant weight and digested at 108°C with 6 mol/L HCl. The filtrate was diluted to an appropriate concentration and the bone Ca, P, Mg, Fe, Zn, and Mn concentrations were detected according to the kit instructions.

3.8 Bone marrow oxidation index detection

After sampling, the left tibial bone marrow cavity was exposed as soon as possible, and the flushing fluid obtained by flushing with normal saline was used to measure bone marrow SOD, GSH-Px and MDA according to the instructions of the ELISA kit.

4. Statistical processing

All data are used SPSS17.0 statistical software was used for one-way analysis of variance, and P < 0.05 was considered statistically significant.

5. Results and Discussion

5.1 Effects of bone polypeptide on appearance, body weight, and diet of osteoporotic rats

As shown in Figure 1, during the experiment, the appearance, diet, and autonomous activities of the rats in the normal group were normal. The model group rats had yellow fur, slow reaction, reduced diet and water intake, and weight loss after one week of modeling. The above conditions did not change significantly until the end of the experiment. The body weight of the model group rats decreased by 13.42%, 15.26%, 15.12%, and 15.70% at 3, 4, 5, and 6 weeks after modeling, respectively, with significant statistical differences (P<0.05). The above conditions improved during the intervention phase of each treatment group. At the end of the experiment, the weight of the rats in the treatment group increased compared with the model group, among which the Fosamax group increased by 11.82%, and the low, medium, and high doses of bone polypeptide groups increased by 11.28%, 13.46%, and 13.77%, respectively; the autonomous activities of the rats in the treatment group increased, and their diet, water intake, and appearance improved slightly.

5.2 Effects of bone polypeptide on organ indexes in osteoporotic rats

As shown in Table 2, compared with the normal group, the organ indexes of the rats in the model group changed significantly (P<0.05 or P<0.01), among which the spleen increased from 1.52g/kg to 1.96g/kg (increase of 29.33%, P<0.01), the liver increased from 23.53g/kg to 31.70g/kg (increase of 34.71%, P<0.01), and the heart, kidney, adrenal gland, prostate, testicle and uterus were significantly reduced; each therapeutic drug had a tendency to reverse the above changes. Compared with the model group, the heart atrophy (3.55g/kg, P<0.05), spleen enlargement (1.73g/kg, P<0.01), and liver enlargement (25.33g/kg, P<0.05) of the rats in the Fosamax group were significantly reduced. The spleen enlargement was significantly reduced after bone polypeptide treatment (P<0.05): 1.80g/kg in the low-dose group, 1.78g/kg in the medium-dose group, and 1.79g/kg in the high-dose group. The above data show that bone polypeptide can reduce gonad damage, spleen, and compensatory hypertrophy of adrenal glands caused by osteoporosis induced by retinoic acid in rats.

Table 2 Effects of bone polypeptide on organ indexes of rat osteoporosis model induced by retinoic acid ( Unit: g/kg·BW)

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

5.3 Effects of bone peptides on serum ALP and blood mineral indexes

Alkaline phosphatase is closely related to bone formation and bone mineralization, and is a specific sensitive indicator for the diagnosis, differential diagnosis, course evolution, dynamic observation and prognosis estimation of metabolic bone disease. As shown in Figure 2, compared with the normal group, the serum alkaline phosphatase ALP content in the model group increased from 56.33IU/L to 151.06IU/L (P<0.05), Ca decreased from 2.38mmol/L to 1.92mmol/L (P<0.01), and P decreased from 2.22mmol/L to 1.62mmol/L (P<0.01). The serum ALP levels of the low, medium and high dose groups of bone polypeptide were 123.55 IU/L, 104.23 IU/L and 98.36 IU/L, respectively, the blood Ca levels were 2.13 mmol/L, 2.17 mmol/L and 2.29 mmol/L, respectively, and the P levels were 1.90 mmol/L, 1.93 mmol/L and 2.09 mmol/L, respectively, which were significantly different from those of the model group (P<0.01 or P<0.05). The above data show that bone polypeptide can reduce serum ALP content, increase the levels of mineral elements Ca and P in the blood, and has a regulatory effect on the bone formation process and bone mineralization in osteoporotic rats.

5.4 Effects of bone polypeptide on the content of bone mineral-related elements

The content of bone mineral elements such as calcium, phosphorus, magnesium, and zinc and bone density directly determine the strength of bones. Calcium and phosphorus are the main components of bone minerals; trace metal elements such as manganese, iron, and zinc regulate and participate in the body's new bone formation and bone transformation. Their content can directly affect bone mineral formation and bone matrix synthesis, and is closely related to the onset of osteoporosis. As shown in Table 3, after modeling, the bone Mn of the model group rats increased from 6.38μg/g to 8.83μg/g (P<0.01), while the bone Ca (242.5mg/g, P<0.01), Fe (0.06mg/g, P<0.05), Mg (10.04mg/g, P<0.01), P (21.12mg/g, P<0.01) and Zn (0.32mg/g, P<0.01) all decreased significantly; the above indicators in each treatment group improved to varying degrees. Compared with the model group, the improvement of Ca, Fe, Mg, P, and Zn in the Fosamax group was statistically significant (P<0.01). The bone Ca content in the low, medium, and high dose groups of bone polypeptide was 338.3 mg/g, 343.1 mg/g, and 361.2 mg/g, respectively. The Zn content in the medium and high dose groups of bone polypeptide was 0.42 mg/g and 0.43 mg/g, respectively. The Mg content in the high dose group of bone polypeptide was 13.86 mg/g, and the P content was 26.44 mg/g, which were significantly higher than the corresponding bone mineral element content in the model group (P<0.01 or P<0.05). This shows that bone polypeptide can regulate the content of bone mineral elements, increase bone mineralization, enhance bone strength, and has a preventive and therapeutic effect on osteoporosis. It has the potential to be further studied and developed as an effective dietary supplement for the prevention of osteoporosis.

