JP2019136035A - Bone density improving food composition, bone density improving agent, precursor osteoblast proliferating food composition, bone differentiation promoting food composition, bone enhancing food composition, anti-osteoporosis food composition, precursor osteoblast proliferation agent, bone differentiation promoting agent, bone enhancing agent, anti-osteoporosis agent, method for producing bone density improving agent, method for producing precursor osteoblast proliferation agent, method for producing bone differentiation promoting agent, method for producing enhancing agent and method for producing anti-osteoporosis agent - Google Patents

Abstract

To provide a bone density improving food composition, a precursor osteoblast proliferating food composition, a bone differentiation promoting food composition, a bone enhancing food composition and an anti-osteoporosis food composition.SOLUTION: The present invention provides a bone density improving food composition, a precursor osteoblast proliferating food composition, a bone differentiation promoting food composition, a bone enhancing food composition and an anti-osteoporosis food composition, containing a Euglena algal body or a Euglena aqueous solvent extract as an active ingredient.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a bone density improving food composition, a bone density improving agent, a precursor osteoblast proliferation food composition, a bone differentiation promoting food composition, a bone reinforcing food composition, an anti-osteoporosis food composition, and a precursor. Osteoblast proliferating agent, osteogenesis promoting agent, bone strengthening agent, anti-osteoporosis agent, bone density improving agent manufacturing method, precursor osteoblast proliferating agent manufacturing method, bone differentiation promoting agent manufacturing method, bone reinforcing agent manufacturing The present invention relates to a method and a method for producing an anti-osteoporosis agent.

  In recent years, an increase in various bone diseases, such as an increase in osteoporosis in middle-aged and older groups and an increase in fracture rate in younger groups, has become a social issue. There may be a bedridden state due to a fracture of the femoral neck and a compression fracture of the spine caused by osteoporosis. From the viewpoint of preventing osteoporosis due to a decrease in bone density with aging, it is important to strengthen bones and increase bone mass.

  Conventionally, dietary calcium intake, exercise, drug therapy, and the like have been performed as methods for strengthening bone. For dietary calcium intake, it is recommended to consume dairy products, small fish, green-yellow vegetables, etc., but the amount of dietary intake is limited. Also, exercise such as walking and walking has been difficult for elderly people who are weak or bedridden. As for drug therapy, it is necessary to take the drug, but caution is required from the viewpoint of side effects and the like.

  As a bone strengthening agent using a natural material, there are known one having a zinc-enriched oyster extract as a main component (Patent Document 1) and one having an egg yolk protein hydrolyzate as an active ingredient (Patent Document 2).

On the other hand, Euglena (genus name: Euglena, Japanese name: Euglena) attracts attention as a biological resource that is expected to be used as food, feed, fuel, and the like.
Euglena has 59 kinds of nutrients that correspond to most of the nutrients necessary for humans to live, such as vitamins, minerals, amino acids, and unsaturated fatty acids, and as a supplement to take a variety of nutrients in a balanced manner. The possibility of use as a food supply source in poverty areas where the necessary nutrients cannot be ingested has been proposed.

Euglena is located in the primary producer of the food chain and is cultivated in large quantities because it is preyed by predators and the culture conditions such as light, temperature conditions, and stirring speed are difficult compared to other microorganisms. However, in recent years, due to the diligent research of the present inventors, a large-scale culture technique has been established, which has opened the way for mass supply of Euglena-derived substances such as Euglena and paramylon extracted from Euglena.
Euglena is a unique organism that possesses the animal nature of flagellar movement and at the same time has chloroplasts as a plant and performs photosynthesis, and Euglena itself and substances derived from Euglena are expected to have many functions. ing.
Therefore, it is desired to develop a method for using Euglena and Euglena-derived substances that can be supplied in large quantities.

JP 2002-80380 A Japanese Patent No. 5213332

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a bone density improving food composition, bone density improving agent, and progenitor osteoblast that can be ingested on a daily basis. Providing a food composition for proliferation, a food composition for promoting bone differentiation, a food composition for reinforcing bone, a food composition for anti-osteoporosis, a precursor osteoblast proliferating agent, a bone differentiation promoting agent, a bone strengthening agent and an anti-osteoporosis agent There is.

