JP2006290813A - Osteogenesis promoting agent, osteoporosis preventing agent and collagen synthesis promoting agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an osteogenesis promoting agent to promote osteogenesis, an osteoporosis preventing agent useful for the prevention of osteoporosis by promoting osteogenesis and a collagen synthesis promoting agent to promote the synthesis of collagen. <P>SOLUTION: The osteogenesis promoting agent, the osteoporosis preventing agent and the collagen synthesis promoting agent contain royal jelly or its extract as an active component. The osteoporosis preventing agent promotes osteogenesis and exhibits the action for the prevention or amelioration of especially senile osteoporosis. The osteoporosis preventing agent preferably further contains a bone resorption suppressing agent and/or a food material having calcium supplementing action. The collagen synthesis promoting agent increases the expression quantity of procollagen 1α1 gene by mixing with ordinary feed and orally administering to mouse (refer to the Example 1 shown in the Figure). The gene is known as a differentiation marker of osteoblast and plays an important role for improving osteogenesis function because the gene codes type-I procollagen which is a precursor of type I collagen constituting the main protein of bones. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an osteogenesis promoter, an osteoporosis preventive agent and a collagen synthesis promoter useful for the prevention and treatment of osteoporosis.

  If osteoporosis is classified based on the underlying disease, primary osteoporosis that does not show any obvious cause of illness and other diseases and illnesses such as administration of steroids, diabetes, and oophorectomy are the secondary causes that children and adolescents have. It is divided into osteoporosis. Primary osteoporosis is further classified into juvenile osteoporosis and regressive osteoporosis (senile / postmenopausal) based on age and the like. Juvenile osteoporosis has a very low incidence rate, and in some cases, it develops in puberty and heals spontaneously, or it occurs in adolescence and repeats fractures, and the cause is currently unknown.

  Regressive osteoporosis is classified into postmenopausal osteoporosis (type I) and senile osteoporosis (type II). Postmenopausal osteoporosis is thought to be caused by a deficiency of female hormone (estrogen) that suppresses calcium absorption (bone fracture) from bone. Senile osteoporosis is caused by a decrease in the function of the intestines due to aging, a decrease in the absorption rate of calcium, a decrease in the function of the kidneys, and a decrease in the amount of active vitamin D synthesis required for calcium absorption. It is considered (see Non-Patent Document 1).

In recent years, with the rapid aging of society, degenerative osteoporosis has become a major social problem.
Bone tissue is constantly undergoing remodeling (bone metabolism) through bone formation and bone resorption in order to change its own morphology and to stabilize blood calcium concentration. Normally, healthy adults have a balance between bone formation and bone resorption, and little change in bone weight is seen. Osteoporosis is a disease that causes a decrease in bone weight due to a decrease in bone formation or an increase in bone resorption, thereby easily causing a fracture. Considering the onset mechanism of osteoporosis from the viewpoint of remodeling of bone tissue, (1) decrease in bone weight due to increased bone resorption (early menopausal osteoporosis, osteoporosis due to hyperthyroidism, etc.), (2) bone formation Reduction of bone weight due to decrease (senile osteoporosis, diabetic osteoporosis, etc.), (3) Bone resorption and bone formation are normal, but due to lack of calcium intake etc., bone resorption amount exceeds bone formation amount There are three types of bone weight reduction.

  As therapeutic agents for (1), estrogen, calcitonin, and bisphosphonate derivatives are mainly used. From the viewpoint of prevention by food, an edible mushroom extract having an estrogen-like action (see Patent Document 1), a pomegranate extract exhibiting a bone resorption inhibitory action (see Patent Document 2), a Kawaraketsumei extract (see Patent Document 3), etc. Has been proposed. Patent Document 4 discloses a royal jelly extract containing 1% by weight or more of a substance having affinity for an estrogen receptor. As a therapeutic agent for (2), paratormon, anabolic agent, and fluoride agent are mainly used. As a treatment for (3), calcium supplementation is performed. From the viewpoint of prevention by food, foods with high calcium content and various materials having calcium absorption promoting action have been proposed.

