CN118717952A - Composition for increasing bone density and application thereof - Google Patents

Abstract

The present invention belongs to the technical field of calcium supplement compositions, and specifically relates to a composition for increasing bone density and its application. The pharmaceutical composition includes by weight: 1-5 parts of amino acid chelated calcium, 5-15 parts of Moringa extract, 5-10 parts of marine collagen peptides, 0.5-2 parts of casein phosphopeptides, 0.0005-0.03 parts of vitamin K2 and 0.0001-0.006 parts of pyrroloquinoline quinone disodium salt. The composition for increasing bone density of the present invention can promote calcium absorption, improve bone density, can be used for preventing and treating female menopause and middle-aged and elderly osteoporosis patients, and is also suitable for sub-healthy groups to eat as a calcium supplement; and it is safe to use, has no toxic side effects when taken for a long time, has a simple and reasonable process design, and is suitable for industrial production.

Description

Composition for increasing bone density and application thereof

Technical Field

The invention belongs to the technical field of calcium supplement compositions, and in particular relates to a composition for increasing bone density and an application thereof.

Background Art

Calcium is present in high levels in the human body, 99% of which is stored in bones and teeth. In addition to being closely related to bone health, calcium can also participate in the activity of nerves and muscles and the release of neurotransmitters, reducing the excitability of nerves and muscles; it can regulate hormone secretion, blood coagulation, enzyme activity, heart rate and blood pressure, reduce vascular permeability, prevent and treat allergic reactions, etc. Various studies have shown that low calcium levels in the human body can cause various diseases.

Osteoporosis is the most common systemic bone disease in the elderly. Its main characteristics are low bone mineral content, bone structure destruction, reduced bone strength and easy fracture. Pain, hunchback, height loss and fractures are the main manifestations of osteoporosis. The international mainstream view is that calcium supplementation through food or drug calcium supplements can prevent or alleviate osteoporosis to a certain extent.

At present, drugs used clinically for the treatment of osteoporosis include calcium and vitamin D, as well as bone absorption inhibitors such as estrogen, bisphosphonates, and calcitonin. However, these calcium preparations are either inorganic salts with poor human absorption, or hormones, which have certain side effects on the human body after long-term use. Therefore, providing a calcium supplement composition with good calcium supplement effect and no side effects on the human body has great economic and social value.

After searching, there are related reports about natural calcium supplementation. For example, Chinese invention patent application CN1559582A discloses a natural calcium supplement powder, which is made of marine organism oyster and natural Chinese herbal medicines such as cassia bark, dragon bone and cordyceps sinensis as raw materials. According to the different characteristics and pharmacological effects of each medicine, powder is made and added to the food of calcium deficiency patients, and the medicine and food are of the same origin. The invention has high calcium content, good calcium supplementation effect, convenient to take, calcium supplementation and health care are integrated, and the preparation is simple. However, the dragon bone and cordyceps sinensis used in the calcium supplement powder are both precious Chinese medicinal materials, which are not easy to obtain and have high cost, which is not conducive to large-scale industrial promotion.

Chinese invention patent application CN1557469A discloses a natural calcium supplement that is easily absorbed and utilized by the human body, and in particular, relates to a method and application for preparing a natural calcium supplement preparation from casein - casein glycomacromolecular calcium and casein phosphopeptide calcium, using casein and its related products as raw materials, performing protein hydrolysis by trypsin and/or chymosin, separating and concentrating by ultrafiltration equipment, preparing a natural calcium supplement with casein phosphopeptide calcium and/or casein-glycomacromolecular peptide calcium as the main components, and preparing it into a food additive or pharmaceutical preparation to help the human body supplement calcium. This calcium supplement only uses casein as raw material, and the calcium exists in a single form, which cannot cover various calcium supplement groups.

In addition, the calcium supplements used in the prior art are in a single form, providing only pure mineral calcium ions. Although calcium is one of the raw materials for bones, it is not all of them. The key to calcium supplementation is how to combine the supplemented calcium with a special protein (osteocalcin) to form bone calcium. Therefore, it is necessary to optimize the composition of the calcium supplement to provide a composition that converts mineral calcium ions into bone calcium, deposit it in the bones, form bone calcium with vitality, and completely improve the problem of osteoporosis.

Summary of the invention

In order to solve the defects of the prior art, the present invention provides a composition for increasing bone density and its application. The pharmaceutical composition adopts components that are both medicinal and edible, has a wide source, and can be industrially produced. At the same time, under the synergistic effect of various components, the mineral calcium ions in the composition can be converted into bone calcium, promote calcium absorption, and increase bone density. It can be used to prevent and treat menopausal women, middle-aged and elderly osteoporosis patients and sub-healthy groups.

