Composition containing eggshell membrane component for increasing bone mass or inhibiting bone marrow adiposity

Abstract

[Problem] To provide an oral composition containing a safe single component as an effective component and capable of preventing or ameliorating bone mass loss or osteoporosis. [Solution] A composition for increasing bone mass or inhibiting bone marrow adiposity according to the present invention comprises an eggshell membrane component as an effective component. The differentiation of bone marrow mesenchymal stem cells is shifted from adipocytes to osteoblasts by the ingestion of eggshell membrane-containing powder or a hydrolyzed eggshell membrane component, whereby bone mass loss or osteoporosis is prevented or ameliorated on the basis of a bone marrow adiposity-inhibiting action or a bone mass-increasing action. A formulation containing said composition can be used in various applications and forms such as a food additive, a food and drink, a supplement, and a pharmaceutical product.

Description

Composition for increasing bone mass or suppressing bone marrow adipogenesis containing eggshell membrane components

[0001] The present invention relates to a composition containing an eggshell membrane component as an active ingredient.

[0002] In modern society, which is an aging society, osteoporosis is a major health problem. Osteoporosis is characterized by a decrease in bone density and changes in the microstructure of bone tissue, significantly increasing the risk of fractures. In particular, its incidence is high in postmenopausal women and the elderly. Also, bone marrow adipogenesis is a phenomenon in which hematopoietic tissue in the bone marrow is replaced by adipose tissue as aging progresses, and is deeply related to a decrease in the number and function of osteoblasts responsible for bone formation. When bone marrow adipogenesis progresses, deterioration of bone quality and reduction of bone mass are promoted, so suppressing this is important for maintaining and improving bone health.

[0003] As measures against osteoporosis and bone loss, supplementation with calcium and vitamin D, exercise, etc. are generally recommended. These methods are essential for bone formation and contribute to maintaining bone density, but have only limited effects on suppressing bone marrow adipogenesis and improving bone quality. Also, in postmenopausal women, estrogen replacement therapy is effective for maintaining bone density, but long-term hormone therapy may increase the risk of breast cancer and cardiovascular disease, so its use is being carefully considered. Furthermore, drugs such as bisphosphonates and denosumab are used for the purpose of suppressing bone resorption, but these drugs have been reported to have side effects such as osteonecrosis of the jaw and pathologic fractures.

[0004] The eggshell membrane is a membrane inside the eggshell of bird eggs such as chicken eggs, consisting of two layers, the outer eggshell membrane adheres closely to the inner surface of the eggshell, and the inner eggshell membrane encloses the egg white, having antibacterial properties to protect the developing embryo from infection. The eggshell membrane is mainly composed of fibrous proteins including type I, type V, and type X collagen, etc., and these proteins contain a lot of cystine and cysteine, and have a network structure composed of tough fibrous proteins.

[0005] It is known that ingesting or applying eggshell membrane-containing components such as eggshell membrane or hydrolyzed eggshell membrane has skin-improving effects such as promoting type III collagen production, leading to beautiful skin and skin regeneration (Patent Documents 1 and 2), as well as general health-enhancing effects such as appetite stimulation and fatigue recovery. These components are already being developed and put into practical use as supplements and cosmetic ingredients. Furthermore, the applicant previously reported that approximately 54% of eggshell membrane-containing powder remains undigested and unabsorbed in the intestines (Non-Patent Document 1), functioning as an indigestible protein. The undigested and unabsorbed components act as prebiotics, preventing the proliferation of harmful bacteria in the intestines and improving the intestinal flora (Patent Document 3).

[0006] Furthermore, Patent Document 4 describes an eggshell powder and powdered eggshell membrane capsule composition for preventing lifestyle-related diseases, strengthening bone density in the human body, and improving human bodily functions, which is a combination of linseed oil, eggshell powder, and powdered eggshell membrane. However, regarding the effect of strengthening bone density in the human body, it only describes the results of another company's research on eggshell calcium, which showed that when osteoporotic rats were given eggshell calcium, seashell calcium, and bovine bone meal calcium respectively, the rats given eggshell calcium showed the greatest increase in bone density, or that it was confirmed that eggshell calcium increases bone mass in humans. There is no mention of how powdered eggshell membrane is involved in strengthening bone density.