Royal jelly is a milky white jelly-like substance made by mixing the secretory secreted from the hypopharyngeal gland and the greater vagina of 3 to 12 days old bees, and has been used as a health food since ancient times. . In recent years, numerous academic reports have been made on the antibacterial action, immune enhancing action, antitumor action, anti-inflammatory action and the like of royal jelly. As described above, royal jelly is widely used as a raw material for health foods, and examples of use related to osteoporosis are disclosed in Patent Documents 4 and 5, for example.
JP 2004-277414 A JP 2000-247896 A JP 2001-72598 A JP 2004-75543 A Japanese Patent Laid-Open No. 11-269078 “About osteoporosis”, [online], Osteoporosis Foundation, [Searched on March 18, 2005], Internet <URL: http://www.jpof.or.jp/content/frame_word.html>

  For the above (1) and (3), daily osteoporosis prevention can be easily carried out by ingesting the above foods, etc., while the above (2) has a preventive effect. At present, no proposals have been made regarding expected food ingredients. Promoting bone formation and activating the entire bone metabolism is effective not only for the prevention and treatment of senile osteoporosis and diabetic osteoporosis, but also for the overall effects of post-menopausal osteoporosis. It is expected to make a significant contribution toward the realization of an information society.

  As a result of intensive studies, the present inventors have found that royal jelly promotes bone formation. Furthermore, the present inventors have also found that royal jelly has an action of enhancing the expression of procollagen 1α1 gene, which is a main protein of bone matrix and is known as an osteoblast differentiation marker. And based on these knowledge, it came to complete this invention.

  The object of the present invention is to provide an osteogenesis promoter capable of promoting bone formation, an osteoporosis prophylactic agent useful for preventing osteoporosis by promoting osteogenesis, and collagen capable of promoting collagen synthesis. It is to provide a synthesis accelerator.

In order to achieve the above object, the osteogenesis promoter according to claim 1 is characterized by comprising royal jelly or an extract thereof as an active ingredient.
The gist of the agent for preventing osteoporosis according to claim 2 is that it comprises royal jelly or an extract thereof as an active ingredient and has a bone formation promoting action.

The gist of the preventive agent for osteoporosis according to claim 3 is that it has an effect of preventing or improving senile osteoporosis in the invention according to claim 2.
The osteoporosis-preventing agent according to claim 4 is characterized in that, in the invention according to claim 2 or claim 3, it further contains a bone resorption inhibitor and / or a food material having a calcium supplementing action.

  The gist of the collagen synthesis promoter according to claim 5 is to use royal jelly or an extract thereof as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, the osteogenesis promoter which can promote bone formation, the osteoporosis preventive agent useful for prevention of osteoporosis by promoting osteogenesis, and the collagen synthesis promoter which can promote the synthesis | combination of collagen. Can be provided.

Hereinafter, an embodiment in which the osteogenesis promoter, osteoporosis preventive agent and collagen synthesis promoter of the present invention are embodied will be described. Hereinafter, royal jelly is abbreviated as RJ.
The osteogenesis promoter of this embodiment contains RJ or an extract thereof as an active ingredient. Since an osteogenesis promoter has an action of promoting bone formation to an administered living body, it is useful for prevention and improvement (treatment) of senile osteoporosis. Furthermore, the osteogenesis promoter can also exert an effect of delaying the onset of osteoporosis by promoting bone formation in, for example, postmenopausal osteoporosis characterized by enhanced bone resorption. That is, the osteogenesis promoter containing RJ or an extract thereof as an active ingredient is also useful for preventing regression stage osteoporosis.

  The collagen synthesis promoter of this embodiment contains RJ or an extract thereof as an active ingredient. The collagen synthesis promoter has an action of increasing the expression of procollagen 1α1 gene in the administered living body. Since the procollagen 1α1 gene is known as an osteoblast differentiation marker, an increase in the expression level of the gene can lead to promotion of bone formation. Further, since the procollagen 1α1 gene is a gene encoding type I procollagen, which is a precursor of type I collagen, which is a major protein of bone, an increase in the expression level of the gene leads to promotion of bone formation. Is possible. Therefore, the collagen synthesis promoter is useful for the prevention and improvement of senile osteoporosis, as well as the osteogenesis osteoporosis as well as the osteogenesis promoter, in order to enhance the bone formation ability of the administered organism. Is also useful.