To achieve the above object, one of the technical solutions of the present invention is:

Provided is a composition for increasing bone density, the composition for increasing bone density comprising: amino acid chelated calcium, moringa extract, marine collagen peptide, casein phosphopeptide, vitamin K2 and pyrroloquinoline quinone disodium salt

Furthermore, the composition for increasing bone density comprises, by weight: 1-5 parts of amino acid chelated calcium, 5-15 parts of Moringa extract, 5-10 parts of marine collagen peptides, 0.5-2 parts of casein phosphopeptide, 0.0005-0.03 parts of vitamin K2 and 0.0001-0.006 parts of pyrroloquinoline quinone disodium salt.

Furthermore, the amino acid chelated calcium is selected from one or more of aspartic acid chelated calcium, lysine chelated calcium, glutamate chelated calcium, threonine chelated calcium, serine chelated calcium, glycine chelated calcium, alanine chelated calcium, cystine chelated calcium, valine chelated calcium, leucine chelated calcium, isoleucine chelated calcium, tyrosine chelated calcium, phenylalanine chelated calcium, histidine chelated calcium, arginine chelated calcium and proline chelated calcium.

Preferably, the amino acid chelated calcium is selected from one or more of aspartate chelated calcium, arginine chelated calcium and glycine chelated calcium.

Furthermore, the weight ratio of vitamin K2 to casein phosphopeptide is 1-3:5-15; preferably 2-3:6-10.

Furthermore, the composition for increasing bone density further comprises one or more of the following nutrients: calcium ascorbate, oligofructose, resistant dextrin, ginger extract, xylitol and trace elements.

Furthermore, the composition comprises, by weight: 1-5 parts of amino acid chelated calcium, 5-15 parts of Moringa extract, 5-10 parts of marine collagen peptides, 0.5-2 parts of casein phosphopeptide, 0.0005-0.03 parts of vitamin K2, 0.0001-0.006 parts of pyrroloquinoline quinone disodium salt, 1-10 parts of calcium ascorbate, 1-5 parts of oligofructose, 5-20 parts of resistant dextrin, 0.5-2 parts of ginger extract, 5-10 parts of xylitol and 0.005-1 parts of trace elements.

Furthermore, the trace elements are selected from one or more of zinc, iron, selenium, magnesium, molybdenum and cobalt.

Preferably, the trace elements are selected from one or more of zinc, iron and magnesium.

In some embodiments of the present invention, the amino acid chelated calcium can be purchased from the market or prepared by methods known in the art. The preparation method preferably uses shells as the calcium source, and the shells include but are not limited to the shells of all mollusks such as oyster shells, scallop shells, clam shells, and abalone shells.

In some embodiments of the present invention, the Moringa extract and the ginger extract can be purchased from the market, or can be prepared using methods known in the art.

The second technical solution of the present invention is:

Provided is the use of any of the above-mentioned compositions in the preparation of anti-osteoporosis drugs, foods or health products.

The third technical solution of the present invention is:

Provided is a preparation comprising any of the above-mentioned pharmaceutical compositions for increasing bone density and a pharmaceutically acceptable carrier or diluent.

The composition for increasing bone density of the present invention can be prepared into granules, tablets, capsules, oral liquids, powders, pills, etc. according to known methods.

The amino acid chelate calcium in the present invention is a calcium with a stable chelate structure, which belongs to a new calcium source with biological activity. It has good solubility and can be fully dissolved in an acidic or alkaline solution with a pH of 1-14, solving the problem that calcium preparations are not easily dissolved due to different gastric acid concentrations, especially the low gastric acid concentration of the elderly, and thus laying a foundation for improving the absorption rate of amino acid chelate calcium in the intestine. Another feature of amino acid chelate calcium is a chelate structure combined with two amino acids and a calcium ion, which is similar to natural dipeptide protein calcium and is a short-chain protein calcium molecule. It does not require the participation of vitamin D3 for absorption, and is very easy to pass through intestinal epithelial cells and directly absorbed by the human intestine, which is inaccessible to general calcium preparations. In addition, the chelate structure is stable, and it is not easy to be combined with substances such as oxalic acid and phytic acid in food in the human intestine and lost. At the same time, the affinity for bone tissue is several times that of other tissues, and it will not cause calculi in the body, which greatly improves the absorption rate of calcium by the human body. It is an ideal, safe and reliable new generation of calcium preparations on the market.

The present invention uses the Moringa extract as an active ingredient, in coordination with amino acid chelated calcium, or in coordination with amino acid chelated calcium and calcium ascorbate, to enhance the absorption of calcium by the human body. Studies have shown that Moringa provides abundant and rare nutrients, such as zeatin, quercetin, beta-sitosterol and kaempferol as well as abundant trace elements. The leaves, roots, seeds, bark, fruits, fresh flowers and immature pods of Moringa can be used as medicines, and are stimulants for the heart and circulatory system, with anti-tumor, antipyretic, anti-epileptic, anti-inflammatory, anti-ulcer, anti-hypertension, cholesterol-lowering, anti-oxidant, anti-osteoporosis, hypoglycemic, liver-protecting, antibacterial and antifungal functions. The various enzyme components in Moringa participate in metabolism, and can also maintain the normal functions of the heart, kidney, muscle and nerve.