Table 3 Effects of bone polypeptide on bone trace elements in rat osteoporosis model induced by retinoic acid

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

5.5 Effect of bone polypeptide on bone density related indicators

Bone density reflects the degree of osteoporosis and is an important indicator for evaluating bone quality and an important basis for predicting the risk of fracture. Bone length, width, bone mass (wet weight, dry weight) and bone density are important indicators for characterizing bone status and the most basic diagnostic basis for osteoporosis in clinical practice. As shown in Table 4, the bone density of the model group decreased from 0.33g/ ^cm2 to 0.26g/ ^cm2 , a decrease of 20.06% (P<0.01), the bone wet weight and bone dry weight decreased by 9.16% and 8.37% respectively, and there was no significant change in bone length and bone width. The above indicators of each treatment group were consistent with the changes in the model group, but the degree was slightly lighter. The bone density of the treatment group was significantly increased compared with the model group, among which the bone density of the low, medium and high dose groups of bone polypeptide increased by 11.79%, 17.11% (P<0.05) and 15.21% (P<0.05) respectively compared with the model group. The above data suggest that bone polypeptide can increase the bone density of osteoporotic rats, improve bone strength and reduce the incidence of fractures.

Table 4 Effects of bone polypeptide on bone mineral density, bone wet weight, bone dry weight, bone length and bone width in rats with osteoporosis induced by retinoic acid

Note: Compared with the model control group: *p<0.05, **p<0.01; compared with the normal group: ^△ p<0.05, ^△△ p<0.01.

5.6 Effects of bone polypeptide on SOD, GSH-Px, and MDA oxidative damage-related indicators in the bone marrow

Oxidative damage can stimulate the FoxOs signaling pathway and inhibit osteoblast differentiation, which is one of the main causes of osteoporosis. Developing anti-osteoporosis drugs from the perspective of oxidative damage repair is a new research idea. As shown in Figure 3, compared with the normal group, the activity of the antioxidant enzyme SOD in the bone marrow of the model group rats decreased by 34.75% from 0.587U·mg ^-1 to 0.383U·mg ^-1 (P<0.01), and the activity of GSH-Px decreased by 39.07% from 0.942U to 0.383U (P<0.01); the content of MDA increased by 48.56% from 0.383nmol·mg ^-1 to 0.569nmol·mg ^-1 (P<0.01). Compared with the model group, the above indexes in each drug-treated group were improved to varying degrees, among which the SOD activity of the high-dose group was 0.514U·mg ^-1 , the GSH-Px activity was 0.825U and the MDA content was 0.456nmol·mg ^-1 , which were 25.61% (P<0.05), 28.46% (P<0.05), 45.30% (P<0.05) and 19.86% (P<0.05) higher than those in the model group, respectively. The above results indicate that bone polypeptide can increase the activities of the bone marrow antioxidant enzymes SOD and GSH-Px in retinoic acid OP rats, reduce the production of lipid peroxidation product MDA, thereby reducing the damage of oxidative stress to bone tissue and playing a bone protective role.

The embodiments described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims (7)

1. The application of bone polypeptide in preparing oral medicine for preventing and treating osteoporosis, and features that the osteoporosis is caused by retinoic acid.

2. The use according to claim 1, wherein the bone polypeptide is a bone polypeptide of porcine origin and the peptide has a relative molecular weight of less than 10000Da comprises 100%.

3. The use of claim 1, wherein the bone polypeptide effects prevention and treatment of osteoporosis by reducing gonadal damage and oxidative damage to bone tissue caused by retinoic acid, increasing bone mineralization, and increasing bone density.

4. The application of bone polypeptide in preparing medicine for relieving gonadal injury and/or bone tissue oxidation injury caused by retinoic acid.

5. Use of a bone polypeptide in the preparation of a medicament for increasing bone mineralization and increasing bone density.

6. The use according to claim 4 or 5, wherein the medicament comprises a bone polypeptide as the only active ingredient and the medicament is an oral medicament.

7. An oral medicament for preventing and treating osteoporosis, wherein the oral medicament comprises a bone polypeptide as the only active ingredient; the bone polypeptide is bone polypeptide derived from pig bone, the peptide with the relative molecular weight lower than 10000Da accounts for 100 percent, and the total nitrogen content of each gram of the bone polypeptide is 0.157 gram.