  As the RJ as an active ingredient, either raw RJ or dry RJ can be used. Further, the production area of RJ may be any region of Asian countries such as China, Taiwan and Japan, European countries, North American countries and South American countries. The RJ extract as an active ingredient is extracted by immersing raw RJ or dried RJ in a polar solvent such as alcohol. As the polar solvent, lower alcohols such as methanol, ethanol, butanol, and propanol, water, or a mixture thereof can be used. However, when taken orally as a food or drink, ethanol, water, or water-containing ethanol is particularly preferable. used. Moreover, you may refine | purify RJ or an RJ extract by a well-known isolation | separation and refinement | purification means as needed.

  Since the osteoporosis-preventing agent of the present embodiment contains RJ or an extract thereof as an active ingredient, it is useful for the prevention and improvement of senile osteoporosis as well as the osteogenesis promoter, and also for the prevention of degenerative osteoporosis. Also useful. In addition to RJ as an active ingredient or an extract thereof, the osteoporosis-preventing agent preferably further includes a bone resorption inhibitor or a food material having a calcium replenishing action. Both the bone resorption inhibitor and calcium are included. It is particularly preferred that it is contained.

  Bone resorption inhibitors include estrogen, calcitonin, bisphosphonate derivatives, isoflavones such as genistein, daidzein, soybean-in, placental extract, mushroom extract, pomegranate extract, Kawaraketsumei extract and other postmenopausal osteoporosis prevention Or any therapeutically effective ingredient is used. Osteoporosis preventive agent containing bone resorption inhibitor promotes bone formation with RJ or its extract, and suppresses bone resorption with bone resorption inhibitor, thereby greatly shifting the balance of bone metabolism to the bone-forming side. It is possible to increase the bone weight, and exerts a preventive and ameliorating effect on the general regressive osteoporosis including senile osteoporosis and postmenopausal osteoporosis.

  Food materials with calcium supplementation include calcium salts such as calcium carbonate and calcium phosphate, calcined calcium (baked shell, eggshell, bone, whey, oyster shell, sea urchin, coral, etc.), uncalcined calcium (unfired Shells, corals, eggshells, bones, pearls, etc.), dolomite ores, gypsum, etc., which can supply bone material to promote bone formation. At this time, it is also possible to suppress a decrease in bone weight due to insufficient intake of calcium (the above-mentioned type (3) osteoporosis). In addition, the osteoporosis-preventing agent of the present embodiment includes, as necessary, minerals such as magnesium, iron, manganese, copper, and zinc, vitamins such as vitamin B group, vitamin D, vitamin E, and ubiquinone, flavonoids, and casein. Peptides such as phosphopeptides, proteins, lipids, saccharides, oligosaccharides and the like are preferably included.

The osteoporosis preventive agent is used as a pharmaceutical, a quasi drug or a food or drink.
In the case of pharmaceuticals, it is administered as an oral or parenteral agent. Examples of oral dosage forms include tablets, capsules, powders, syrups, and drinks. Examples of parenteral dosage forms include injections in addition to external preparations such as ointments, creams, and liquids. In the case of quasi drugs, it is used in dosage forms such as tablets, capsules, drinks and the like. On the other hand, in the case of food and drink, it is taken orally as a beverage such as a drink, food such as rice cake, rice cracker, and cookies, or a food preparation. The food and drink may contain base materials, carriers, excipients, additives, auxiliary materials, extenders, colorants, fragrances and the like that are acceptable for food production. Examples of dosage forms of food preparations include powders, tablets, drinks, capsules and the like.