Casein phosphopeptide is derived from natural high-quality protein - bovine milk casein, which can promote the absorption of calcium, zinc, and iron, resist tooth decay, improve animal fertilization ability, regulate blood pressure, etc.; compared with vitamin D, it has a higher absorption rate, no toxic side effects, and can improve the absorption rate of other divalent minerals such as zinc and iron.

Vitamin K is a vitamin that depends on multiple proteins in bones. It increases the synthesis of osteocalcin in osteoblasts. The γ-carboxylation of glutamic acid in osteocalcin has biological effects. Carboxylated osteocalcin can combine with Ca and hydroxyapatite to mineralize bones. This carboxylation must be done with vitamin K. Vitamin K2 can help carboxylate the primary osteocalcin secreted by osteoblasts and turn it into active osteocalcin, thereby promoting the deposition of calcium ions in the blood (i.e., blood calcium, mineral calcium ions in diet and calcium supplements are digested in the digestive tract to form intestinal calcium, which is absorbed into the blood by the villi of the small intestine) into bones. If there is enough vitamin K2 in the body, osteocalcin can be activated. Activated osteocalcin has a unique affinity for calcium ions, which can lead calcium into bones and deposit calcium salts, thereby promoting bone mineralization.

Pyrroloquinoline quinone disodium salt (pQQ) is known as the "fourteenth vitamin" and the "king of immunity". It is the most powerful bioactive substance discovered by humans so far. Studies have shown that dietary pQQ supplements can prevent osteoporosis caused by natural aging.

The casein phosphopeptide of the present invention cooperates with vitamin K2 and pyrroloquinoline quinone disodium salt to further improve the intestinal microbial environment, promote the carboxylation degree of osteocalcin, increase the conversion of osteocalcin, and further improve the calcium absorption rate.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The bone density-enhancing composition provided by the present invention can promote calcium absorption and improve bone density, and can be used to prevent and treat osteoporosis in women in menopause and middle-aged and elderly patients, and is also suitable for consumption by sub-healthy groups as a calcium supplement; under the synergistic effect of various components, especially the synergistic effect of amino acid chelated calcium and Moringa oleifera extract, and the synergistic effect of casein phosphopeptide and vitamin K2 and pyrroloquinoline quinone disodium salt, the effect of promoting calcium absorption and improving bone density is significantly enhanced.

(2) The synergistic effect of amino acid chelated calcium, Moringa extract and calcium ascorbate further enhances the effect of the composition in increasing bone density and promoting calcium absorption.

(3) The bone density-enhancing drug composition uses ingredients that are both medicinal and edible, is safe to use, and has no toxic side effects when taken for a long time.

(4) The process design of the drug composition for increasing bone density is simple and reasonable, and is suitable for industrial production.

DETAILED DESCRIPTION

In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work are all within the protection scope of the present invention.

Unless otherwise specified, the raw materials used are common commercially available products, the operating methods used are conventional operating methods, and the equipment used are conventional equipment.

The following are the sources of raw materials used in the embodiments of the present invention:

Calcium Aspartate, Calcium Arginine, Calcium Bisglycinate and Calcium Glutamate were purchased from Ningbo Yingqian Technology Co., Ltd.

Moringa extract (item number: SNT069), ginger extract (item number: SNT022), calcium ascorbate, marine collagen peptides (specifically fish collagen peptides), and casein phosphopeptide were purchased from Fufeng Snot Biotechnology Co., Ltd.

Example 1

The formula of each composition in this embodiment (unit: kg) is shown in Table 1:

Table 1 Formula of each composition in this example

Note: / indicates not added.

The preparation method of the composition for increasing bone density comprises the following steps: weighing various raw materials according to the formula, and mixing them evenly to obtain the powder of the composition with the effect of increasing bone density.

Effect example 1: Bone density increase effect test

(1) Experimental animals: 50 weaned SD rats, SpF grade, female, 64-80 g, four weeks old, provided by Shanghai Jiesijie Experimental Animal Co., Ltd. Production license number: SCXK (Shanghai) 2013-0006; breeding room temperature 20-25°C, relative humidity 40-80%, experimental animal use license number: SYXK (Shanghai) 2013-2008; experimental basic feed was provided by Shanghai Fubei Pet Products Co., Ltd., registration certificate number: Shanghai Feed Certificate (2014) 04002.