  As a result of diligent research to solve the above problems, the present inventors have found that Euglena improves (increases and improves) bone density, proliferates progenitor osteoblasts, promotes bone differentiation, As a result, the present invention has been completed.

Therefore, according to this invention, the said subject is solved by the food composition for bone density improvement characterized by containing the Euglena alga body or the Euglena aqueous solvent extract as an active ingredient.
At this time, the Euglena aqueous solvent extract may be at least one selected from the group comprising Euglena water extract, Euglena hot water extract and Euglena ethanol extract.

  Further, according to the present invention, the above-described problem is that, according to the present invention, a bone density improver, a food composition for progenitor osteoblast proliferation, a bone composition containing Euglena-derived substance, for example, Euglena alga body or Euglena aqueous solvent extract, It is solved by a food composition for promoting differentiation, a food composition for reinforcing bone, a food composition for anti-osteoporosis, a precursor osteoblast proliferating agent, a bone differentiation promoting agent, a bone reinforcing agent, and an anti-osteoporosis agent.

In addition, according to the present invention, the above-described problem is a dispersion preparation step in which an aqueous solvent is added to Euglena and shaken to obtain a dispersion; the dispersion is centrifuged, and the supernatant is extracted with Euglena aqueous solvent. And a method for producing the bone density improving agent, characterized by comprising an extraction step obtained as a product.
At this time, it is preferable to perform a heating step of heating the dispersion after the dispersion preparation step and before the extraction step.
At this time, the aqueous solvent may be at least one selected from the group including water, hot water, and ethanol.

  In addition, according to the present invention, the above-described problem is a dispersion preparation step in which an aqueous solvent is added to Euglena and shaken to obtain a dispersion; the dispersion is centrifuged, and the supernatant is extracted with Euglena aqueous solvent. And a method for producing a bone differentiation promoting agent characterized by comprising an extraction step obtained as a product.

  In addition, according to the present invention, the above-described problem is a dispersion preparation step in which an aqueous solvent is added to Euglena and shaken to obtain a dispersion; the dispersion is centrifuged, and the supernatant is extracted with Euglena aqueous solvent. And an extraction step obtained as a product.

  In addition, according to the present invention, the above-described problem is a dispersion preparation step in which an aqueous solvent is added to Euglena and shaken to obtain a dispersion; the dispersion is centrifuged, and the supernatant is extracted with Euglena aqueous solvent. And a method for producing an anti-osteoporosis agent characterized by comprising an extraction step obtained as a product.

According to the present invention, a novel bone density improving agent, food composition for progenitor osteoblast proliferation, food composition for progenitor osteoblast proliferation, food composition for promoting bone differentiation, food composition for bone strengthening, anti-osteoporosis Food composition, progenitor osteoblast proliferation agent, bone differentiation promoter, bone strengthening agent and anti-osteoporosis agent can be provided.
Bone density improving agent, food composition for progenitor osteoblast proliferation, food composition for progenitor osteoblast proliferation, food composition for promoting bone differentiation, food composition for bone strengthening, food composition for anti-osteoporosis according to the present invention , Progenitor osteoblast proliferators, osteodifferentiation promoters, bone strengtheners and anti-osteoporosis agents have no reports of side effects so far, and Euglena with a level of safety consistent with the Food Sanitation Act is the active ingredient Therefore, long-term continuous administration and continuous intake are possible.

It is a flowchart which shows the manufacturing method of the bone density improving agent of this embodiment, the manufacturing method of a progenitor osteoblast proliferation agent, the manufacturing method of a bone differentiation promoter, the manufacturing method of a bone strengthening agent, and the manufacturing method of an anti-osteoporosis agent. It is a graph which shows the result of the progenitor osteoblast proliferation test of the Euglena water extract of Example 1 verified in Test 1. It is a graph which shows the result of the progenitor osteoblast proliferation test of the Euglena hot water extract of Example 2 verified in Test 1. It is a graph which shows the result of the progenitor osteoblast proliferation test of the Euglena ethanol extract of Example 3 verified in Test 1. It is a graph which shows the result of the bone differentiation promotion test of the Euglena water extract of Example 1 verified in Test 2. It is a graph which shows the result of the bone differentiation promotion test of the Euglena hot water extract of Example 2 verified in Test 2. It is a graph which shows the result of the bone differentiation promotion test of the Euglena ethanol extract of Example 3 verified in Test 2. It is a graph which shows the result of the examination of the influence which the ingestion of Euglena which performed verification in the test 3 has on a human bone.