[0007] Japanese Patent Publication No. 2003-246741, Japanese Patent Publication No. 2012-153614, Japanese Patent No. 7086517, Japanese Patent Publication No. 2020-184983

[0008] Mol. Nutr. Food Res. (2013) Vol.57, p.291-306

[0009] The present invention aims to provide an oral preparation that is highly safe, can be used daily as a food or beverage, supplement, or pharmaceutical, and can suppress myelostosis and increase bone mass with a single ingredient. As myelostosis progresses, the deterioration of bone quality and the decrease in bone mass are accelerated, so suppressing this is important for maintaining and improving bone health. The present invention aims to provide a safe and effective composition for maintaining bone health that can prevent the decrease and decline in function of osteoblasts, which are responsible for bone formation, by suppressing myelostosis.

[0010] The inventors focused on the possibility that eggshell membrane components may suppress the differentiation of adipocytes and promote the differentiation and function of osteoblasts in the bone marrow environment, and conducted animal experiments to verify this, leading to the completion of the present invention.

[0011] The present invention relates to the compositions (1) to (4) below, the tablets, granules, powders, capsules, jellies, or drinks of (5), and the food, supplements, or pharmaceuticals of (6). (1) A composition for increasing bone mass or inhibiting myelostosis, comprising an eggshell membrane component as an active ingredient. (2) The composition according to (1) above, wherein the eggshell membrane component is an eggshell membrane-containing powder. (3) The composition according to (2) above, wherein the eggshell membrane-containing powder has a volume average particle diameter of 6 μm or less and / or a volume maximum particle diameter of 20 μm or less, as determined by laser diffraction. (4) The composition according to any one of (1) to (3) above, which is an orally administered preparation. (5) A tablet, granules, powder, capsule, jellies, or drink containing the composition according to any one of (1) to (3) above, for increasing bone mass or inhibiting myelostosis. (6) Foods, food additives, supplements, or pharmaceuticals for increasing bone mass or inhibiting myelostosis, comprising the composition described in any of (1) to (3) above. The present invention also relates to the methods described in (7) and (8), the use described in (9), and the eggshell membrane component described in (10). (7) A method for increasing bone mass or inhibiting myelostosis, comprising the step of administering an eggshell membrane component. (8) A method for treating osteoporosis, comprising the step of administering an eggshell membrane component. (9) Non-therapeutic use of an eggshell membrane component for increasing bone mass or inhibiting myelostosis. (10) An eggshell membrane component for treating osteoporosis.

[0012] In experiments using rats, femoral samples were histologically and biochemically analyzed after an 8-week period in which rats were fed a normal solid diet and finely ground eggshell membrane. Histological analysis showed a significant increase in bone mass and suppression of bone marrow steatosis compared to the control group. Furthermore, gene expression analysis revealed increased expression of osteoblast-related factors and decreased expression of adipocyte-related factors compared to the control group. These findings confirm the effectiveness of eggshell membrane-containing powder in preventing or improving bone loss and osteoporosis, based on its effect of suppressing bone marrow steatosis or increasing bone mass by shifting the differentiation of bone marrow mesenchymal stem cells from adipocytes to osteoblasts.

[0013] According to the present invention, a composition for preventing or improving osteoporosis can be provided that can be taken daily in various forms such as food additives, foods and beverages, supplements, and pharmaceuticals. Because it has the effect of increasing bone mass and inhibiting fatty degeneration of bone marrow, it has the advantage of being usable as a supplement for a wide range of age groups, both for the prevention of osteoporosis before bone mass decreases and for the improvement of osteoporosis after bone mass decreases. Furthermore, the composition of the present invention is non-irritating, can be used in combination with various ingredients in various forms, and can be easily made into tablets.

[0014] The macroscopic image of a cross-section of the lower femur, showing the effects of eggshell membrane intake on bone marrow adipogenesis and bone mass increase in the eggshell membrane intake group (EM) compared with the control group (CO), as a result of animal experiments in the examples. The microscopic image of a cross-section of the black-framed area of ​​the lower femur in Figure 1, also a result of animal experiments in the examples. The upper row is a bright-field image, and the lower row shows adipocytes colored with a blue fluorescent dye. The arrows a in the upper row and b in the lower row indicate trabeculae. The images also show the results of animal experiments in the examples: (a) bone mass of cancellous bone (BV), (b) number of adipocytes in the bone marrow, (c) gene expression of osteoblast-related factor (Sp7) (n=6), and (d) gene expression of adipocyte-related factor (Pparg) (n=6).