(2) Experimental groups and dosage: This animal experiment set up three dose groups, namely, low, medium and high dose groups, i.e., 0.2 g/kg, 0.4/kg and 1.2/kg (0.2 g, 0.4 g and 1.2 g of the powder prepared by combination 1 were taken, respectively, added with deionized water to 10 mL, and the test solutions for the low, medium and high dose groups were prepared by uniform mixing according to this ratio), which was equivalent to 5, 10 and 30 times the recommended human dose of the sample (the recommended human dose and calcium content: 2.4 g/60 kg body weight per day, the calcium content of the sample was 18.02%). At the same time, a low calcium control group (an equal volume of deionized water was used instead of the test solution) and a calcium carbonate control group (the calcium content was equivalent to that of the high dose group, 541 mg of calcium carbonate with a calcium content of 40% was taken, added with deionized water to 10 mL, and the test solution for the calcium carbonate control group was prepared by uniform mixing according to this ratio); the sample was administered by gavage, and the gavage volume was 10 mL/kg BW.

(3) Experimental methods: After a one-week adaptation period, weaned rats aged 4 weeks were fasted overnight, weighed, and randomly divided into groups according to their weight, with 10 rats in each group and housed in separate cages (5 rats/cage). They drank deionized water to avoid obtaining calcium from drinking water. Each group was gavaged with samples according to the dosage design for 3 months (12 weeks). The weight of each group of animals was weighed and recorded every week, and the height was measured once a week in the first 4 weeks.

(4) Calcium metabolism experiment: During the first three days of the fourth week of the experiment, the rats were placed in metabolic cages for a three-day calcium metabolism experiment. Each dose group was still gavaged with the corresponding dose of the test substance every day. The food intake was recorded for three days, and feces were collected for 72 hours to measure the calcium content in the feed and feces. The fecal samples were dried and digested in an 80°C oven and then measured by an atomic absorption spectrophotometer. Calculation was performed according to the following formula:

Calcium intake (mg) = calcium content in feed (mg/g) × 3-day feed consumption + calcium content given by gavage

Fecal calcium (mg) = fecal calcium content (mg/g) × fecal excretion in 3 days

Calcium absorption rate % = (calcium intake - fecal calcium) / calcium intake × 100%

(5) Weighing the dry weight of the femur: dissect the rat, peel off the right femur, dry the femur in an oven at 105°C until constant weight, and weigh the dry weight of the femur.

(6) Femoral bone density measurement: The bone density of the midpoint and distal end of the femur of rats was measured using a discovery-wi bone densitometer.

(7) Determination of bone calcium content: The left femur of the rat was excised and measured by atomic absorption spectrometry after digestion. The calculation formula is as follows:

Where: C: calcium concentration obtained on the instrument (mg/L);

W: femur dry weight (g);

V: Sample fixed volume (10mL).

Principle for determining results: If the bone calcium content or bone density in any dose group is significantly higher than that in the low calcium control group and not lower than that in the corresponding dose calcium carbonate control group, and the calcium absorption rate is not lower than that in the calcium carbonate control group, it can be determined that the test substance has the function of increasing bone density.

(8) Experimental results:

8.1 The results of the effects of the samples on rat body weight are shown in Table 2.

Table 2 Effects of samples on rat body weight

As shown in Table 2, there were no significant differences in the initial weight, weight at the end of each week, and weight gain between the various groups and the low-calcium control group (p>0.05). There were no significant differences in the initial weight, weight at the end of each week, and weight gain between the high-dose group and the calcium carbonate control group (p>0.05).

8.2 The effects of the composition on the weight, bone calcium and femoral bone density of rats are shown in Table 3.

Table 3 Effects of samples on rat femur weight, bone calcium and bone density

Note: ^* Compared with the low calcium control group, p < 0.05, ^** Compared with the low calcium control group, p <0.01;^#Compared with the calcium carbonate control group, p < 0.05.

As can be seen from Table 3, compared with the low-calcium control group, the femoral dry weight and bone calcium content of the low-dose group increased significantly (p < 0.05), and the femoral dry weight, bone density and bone content of the medium-dose group and the high-dose group were significantly higher than those of the low-calcium control group (p < 0.01); compared with the calcium carbonate control group, the femoral dry weight, bone calcium content and bone density of the low-dose group were higher than those of the calcium carbonate control group with the same calcium level, and the bone density and bone calcium content of the medium and high-dose groups were significantly higher than those of the calcium carbonate control group with the same calcium level (p < 0.05).

The results of the effect of 8.3 composition on calcium metabolism in rats are shown in Table 4.

Table 4 Effects of samples on calcium metabolism in rats during the experimental period

Note: ^* Compared with the low calcium control group, p <0.05;^#Compared with the calcium carbonate control group, p < 0.05.