Hereinafter, embodiments of the present invention will be described with reference to FIGS.
The present embodiment is a bone density improving agent containing a Euglena-derived substance as an active ingredient, a food composition for progenitor osteoblast proliferation, a food composition for progenitor osteoblast proliferation, a food composition for promoting bone differentiation, and for bone strengthening Food composition, food composition for anti-osteoporosis, progenitor osteoblast proliferator, bone differentiation promoter, bone strengthening agent, anti-osteoporosis agent, method for producing bone density improving agent, method for producing progenitor osteoblast proliferator, bone The present invention relates to a method for producing a differentiation promoting agent, a method for producing a bone strengthening agent, and a method for producing an anti-osteoporosis agent.

<Osteblast differentiation>
In the present specification, an osteoprogenitor cell refers to a cell that has acquired the ability to differentiate into an osteoblast, and a precursor osteoblast (preosteoblast) is also referred to as a preosteoblast, A cell that has the same ability as a cell, but is in a proliferative phase, and an osteoblast is an osteoblast-like cell that has the ability to form bone but does not proliferate. ) Refers to a cell line with the same ability as osteoblasts.

  Osteoblasts differentiate from mesenchymal stem cells. In addition to osteoblasts, mesenchymal stem cells differentiate into chondrocytes, muscle cells, adipocytes, etc., but differentiation from mesenchymal stem cells to osteoblasts involves hormones, cytokines, extracellular matrix, etc. It is known to be induced by stimuli.

  Mesenchymal stem cells are differentiated in the order of osteoprogenitor cells, preosteoblasts, and osteoblasts, and mature osteoblasts eventually become bone cells. (Bone cell, osteocyte). Mature osteoblasts secrete a large amount of osteocalcin, and a strong bone matrix is formed by calcification of the extracellular matrix (Tetsuya Matsuguchi, “Osteblast differentiation and JNK signal”, Biochemistry, 81st Vol. 8, No. 703-707).

<Osteoporosis>
Osteoporosis is a condition in which the risk of fracture is increased. The World Health Organization (WHO) defines "Osteoporosis is a disease characterized by low bone mass and abnormalities in the fine structure of bone tissue that increases bone vulnerability and increases the risk of fractures" Osteoporosis is a disease, a pathological process leading to fractures, and fractures are one of the resulting complications (“Osteoporosis Prevention and Treatment Guidelines, Committee for Prevention and Treatment of Osteoporosis” 2015 ", p. 2).

  Diagnosis of osteoporosis is based on diagnostic criteria for primary osteoporosis, including medical interviews, physical examinations, diagnostic imaging, blood and urine tests, and bone evaluation (measurement of bone density). And spinal X-ray), differential diagnosis and diagnosis criteria for primary osteoporosis are applied and confirmed ("Osteoporosis Prevention and Treatment Guidelines 2015 Edition", page 18).

<Locomotive syndrome>
According to the Japanese Orthopedic Association, the locomotive syndrome is a function of “standing” or “walking” when any or more of the exercise devices such as bones, joints, cartilage, intervertebral discs, and muscles are damaged. It is defined as being in a lowered state (Non-Patent Document 2).
Locomotive syndrome is sometimes abbreviated as locomo, and is sometimes called locomotive syndrome or musculoskeletal syndrome.
Hereinafter, diagnostic criteria for locomotive syndrome will be described (Noriko Yoshimura, “Clinical Diagnostic Value and Prevalence of Locomotive Syndrome”, Journal of the Japan Geriatrics Society Vol. 52, No. 4, pp. 350-353 (2015)).

(Locomo test)
The locomotive degree test, which is one of diagnostic criteria for the locomotive syndrome, includes (1) a rising test, (2) a two-step test, and (3) locomo 25.
(1) The standing-up test is a test in which four heights of 10 cm, 20 cm, 30 cm, and 40 cm are prepared, and leg strength is measured based on whether or not one leg or both legs can stand up.
(2) In the two-step test, a two-step value is calculated by walking two steps with as much thigh as possible, measuring the step length for two steps, and dividing by the height. The walking ability including lower limb muscle strength, balance ability, flexibility, etc. is evaluated by two-step values.
(3) Locomo 25 evaluates whether there has been any physical pain or difficulty in daily life in the past month with a 25-item questionnaire.