[0015] The eggshell membrane used in this invention can be any membrane found on the inside of the eggshell of any terrestrial oviparous animal egg, especially bird eggs. Chicken eggshell membrane is particularly preferred in terms of availability and cost. The eggshell membrane component used in this invention is either an eggshell membrane-containing powder or a hydrolyzed eggshell membrane component. The eggshell membrane-containing powder is not particularly limited as long as it contains at least eggshell membrane, and powder refers to any powder regardless of particle size. Peeled eggshell membrane or raw material with eggshell membrane attached to the eggshell may be powdered using any known method. Alternatively, commercially available eggshell membrane-containing powder may be used.

[0016] The hydrolyzed eggshell membrane component used in this invention is obtained by hydrolyzing eggshell membrane or the aforementioned eggshell membrane-containing powder in a solution having a protein-degrading effect, such as an acidic aqueous solution, an alkaline aqueous solution, an alkaline organic solvent solution, or a proteolytic enzyme-containing solution. It can also be used as an aqueous solution or extract, but for ease of handling and storage stability, a powder form obtained by drying means such as spray drying or freeze-drying is preferred. As hydrolyzed eggshell membrane powder, a powder with a pH of 3 to 6 is particularly preferred from the viewpoint of storage stability and safety.

[0017] The eggshell membrane-containing powder used in this invention refers to a powder whose maximum volume particle diameter and / or volume average particle diameter is generally less than 100 μm. In this specification, the "volume average particle diameter" and "maximum volume particle diameter" of the powder refer to values ​​measured using a laser diffraction particle size distribution analyzer (Seishin Co., Ltd., LMS-30). When measuring the particle diameter, a sample prepared by dispersing eggshell membrane-containing fine powder in water using a surfactant is used. Furthermore, "volume average particle diameter" refers to the particle diameter at which the cumulative value from the small particle size side in the particle size distribution reaches 50%.

[0018] The eggshell membrane-containing powder used in this invention is preferably one with a volume-average particle diameter of 6 μm or less, or a volume-maximum particle diameter of 20 μm or less, from the viewpoint of digestion and absorption efficiency. Eggshell membrane-containing raw materials, such as commercially available eggshell membrane-containing powder, can be produced by a fine grinding process using a jet mill that causes particles to collide with each other in a gas atmosphere. More preferably, the material is finely ground in a ball mill until the volume-average particle diameter is 6 μm or the volume-maximum particle diameter is 20 μm or less. By grinding with a ball mill, although the overall volume-average particle diameter does not change, fine powder molecules that are smaller and more uniform in size are obtained compared to conventional fine powders, resulting in an eggshell membrane-containing fine powder that is more uniformly dispersed and more solubilized.

[0019] Osteoporosis is a disease in which the metabolic balance of bone is disrupted, and bone destruction continues to outweigh bone formation, making bones brittle and prone to fractures. The bones in the body are living tissue, and even though they may appear the same, bone formation (new bone creation) and bone resorption (bone breakdown) are constantly occurring. In osteoporosis, this balance is disrupted, causing the bones to become porous. Osteoporosis is overwhelmingly more common in women, especially postmenopausal women, and is thought to be closely related to the decrease in female hormones and aging.

[0020] The inventors used 7-week-old rats, whose fat content gradually increased after preliminary rearing, and divided them into an EM group (containing 120 mg / kg of regular solid feed and finely ground eggshell membrane per day) and a control group CO (containing only regular solid feed). The EM group was given eggshell membrane every 5 days / week for 8 weeks. After that, femurs were removed from the euthanized rats, and the removed samples were analyzed macroscopically, histologically, and biochemically. Macroscopically, the EM group showed more abundant trabecular formation and a significant increase in the mesiodistal width of the cancellous bone.

[0021] Histological analysis revealed that compared to CO, EM showed a significant increase in bone mass, a significantly higher osteoblast surface area per trabecular bone, a significantly lower number of adipocytes in the bone marrow, and suppressed steatosis in the bone marrow. Furthermore, gene expression analysis showed increased expression of osteoblast-related factors and decreased expression of adipocyte-related factors in EM. These results suggest that eggshell membrane intake contributes to suppressing steatosis in the bone marrow and increasing bone mass by suppressing the differentiation of mesenchymal stem cells into adipocytes and promoting their differentiation into osteoblasts in the bone marrow.