  The content of active ingredients in pharmaceuticals, quasi drugs and foods and beverages is preferably 0.001 to 100% by mass, more preferably 0.001 to 50% by mass, and still more preferably 0.01 to 30% by mass. %. If the content of the active ingredient is less than 0.001% by mass, bone formation cannot be sufficiently promoted. Moreover, it is preferable that a food / beverage product is orally ingested regularly for prevention of osteoporosis, and it is especially preferable that it is ingested orally several times a day. The daily intake is not particularly limited, but is preferably 0.001 to 30 g, more preferably 0.001 to 20 g, and still more preferably 0.01 to 10 g in terms of weight as royal jelly. . When the daily intake of food and drink is less than 0.001 g, the preventive effect against osteoporosis is not sufficiently exhibited. Conversely, if the daily intake of food and drink exceeds 30 g, the preventive effect against osteoporosis is sufficiently exerted, but the increase in the preventive effect commensurate with the increase in intake is not seen, which is uneconomical. It is.

The effects exhibited by the embodiment will be described below.
-The osteogenesis promoter, osteoporosis preventive agent, and collagen synthesis promoter of this embodiment all have RJ or an extract thereof as an active ingredient. Since the active ingredient promotes bone formation and collagen synthesis in the administered organism, it exhibits an excellent effect in the prevention and improvement of senile osteoporosis, as well as various types of osteoporosis including postmenopausal osteoporosis. It can exert an excellent effect on prevention. In particular, in preventing osteoporosis, promoting bone formation is extremely effective in increasing the maximum bone mass before the bone mass begins to decrease and in delaying the decrease in bone mass. Also, since senile osteoporosis is a disease that can affect both men and women, it is also important that it is more versatile than an osteoporosis preventive agent that is effective only for postmenopausal osteoporosis.

  -In addition to the active ingredient of the present embodiment, by containing a bone resorption inhibitor, the balance of bone metabolism can be shifted to the bone forming side through both the effects of promoting bone formation and suppressing bone resorption. And synergistic effects on the prevention and improvement of osteoporosis can be expected. Moreover, in addition to the active ingredient of this embodiment, by containing a food material having a calcium replenishing action, an effect of suppressing osteoporosis of the above type (3) is expected while supplying an osteogenic material.

Examples are shown below, but these examples are for better understanding of the present invention and do not limit the scope of the present invention.
<Evaluation of bone formation ability by RJ>
Example 1
RJ mixed feed was prepared by adding 4% of Chinese RJ lyophilized powder to powdered feed CRF-1 for mouse, rat and hamster (manufactured by Oriental Yeast Co., Ltd.).

(Example 2)
After adding 2.5 L of 95% volume ethanol to 1 kg of Chinese-made RJ and stirring at room temperature for 2 hours, suction filtration was performed. This filtrate is used as a primary extract. On the other hand, 2 L of 95% volume ethanol was added to the residue after collecting the primary extract and stirred at room temperature for 2 hours, followed by suction filtration. This filtrate is used as a secondary extract. The primary extract and the secondary extract were mixed, the solvent was removed by distillation under reduced pressure, and 4% was added to CRF-1, thereby preparing an RJ extract mixed feed.

(Test Example 1: Evaluation of bone formation ability for normal mice)
C57BL mice (9-week-old female) were purchased from Japan SLC. After one week of preliminary breeding with CRF-1, the mice were divided into 4 groups (10 mice in each group), of which 3 groups of mice were fed with normal feed consisting of CRF-1 and 4% RJ mixed feed (Examples). 1) or 4% RJ extract mixed feed (Example 2) was given for 2 months and allowed to ingest freely. There was no significant difference in food intake between groups during the study period. As a positive control, a group in which 17β estradiol (manufactured by Sigma, # E8875) was subcutaneously administered at a dose of 3 μg / kg / day for 2 months (5 days / week) was also provided.

  Two months later, each group of mice was sacrificed under ether anesthesia, and the tibia was removed and vacuum-dried, and then the dry bone weight was measured. Further, the dried bone was ashed at 600 ° C. for 3 hours, and the ashed weight was measured. The average value ± standard deviation of dry bone weight and ashing weight was determined for each group of mice (10 mice per group), and the results are shown in Table 1 below. Table 1 also shows the results of t-test for each group with respect to the normal feed group.