The determination based on the locomotive degree test is made up of two, the locomotive degree 1 and the locomotive degree 2.
The clinical judgment value of Locomo degree 1 is as follows.
1) Stand-up test: Cannot stand up from a height of 40 cm with either one leg 2) 2-step test: 2-step value is less than 1.3 3) Locomo 25: Locomo 25 result is 7 points or more 1) to above If any one of 3) applies, it is determined that the degree of locomotive is 1, and it is determined that the deterioration of the mobile function has started.

The clinical judgment value of Locomo degree 2 is as follows.
1) Stand-up test: Cannot stand up from 20cm height on both legs 2) 2-step test: 2-step value is less than 1.1 3) Locomo 25: Locomo 25 result is 16 points or more Any of 1) to 3) above If even one of them is applicable, it is determined that the degree of locomotive is 2, and it is determined that the deterioration of the mobile function is in progress.

<Euglena>
In the embodiment, “euglena” includes taxonomically classified microorganisms classified as Euglena, variants thereof, variants thereof, and closely related species of Euglenaae.
Here, the Euglena genus (Euglena) is a group of organisms belonging to the Excavator, Euglenozoa, Euglena algae, Euglena, Euglena family among eukaryotes.

Specific examples of the genus Euglena include Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena mutabilis, Euglena proxima, Euglena spirogyra, Euglena viridis, and the like.
As Euglena, Euglena gracilis (E. gracilis), particularly E. gracilis Z strain, can be used. It may be a chloroplast-deficient strain, a variant E. gracilis var. Bacillaris, a gene mutant such as a chloroplast mutant of these species, or other Euglena such as Astasia longa.

Euglena is widely distributed in fresh water such as ponds and swamps, and may be used separately from these, or any Euglena already isolated may be used.
Euglena includes all its mutants. These mutant strains also include those obtained by genetic methods such as recombination, transduction, transformation and the like.

In the culture of Euglena cells, examples of the culture solution include a culture solution to which nutrient salts such as a nitrogen source, a phosphorus source, and a mineral are added, for example, a modified Cramer-Myers medium ((NH ₄ ) ₂ HPO ₄ 1.0 g / L. , KH ₂ PO ₄ 1.0 g / L, MgSO ₄ · 7H ₂ O 0.2 g / l, CaCl ₂ · 2H ₂ O 0.02 g / l, Fe ₂ (SO ₂ ) ₃ · 7H ₂ O 3 mg / l, MnCl ₂ · 4H ₂ O 1.8 mg / l, CoSO ₄ · 7H ₂ O 1.5 mg / l, ZnSO ₄ · 7H ₂ O 0.4 mg / l, Na ₂ MoO ₄ · 2H ₂ O 0.2 mg / l, CuSO ₄ .5H ₂ O 0.02 g / l, thiamine hydrochloride (vitamin B1) 0.1 mg / l, cyanocobalamin (vitamin B12), (pH 3.5)) can be used. Note that (NH ₄ ) ₂ HPO ₄ can also be converted to (NH ₄ ) ₂ SO ₄ or NH ₃ aq. In addition, a known Hutner medium or Koren-Hutner medium prepared based on the description of Euglena Physiology and Biochemistry (edited by Shozaburo Kitaoka, Society Publishing Center, Inc.) may be used.

The pH of the culture solution is preferably 2 or more, and the upper limit thereof is preferably 6 or less, more preferably 4.5 or less. By setting the pH to the acidic side, the photosynthetic microorganisms can grow more dominantly than other microorganisms, so that contamination can be suppressed.
Euglena cells may be cultured by an open pond method using sunlight directly, a light collecting method in which sunlight condensed by a light concentrator is sent through an optical fiber, etc., and irradiated in a culture tank and used for photosynthesis.
Euglena cells can be cultured using, for example, a fed-batch method, but flask culture, fermentation using a fermentor, batch culture, semi-batch culture (fed-batch culture), continuous culture (perfusion) Any liquid culture method such as a culture method may be used.
Euglena cells are separated, for example, by centrifugation, filtration or simple sedimentation of the culture medium.