As shown in Table 4, the growth and calcium absorption rate of each dose group were higher than those of the low calcium control group and the calcium carbonate control group. There was no significant difference in growth and development between each dose group and the low calcium control group (p>0.05), and there was a significant difference in calcium absorption rate between the medium and high dose groups (p<0.05); there was a significant difference in calcium absorption rate between the high dose group and the calcium carbonate control group with the same calcium level (p<0.05).

In summary, according to the result judgment principle of the "bone density increasing function" test in the "Technical Specifications for Inspection and Evaluation of Health Food" (Ministry of Health 2003 edition), the composition of the present invention has the function of increasing the bone density of rats.

Effect Example 2

To further verify the effect difference of the composition in each combination, 110 SD rats, female, were randomly divided into 11 groups, 10 in each group, i.e., low calcium control group, model control group, positive control group and experimental group 1-8 (i.e., combination 1-8). Except that the low calcium control group was given equal volume of deionized water, all other groups were given dexamethasone 1mg/kg, injected once every three days, intramuscular injection, and at the same time, combined with retinoic acid gavage, daily dosage 70mg/kg, for two consecutive weeks. Then, gavage was adopted, three times a day, and continuous administration was given for two weeks, wherein experimental group 1-8 was respectively given the powder 0.4g/kg (dosage in the above-mentioned test), positive control group was given 0.4g/kg Xianling Gubao capsule content, and low calcium control group and model control group were given equal volume of distilled water. After the administration, the animals were anesthetized with pentobarbital, dissected animals, femurs were taken to measure bone density and bone calcium content, and blood was taken to measure serum calcium content. The results are shown in Table 5.

Table 5 Average femoral bone density, bone calcium content and serum calcium content of rats in each group

Note: ^* Compared with the low calcium control group, p <0.05;^#Compared with the model control group, p < 0.05, ^## Compared with the model control group, p < 0.01.

The results in Table 5 show that compared with the low calcium control group, the average femur density and bone calcium content of the model control group were significantly reduced, and the serum calcium content was significantly increased, indicating that the modeling was successful. The composition 1-8 for increasing bone density of the present invention and the positive control group drug can both increase femur density and bone calcium content and reduce serum calcium content, wherein the effect of the experimental group 1 (composition 1) of the present invention is better than that of the positive control group; at the same time, compared with the experimental group 1 (composition 1), the femur density and bone calcium content of experimental groups 7 and 8 (compositions 7 and 8) were reduced, and serum calcium was increased, which fully proves that the use of amino acid chelated calcium and Moringa extract together has a synergistic effect, and the combination of the two can further increase bone density, promote calcium absorption, and reduce calcium content in serum.

Example 2

The formula of each composition in this embodiment (unit: kg) is shown in Table 6:

Table 6 Formula of each composition in this example

The preparation method of the composition powder having the effect of increasing bone density is the same as that of Example 1.

Effect Example 3

The bone density increasing effect of the composition of Example 2 was evaluated according to the test method of Effect Example 2. The experimental results are shown in Table 7.

Table 7 Average femoral bone density, bone calcium content and serum calcium content of rats in each group

Note: ^* Compared with the low calcium control group, p <0.05;^#Compared with the model control group, p < 0.05, ^## Compared with the model control group, p < 0.01.

The results in Table 7 show that compared with the model control group, the bone density increasing compositions of combination 1 and combination 9-14 of the present invention can increase femoral bone density and bone calcium content, and reduce serum calcium content. Compared with experimental group 1 (combination 1), experimental groups 9-14 (combination 9-14) are insufficient in increasing femoral bone density and bone calcium content and reducing serum calcium content. This fully proves that the combination of amino acid chelated calcium, Moringa extract and calcium ascorbate has a synergistic effect, can more significantly increase bone density, promote calcium absorption, and reduce serum calcium content.

Example 3

The formula of each composition in this example (unit: kg) is shown in Table 8:

Table 8 Formula of each composition in this example

The preparation method of the composition powder having the effect of increasing bone density is the same as that of Example 1.

Effect Example 4

The bone density increasing effect of the composition of Example 3 was evaluated according to the test method of Effect Example 2. The experimental results are shown in Table 9.

Table 9 Average femoral bone density, bone calcium content and serum calcium content of rats in each group

Note: ^* Compared with the low calcium control group, p <0.05;^#Compared with the model control group, p < 0.05, ^## Compared with the model control group, p < 0.01.