(Comparative Example 1: Evaluation of bone forming ability for ovariectomized mice)
C57BL mice (7-week-old female) were purchased from Japan SLC. After pre-breeding for 1 week with CRF-1, both mouse ovaries were surgically removed to produce postmenopausal osteoporosis model mice (ovariectomy group). In addition, as a sham operation group, only a sham operation (open surgery) in which the ovaries were not removed was performed. The mice in the ovariectomized group were each raised on CRF-1 for 2 weeks, and then fed with a normal diet consisting of CRF-1 or a 4% RJ mixed diet (Example 1) for 2 months. Similarly, mice in the sham-operated group were reared on CRF-1 for 2 weeks, and then a normal diet comprising CRF-1 was given for 2 months and allowed to freely ingest. There was no significant difference in food intake between groups during the study period. As a positive control, there was also provided a group in which 17β estradiol was subcutaneously administered (5 days / week) at a dose of 3 μg / kg / day for 2 months while giving a normal feed.

  Two months later, each group of mice was sacrificed under ether anesthesia, the tibia was removed, and the dry bone weight and ash weight were measured. The average value ± standard deviation of dry bone weight and ashing weight was determined for each group of mice (10 mice per group), and the results are shown in Table 1 below. Table 1 also shows the results of t-test for each group with respect to the normal feed group.

  As shown in Table 1, in Comparative Example 1, which is a model of postmenopausal osteoporosis, no increase in ashing weight (weight of mineral components such as calcium) was observed even when the RJ mixed feed (Example 1) was given. It was. On the other hand, in Test Example 1, an increasing tendency of the ashing weight was recognized by giving the RJ mixed feed (Example 1), and the ashing weight was significantly increased by giving the RJ extract mixed feed (Example 2). Increase was observed. Therefore, the RJ mixed feed of Example 1 and the RJ extract mixed feed of Example 2 are not strong in suppressing bone resorption (that is, postmenopausal osteoporosis), but rather in suppressing bone formation, that is, senile. It was excellent in action to prevent and ameliorate osteoporosis. As described above, both RJ and RJ extract can exert an excellent effect on prevention of osteoporosis and an effect on prevention and improvement of senile osteoporosis through promotion of bone formation.

  Further, in this example, the dry bone weight and the ashing weight of the tibia were measured, in which the variation in bone weight at the time of measurement was smaller than that of the femur and the like, and the increase and decrease of the bone weight of the whole individual could be accurately reflected. For this reason, an increase in the bone weight (especially ashing weight) of the tibia in Examples 1 and 2 of Test Example 1 does not mean that only the weight of a specific bone has increased. This is very important in that it means that the weight has increased physiologically. The dry bone weight shown in Table 1 is the sum of the ashed weight and the weight of organic matter such as protein. In addition to the fact that large variation was observed among individuals in the same group, in osteoporosis, Since bone density (ashed weight) is a problem, it should be considered as a reference level.

<Evaluation of expression level of procollagen 1α1 gene by RJ>
SAMR mice (9 weeks old female) were purchased from Japan SLC. After 1 week of preliminary breeding with CRF-1, the mice were divided into 3 groups (8 animals per group), of which 2 groups of mice were each fed with a normal feed consisting of CRF-1 or a 4% RJ mixed feed (Example 1). ) Was given for 2 months and taken freely. There was no significant difference in food intake between groups during the study period. As a positive control, there was also provided a group in which 17β estradiol was administered subcutaneously (5 days / week) for 2 months at a dose of 3 μg / kg / day while giving a normal feed.

  Two months later, each group of mice was sacrificed under ether anesthesia, and the femur was removed and frozen in liquid nitrogen. Subsequently, after extracting total RNA from the femur using Trizol reagent (Invitrogen), it was converted to cDNA using a reverse transcription kit (SuperScript First-Strand Synthesis System for RT-PCR manufactured by Invitrogen) Quantitative PCR was performed.