[0022] The amount of eggshell membrane-containing powder in the oral composition of the present invention is not particularly limited, but from the viewpoint of smooth granulation and tableting and cachexia-improving effects when tablets are ingested, it is contained in a proportion of 5 to 40% by weight, more preferably 10 to 35% by weight, relative to the total weight of the tablets. By setting the content to 5% by weight or more, it becomes unnecessary to ingest a large number of tablets, while by setting it to 35% by weight or less, granulation and tableting become easier, making it easier to manufacture tablets.

[0023] The oral composition of the present invention may contain various known additives for forming tablets, granules, powders, capsules, jellies, or drinks, and may also contain excipients, binders, disintegrants, lubricants, and other nutrients. As excipients, known excipients can be used as appropriate, but it is preferable to use at least one of modified starch and lactose in an amount of 0.3 to 3 times, and more preferably 1 to 2.5 times, relative to the weight of the eggshell membrane component, from the viewpoint of excipient properties. As modified starch, one or more types of dextrin such as roasted dextrin, oxidized starch, and low-viscosity modified starch can be used. When modified starch and lactose are used in combination as excipients, the usage ratio (weight ratio) is preferably 1:5 to 5:1. As binders, known binders can be used as appropriate, such as starch paste, gum arabic paste, and hydroxypropyl cellulose. As disintegrants, known disintegrants can be used as appropriate, such as celluloses. As a lubricant, any known lubricant can be used as appropriate, such as magnesium stearate, sucrose fatty acid esters, waxes, talc, and vitamin C.

[0024] Furthermore, in order to increase the hardness of the tablets, prevent deformation and scratching, and improve handling, it is preferable to include eggshell calcium as a hardness improver, and the eggshell calcium content in the tablets is preferably 0.1 to 20% by weight, more preferably 0.5 to 15% by weight. In addition, in order to prevent deterioration and decomposition of the components contained in the tablets and to improve the scratch resistance of the tablet surface, it is preferable to cover them with a coating film. Any known coating film can be used as appropriate, but for example, the product name "Cellar" (manufactured by Gifu Shellac Co., Ltd.) can be used, and the tablets of the present invention may be covered with a sugar coating, may be colored, and may be given a gloss treatment after coloring.

[0025] The size of the tablets of the present invention is not particularly limited, but generally, a circular or oval shape with a diameter of about 7 to 10 mm is preferred for ease of handling and administration. The weight of one tablet is about 350 to 600 mg, and the active ingredient is preferably about 10 to 240 mg, more preferably about 20 to 150 mg. The tablets of the present invention can be manufactured by known tablet manufacturing methods using a tableting raw material containing at least eggshell membrane powder. For example, after a tableting step in which the tableting raw material is compressed to form uncoated tablets, a granulation step, a protective coating step, a sugar coating step, etc. may be performed as needed, and further coloring and glossing may be performed. In addition, granules and powders may be used as solid preparations other than tablets.

[0026] Furthermore, the effective dose of the pharmaceutical composition for osteoporosis of the present invention varies depending on the severity of the symptoms to be treated or prevented, the condition of the recipient (including age and sex), and the dosage form. Based on animal experiments, it is desirable to ingest 100 mg / kg of eggshell membrane powder per day. The oral dose of such a pharmaceutical for a human (an adult weighing 60 kg) is preferably 100 to 10,000 mg per day, converted to eggshell membrane-containing powder. For example, the effective dose can be 1,000 to 10,000 mg of eggshell membrane-containing powder per day in total. The eggshell membrane-containing powder of the present invention is highly safe and has no concerns about side effects, so as long as other components are appropriately selected, there is no problem even if the intake or application amount exceeds the above range. It is desirable to take it during or after meals to improve absorption efficiency. Furthermore, long-term intake is effective.

[0027] Furthermore, the formulations of the present invention can be used as food additives in confectionery, beverages, health foods, preserved foods, processed foods, and the like. Here, the terms "pharmaceuticals" and "foods and beverages" are not limited to humans, but also include pharmaceuticals and feeds for mammals such as pets and livestock. In addition, the concept of "foods and beverages" includes not only ordinary foods and beverages, but also enteral nutrition foods, nutritional functional foods, foods with functional claims, and foods for specified health uses.