The results in Table 9 show that compared with the model control group, the bone density increasing compositions of combination 1 and combination 15-17 of the present invention can improve femoral bone density. Compared with experimental group 1 (combination 1), experimental groups 15-16 (combination 15-16) are insufficient in improving femoral bone density and bone calcium content and reducing serum calcium content, which fully proves that vitamin K2 and pyrroloquinoline quinone disodium salt act synergistically and combine with casein phosphopeptide to convert it into bone calcium, which is more effective; the lack of any one of them has no obvious effect. Experimental group 17 (combination 17) is based on experimental group 1 (combination 1), after casein calcium phosphate is replaced with casein calcium peptide with similar function, even if vitamin K2 and pyrroloquinoline quinone disodium salt are also present, its effect of improving femoral bone density and bone calcium content and reducing serum calcium content is still not ideal, which fully illustrates that the three work together and cooperate with each other, and the two effectively combine with osteocalcin (especially casein calcium phosphate, and also include mineral calcium ions present in Moringa extract, etc.) to convert it into bone calcium to achieve an increase in bone density.

Example 4

The formula of each composition in this example (unit: kg) is shown in Table 10:

Table 10 Formula of each composition in this example

The preparation method of the composition powder having the effect of increasing bone density is the same as that of Example 1.

Effect Example 5

The bone density increasing effect of the composition of Example 4 was evaluated according to the test method of Effect Example 1. The experimental results are shown in Table 11.

Table 11 Average femoral bone density, bone calcium content and serum calcium content of rats in each group

Note: ^* Compared with the low calcium control group, p <0.05;^#Compared with the model control group, p < 0.05, ^## Compared with the model control group, p < 0.01.

The results in Table 13 show that compared with the model control group, the bone density increasing compositions of combination 1 and combination 18-21 of the present invention can improve femoral density. Compared with combination 1, femoral density and bone calcium content of combination 18-21 decreased, and serum calcium content increased, which indicates that the dosage of amino acid chelated calcium and Moringa extract in the composition of the present invention also has a great influence on improving bone density. The present invention achieves a better effect of improving bone density by using the key components of the composition, amino acid chelated calcium and Moringa extract, in a suitable dosage ratio.

Effect Example 6: Safety Evaluation Test

According to the Technical Specifications for Inspection and Evaluation of Health Food (2003 edition) of the Ministry of Health, the pharmaceutical composition of the present invention was subjected to acute toxicity, bone marrow cell micronucleus, mouse sperm abnormality and Ames tests.

(1) Experimental animals and feeding: 50 SpF grade Kunming mice, half male and half female, weighing 25-27 g, were provided by Shanghai Jiesijie Experimental Animal Co., Ltd., with experimental animal production license number: SCXK(Shanghai)2013-0006; experimental animal use license number: SYXK(Shanghai)2013-0008; mouse feed was provided by Shanghai Fubei Pet Products Co., Ltd., with registration number: Shanghai Feed Certificate (2014)04002; experimental environment: the temperature of the feeding room was 20-21°C, and the relative humidity was 57-58%.

(2) Test sample: the composition prepared by combination 1 of the present invention.

(3) Strain source and preservation: The test strains TA97, TA98, TA100, and TA102 were provided by MOLECULAR TOXICOLOGY, INC., USA. The batch numbers of the product quality assurance certificates are 4584D, 4599D, 4600D, and 4604D, respectively. The biological characteristics of the strains were confirmed to meet the experimental requirements.

(4) In vitro metabolic activation system: S ₉ was produced by American Molecular Toxicology Co., Ltd. and was prepared by inducing liver tissue of adult male SD rats with Aroclor 1254. The S ₉ mixed solution contained 10% of the S ₉ component and supplemented with auxiliary factors, and was used as the in vitro metabolic activation system of this experiment.

1. Acute toxicity test

1.1 Preparation of test samples: Weigh 20 g of the test sample, add distilled water to 40 mL, mix thoroughly, and prepare the test solution according to this ratio.

1.2 Experimental method: After the animals were fasted (but not watered) for 16 hours, 10 female and 10 male mice were selected according to the weight requirements and placed in two cages. The weight difference between mice of the same sex should not exceed 3g. The mice were weighed one by one and exposed to the poison by oral gavage. The gavage volume was 20mL/kg BW each time. The mice were gavaged twice within 24 hours with an interval of 4 hours. After exposure, the general condition, weight change, poisoning symptoms and death of the animals were observed for two weeks. The animals were weighed once a week, and the dead animals and animals killed at maturity were dissected to observe the gross pathological changes with the naked eye. The whole experimental process and observation content were recorded in detail, and the acute oral LD50 results of mice were obtained according to the maximum tolerated dose method test results, see Table 12.

Table 12 Results of acute oral toxicity test in male and female mice

1.3 Main symptoms:

During the experiment, the animals in each group moved normally, their fur was shiny and glossy, and no poisoning symptoms or death were observed. When the experiment ended, the animals were killed and their organs were observed by gross dissection and no abnormalities were found.

1.4 LD50 results:

Female mice: LD50>20g/kg; male mice: LD50>20g/kg; it can be seen that the LD50 of the composition of the present invention in the acute oral toxicity test on male and female mice is greater than 20g/kg, which is non-toxic.