  The sequences of the primers used are shown in SEQ ID NOs: 1 to 4. The sequences represented by SEQ ID NOs: 1 and 2 represent the base sequences of the sense primer and antisense primer that bind to the glyceraldehyde triphosphate dehydrogenase (GAPDH) gene, and the sequences represented by SEQ ID NOs: 3 and 4 are The base sequences of the sense primer and antisense primer that bind to the procollagen 1α1 gene are shown. A PCR reagent (Platinum SYBR Green qPCR SuperMix UDG manufactured by Invitrogen) was used for the PCR reaction, and a quantitative PCR apparatus (7300 Real Time PCR System manufactured by Applied Biosystems) was used for the reaction and detection. The PCR reaction conditions were 50 ° C., 2 minutes, 95 ° C., 10 minutes, followed by 40 reaction cycles (95 ° C., 15 seconds → 60 ° C., 1 minute).

  The relative amount of mRNA of the procollagen 1α1 gene was calculated by the comparative Ct method described in the manual attached to the quantitative PCR device (Applied Biosystems, User Bulletin # 2). The mRNA amount of the procollagen 1α1 gene in each group was standardized by measuring the mRNA amount of the internal standard GAPDH. With respect to the amount of mRNA (expression level) of the procollagen 1α1 gene after standardization, the group fed with the normal feed was set to 1.00, and the relative value was shown. The results are shown in FIG. Moreover, t-test of each group was performed with respect to the group which received the normal feed, and * mark was attached in the figure when the risk rate was less than 5%.

  As shown in FIG. 1, when 4% RJ mixed feed (Example 1) was given, it was shown that the expression level of procollagen 1α1 gene was significantly increased. Procollagen 1α1 gene is not only known as a differentiation marker for osteoblasts, but also encodes type I procollagen, which is a precursor of type I collagen, which is a major protein of bone, and therefore bone formation It plays an important role in enhancing performance.

In addition, this embodiment can also be changed and embodied as follows.
-The osteoporosis preventive agent can be used for juvenile osteoporosis and secondary osteoporosis.
-An osteoporosis-preventing agent may contain paratormon, anabolic agent, fluoride agent and the like. Moreover, you may make the osteoporosis preventive agent contain the raw material (calcium absorption promoter) which has a calcium absorption promotion effect.

Further, the technical idea that can be grasped from the embodiment will be described below.
The osteoporosis preventive agent according to any one of claims 2 to 4, which has a collagen synthesis promoting action.

The osteogenesis promoter according to claim 1, wherein the extract is a polar solvent extract. The osteogenesis promoter according to claim 1, wherein the extract is an ethanol extract.
-A food or drink containing royal jelly or an extract thereof and having an osteoporosis-preventing action. A food preparation comprising a royal jelly or an extract thereof as an active ingredient and having an effect of preventing osteoporosis. A pharmaceutical product comprising a royal jelly or an extract thereof as an active ingredient and having an effect of preventing osteoporosis. A pharmaceutical product comprising royal jelly or an extract thereof as an active ingredient and having an effect of preventing and improving senile osteoporosis. A quasi-drug that has royal jelly or an extract thereof as an active ingredient and has an osteoporosis-preventing action. In these cases, foods and drinks, food preparations, pharmaceuticals or quasi drugs useful for the prevention of osteoporosis and the like can be provided.

The graph which shows the result of having investigated the expression level of the procollagen 1 (alpha) 1 gene of an Example.

Claims (5)

  An osteogenesis promoter comprising royal jelly or an extract thereof as an active ingredient.   An agent for preventing osteoporosis, which comprises royal jelly or an extract thereof as an active ingredient and has a bone formation promoting action.   The osteoporosis preventive agent according to claim 2, which has an effect of preventing or improving senile osteoporosis.   Furthermore, the osteoporosis preventive agent of Claim 2 or Claim 3 containing the foodstuff material which has a bone resorption inhibitor and / or a calcium replenishment effect | action.   A collagen synthesis promoter comprising royal jelly or an extract thereof as an active ingredient.

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