[0028] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0029] [Animal Experiment] Sixteen 6-week-old (W) male Wistar rats were used. All rats were divided into two groups: EM (n=8), which was fed a normal solid diet and finely ground eggshell membrane (120 mg / kg per day) after a one-week pre- rearing period, and CO (n=8), which was fed only a normal solid diet. Eggshell membrane administration to the EM group was performed every 5 days / week, safely using an oral tube for rodents under light anesthesia by isoflurane inhalation. The experiment lasted for 8 weeks. After the experiment, rats in each group were euthanized by carbon dioxide inhalation, and their femurs were removed. The removed samples were analyzed macroscopically, histologically, and biochemically.

[0030] [Gross Image] Figure 1 shows cross-sectional images of the lower part of the femur from CO and EM. In EM, compared to CO, a wider area of ​​cancellous bone (indicated by arrows) with abundant trabeculae was observed. While a decrease in cancellous bone is diagnosed as osteoporosis, in EM, the mesiodistal width (longitudinal direction) of the cancellous bone was significantly increased. Bone marrow is present between the trabeculae.

[0031] [Measurement of bone mass in trabecular bone] Figure 3(a) shows the results of bone mass (BV) analysis of trabecular bone using μCT (Sky Scan 1276: Bruker). In EM, the BV of trabecular bone was increased by approximately 16% compared to CO.

[0032] [Histological Analysis] Figure 2 shows photographs of microscopic observations of a portion of the lower femur cross-section shown in Figure 1 (the black-framed area in EM, and the same area in C0) that was further cut into thin sections of approximately 150 μm. The upper row is a bright-field photograph, and the lower row shows adipocytes colored with a blue fluorescent dye (since Figure 2 is a monochrome photograph, adipocytes are shown as white dotted structures). In the upper row of Figure 2, the white trabeculae (arrow a) are large in EM, and osteoblasts are observed on the surface of the trabeculae, whereas in CO, the white trabeculae (arrow a) are small. In the lower row of Figure 2, in EM, the number of adipocytes in the bone marrow located between the trabeculae (arrow b) (white dotted structures) is significantly reduced compared to CO.

[0033] [Measurements related to trabeculae, and measurement of osteoblast and adipocyte counts] Using sections, within a specified ROI, bone mineral density (BV / TV: %), trabecular width (Tb.Th: μm), number of trabeculae (Tb.N: n / mm), osteoblast surface area per trabecular surface (Ob.S / BS: %), and number of adipocytes in bone marrow (Ad.N / BM: n / mm) were measured. ２ The following five parameters were calculated. Ad.N / BM was calculated by measuring the number of adipocytes present in the bone marrow portion, excluding the trabeculae and vascular lumen. All parameters were blinded between groups and calculated after bias elimination by external contractor.

[0034] Compared to CO, EM showed significantly higher values, with BV / TV increasing from approximately 24% to 35%, Tb.Th from approximately 70 μm to 90 μm, Tb.N from approximately 3.3 n / mm to 4 n / mm, and Ob.S / BS from approximately 27% to 42%. On the other hand, as shown in Figure 3(b), Ad.N / BM was significantly lower in EM, being less than half that of CO.

[0035] [Gene Expression Measurement of Osteoblast-Related Factors and Adipocyte-Related Factors (n=6)] Gene expression of osteoblast-related factors (Sp7, Rn02769744_S1, Invitrogen) and adipocyte-related factors (Pparg, Rn00440945_m1, Invitrogen) in 6 EM and 6 CO individuals was analyzed by qRT-PCR.

[0036] The results showed that Sp7 expression was significantly higher in EM compared to CO, as shown in Figure 3(c). On the other hand, Pparg expression was significantly lower in EM, as shown in Figure 3(d). At the gene level, it was confirmed that EM increased bone mass and suppressed myeloadipopathy compared to CO.

Claims

1. A composition for increasing bone mass or inhibiting myelolipidemia, containing eggshell membrane components as an active ingredient.

2. The composition according to claim 1, wherein the eggshell membrane component is an eggshell membrane-containing powder.

3. The composition according to claim 2, wherein the eggshell membrane-containing powder has a volume-average particle diameter of 6 μm or less and / or a volume-maximum particle diameter of 20 μm or less, as determined by laser diffraction.

4. The composition according to any one of claims 1 to 3, which is an orally administered preparation.

5. A tablet, granule, powder, capsule, jelly, or drink containing the composition described in any one of claims 1 to 3, for increasing bone mass or inhibiting myelolipidemia.

6. Foods, food additives, supplements, or pharmaceuticals for increasing bone mass or inhibiting bone marrow steatosis, comprising the composition described in any one of claims 1 to 3.

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