2. Bone marrow cell micronucleus test

1.1 Sample processing and preparation: According to the results of acute oral toxicity test, low, medium and high dose groups of test samples were set up with 2.5g/kg, 5.0g/kg and 10.0g/kg respectively. Weigh 2.5g, 5.0g and 10.0g of test samples, add distilled water to 20mL respectively, mix well as test samples, and prepare low, medium and high dose group test solutions in this proportion; set up solvent (distilled water) control group, with a dosage of 20mL/kg; weigh 40mg of cyclophosphamide, add physiological saline to 20mL, dissolve as positive control test solution, with a dosage of 20mg/kg.

1.2 Experimental methods: The animals were randomly divided into 5 groups, each with 10 animals, half of which were male and half were female, and they were used as three dose groups of samples, a negative control group and a cyclophosphamide positive control group; the animals were gavaged twice every 30 hours, and the animals were weighed. The prepared samples of different concentrations were gavaged at 0.4 mL/20 kg BW to each group of animals; 6 hours after the second gavage, the animals were killed by cervical dislocation, and the femoral bone marrow was mixed with calf serum, and the slides were smeared, fixed and stained with Giemsa; under microscopic observation, the number of micronuclei of 1000 polychromatic erythrocytes (pCE) was counted for each mouse, and the micronucleus rate was expressed in per thousand. When counting polychromatic erythrocytes (counting 200 pCE), the number of mature erythrocytes (NCE) was also counted, and the pCE/NCE value was calculated.

1.3 The results are shown in Table 13.

Table 13 Incidence of micronuclei in polychromatic erythrocytes in animal bone marrow

Note: **Compared with the negative control group (by chi-square test), p<0.01.

There was no significant difference in the pCE/NCE ratio and micronucleus rate between the test substance in each dosage group and the negative control group, and the ratio of pCE to the total number of RBCs in the test substance group was not less than 20% of that in the solvent control group, indicating that the test substance had no inhibitory effect on mouse bone marrow cells; after statistical analysis, the result of the bone marrow polychromatic erythrocyte micronucleus test of the composition of the present invention was negative.

3. Mouse sperm abnormality test

1.1 Sample processing and preparation: According to the results of acute oral toxicity test, low, medium and high dose groups of test samples were set up with 2.5g/kg, 5.0g/kg and 10.0g/kg respectively. Weigh 2.5g, 5.0g and 10.0g of test samples, add distilled water to 20mL respectively, mix well as test samples, and prepare low, medium and high dose group test solutions in this proportion; set up solvent (distilled water) control group, with a dosage of 20mL/kg; weigh 40mg of cyclophosphamide, add physiological saline to 20mL, dissolve as positive control test solution, with a dosage of 40mg/kg.

1.2 Experimental methods: The animals were randomly divided into 5 groups, 5 in each group, as three sample dose groups, distilled water negative control group and cyclophosphamide positive control group; after the animals were weighed, the prepared samples of different concentrations were gavaged into each group of animals at 0.4mL/20kgBW, once a day for 5 consecutive days; on the 35th day after the first sampling, the animals were killed by cervical dislocation, the epididymis on both sides were taken and placed in physiological saline, cut into pieces, filtered through four layers, the filtrate was taken for smear, fixed, and stained with 2% eosin for slide preparation; microscopic observation, the number of sperm deformities of 1000 sperms per mouse was counted, the incidence of sperm deformity was calculated, and statistical analysis was performed.

1.3 The results are shown in Table 14.

Table 14 Incidence of sperm abnormalities in animals

Note: ^** Compared with the negative control group (by chi-square test), p<0.01.

According to statistical analysis, the results of the mouse sperm deformity test of the pharmaceutical composition of the present invention and the sample of the composition of the present invention were negative.

4. Ames test

1.1 Test strains: Take out a set of liquid nitrogen-preserved strains, make a master plate, select a well-isolated single colony and suspend it in 15mL/bottle of nutrient broth, and culture it in the dark at 37℃ with shaking at 120 times/min for 12 hours. The bacterial solution concentration should be 1.1-1.6×10 ⁹ /mL.

1.2 Sample treatment and preparation: The sample contents were sterilized at 121℃ for 20min under high pressure. 2.5g of the sterilized sample was added with sterile distilled water to 20mL and mixed. The sample was slowly added to the pretreated XAD-II resin column and adsorbed at a flow rate of 20mL/h to remove histidine. Air was then blown into the ear bulb to drive away moisture. 50mL of redistilled acetone was added and eluted at a flow rate of 20mL/h. The eluate was collected. After the acetone was evaporated at 60℃, 50mL of DMSO was added. The sample content in the collected solution was 50mg/mL. According to the dosage of 8μg/dish, 40μg/dish, 200μg/dish, 1000μg/dish, and 5000μg/dish, the collected solution was diluted with MDSO at a ratio of 1:4 to obtain the test substance of each test dose group. The solvent control was DMSO. Positive control: -S ₉ :TA97 ICR (1 μg/dish), TA98 p-nitroquinoline (200 μg/dish), TA100, TA102 MMS (1 μL/dish); +S ₉ : TA97, TA98, TA100, 2-aminofluorene (10 μg/dish), TA102 1,8-dihydroxyanthraquinone (50 μg/dish).

1.3 Test method: According to the plate incorporation method in the Salmonella typhimurium/mammalian microsomal enzyme test in the Technical Specifications for Inspection and Evaluation of Health Food (2003 edition), add 0.1 mL of the test substance and 0.1 mL of the strain enrichment solution to 2 mL of the top culture medium kept at 45°C, add 0.5 mL of S ₉ mixed solution when activation is required, mix thoroughly and quickly pour the bottom plate to make three parallel plates; culture at 37°C for 48 hours, count the number of revertant colonies, and repeat the test once.

1.4 The results are shown in Table 15 (first test) and Table 16 (second test).

Table 15 Test results of the first test

Note: ICR dose 1μg/dish, p-nitroquinoline dose 200μg/dish, methyl methane sulfonate dose 1μ/dish, aminofluorene dose 10μg/dish, 1.8-dihydroxyanthraquinone dose 50μg/dish (the same below).

Table 16 Test results of the first test

In the secondary test under the experimental conditions, the test substance was 8 μg to 5000 μg per dish, and the number of recovered colonies detected by the plate incorporation method with and without the S ₉ activation system was less than 2 times that of the solvent control, and the Ames test result of the drug composition for increasing bone density (the composition of the present invention) was negative.

Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. A composition for increasing bone density, wherein the composition for increasing bone density comprises: amino acid chelated calcium, moringa oleifera extract, marine collagen peptide, casein phosphopeptide, vitamin K2, and pyrroloquinoline quinone disodium salt.

2. The bone mineral density increasing composition of claim 1, wherein the pharmaceutical composition comprises, in parts by weight: 1-5 parts of amino acid chelated calcium, 5-15 parts of moringa oleifera extract, 5-10 parts of marine collagen peptide, 0.5-2 parts of casein phosphopeptide, 0.0005-0.03 part of vitamin K2 and 0.0001-0.006 part of pyrroloquinoline quinone disodium salt.

3. The bone mineral density increasing composition of claim 1, wherein the amino acid chelate calcium is selected from one or more of aspartic acid chelate calcium, lysine chelate calcium, glutamic acid chelate calcium, threonine chelate calcium, serine chelate calcium, glycine chelate calcium, alanine chelate calcium, cystine chelate calcium, valine chelate calcium, leucine chelate calcium, isoleucine chelate calcium, tyrosine chelate calcium, phenylalanine chelate calcium, histidine chelate calcium, arginine chelate calcium, and proline chelate calcium; preferably one or more of aspartic acid chelate calcium, arginine chelate calcium and glycine chelate calcium.

4. The bone mineral density increasing composition of claim 1, wherein the weight ratio of vitamin K2 to casein phosphopeptide is 1-3:5-15; preferably 2-3:6-10.

5. The bone mineral density increasing pharmaceutical composition according to any one of claims 1-4, further comprising one or more of the following nutritional ingredients: calcium ascorbate, fructo-oligosaccharides, resistant dextrins, ginger extract, xylitol and trace elements.

6. The bone mineral density increasing composition of claim 5, wherein the pharmaceutical composition comprises, in parts by weight: 1-5 parts of amino acid chelated calcium, 5-15 parts of moringa extract, 5-10 parts of marine collagen peptide, 0.5-2 parts of casein phosphopeptide, 0.0005-0.03 part of vitamin K2, 0.0001-0.006 part of pyrroloquinoline quinone disodium salt, 1-10 parts of calcium ascorbate, 1-5 parts of fructo-oligosaccharide, 5-20 parts of resistant dextrin, 0.5-2.0 parts of ginger extract, 5-10 parts of xylitol and 0.005-1 part of trace elements.

7. The bone mineral density increasing composition of claim 5 wherein the trace elements are selected from one or more of zinc, iron, selenium, magnesium, molybdenum, and cobalt; preferably one or more of zinc, iron and magnesium.

8. Use of a composition according to any one of claims 1 to 7 for the preparation of an anti-osteoporosis medicament, food or health product.

9. A formulation comprising the bone density increasing composition of any one of claims 1-8, and a pharmaceutically acceptable carrier or diluent.

10. The formulation of claim 9, wherein the formulation is a granule, tablet, capsule, oral liquid, powder, or pill.