(Euglena algae)
In the present embodiment, viable Euglena cells separated by centrifugation, filtration, sedimentation, or the like can be used as they are as Euglena alga bodies. Euglena viable cells can be used as they are after harvesting from the culture tank, but are preferably washed with water or physiological saline. Moreover, you may use in the state of the dispersion liquid which Euglena alga body disperse | distributed to liquids, such as water. In the present embodiment, it is preferable to use Euglena dry algae obtained by freeze-drying or spray-drying viable Euglena cells as Euglena algae.
Furthermore, a mechanically treated product of algal bodies obtained by subjecting viable Euglena cells to ultrasonic treatment or mechanical processing such as homogenization may be used as Euglena alga bodies. Moreover, you may use the mechanically processed material dried material which performed the drying process to the mechanically processed material as Euglena alga.

(Euglena aqueous solvent extract)
In the present embodiment, “Euglena aqueous solvent extract” means an extract extracted from Euglena using an aqueous solvent, and in particular, water is used as an aqueous solvent at 5 ° C. to 600 ° C. for several seconds to several times. It is preferable to use an Euglena water extract or hot water extract extracted for 10 hours.
The water used for extraction does not necessarily need to be distilled water, pure water, or ultrapure water. For example, it may contain tap water or impurities, but it contains components that hinder the extraction of active ingredients. No water is preferred.

In the present embodiment, the “water extract” means an extract with water of 0 to 50 ° C. (excluding 0 ° C.).
Here, “water” means water at 0 to 50 ° C. (excluding 0 ° C.).
The temperature of water is not particularly limited as long as it is within a range in which the active ingredient can be sufficiently extracted without affecting the active ingredient, but preferably 1 to 40 ° C, more preferably 5 to 35 ° C, Especially preferably, it is 10-30 degreeC.

In the present embodiment, the “hot water extract” means an extract with water having a temperature higher than 50 ° C., and can also be referred to as a “hot water extract”.
Here, “hot water” means water having a temperature higher than 50 ° C., and is a concept including “hot water”, and includes water in a boiling state. Moreover, it is not limited to hot water in a liquid state, and includes hot water in a gas state and a supercritical state.
The temperature of the hot water is not particularly limited as long as it is within a range in which the active ingredient can be sufficiently extracted without affecting the active ingredient, but is preferably higher than 50 ° C. and 120 ° C. or lower, more preferably 50 Higher than 100 ° C. and lower than 100 ° C.

  The pH of water used for extraction is not particularly limited as long as the active ingredient can be sufficiently extracted without affecting the active ingredient, but is preferably 4 to 10, more preferably 5 to 9. Particularly preferably, the pH is 6-8.

  In this embodiment, water is used alone as the aqueous solvent, but the active ingredient can be sufficiently extracted without affecting the active ingredient, and usually one kind of solvent that can be used for extraction is used. Or you may select and use 2 or more types. For example, water, alcohols, glycols and the like can be mentioned, but are not limited thereto. Examples of alcohols include ethanol, methanol, n-propanol, and isopropanol. Examples of glycols include butylene glycol and propylene glycol. Other aqueous solvents include acetone. These solvents may be used alone or as an aqueous solution, and may be used as any two or three or more mixed solvents.

The temperature of the aqueous solvent used for extraction is, for example, 0 ° C. or higher, and is not particularly limited as long as it does not affect the active ingredient. Although an aqueous solvent in a boiling state or a supercritical state can be used, it is preferable to use an aqueous solvent at 5 ° C to 600 ° C, and more preferable to use an aqueous solvent at 10 ° C to 200 ° C.
Therefore, the aqueous solvent for extraction includes an aqueous solvent in a boiling state or a supercritical state. The amount of the aqueous solvent used for the extraction is preferably an amount that can sufficiently dissolve the water-soluble active ingredient contained in Euglena.

The extraction method is also not particularly limited. For example, extraction can be performed by the following method, but the extraction method is not limited to this, and a normal extraction method can be freely selected and used. For example, a method of centrifuging or filtering Euglena alga body dry powder in an aqueous solvent for a predetermined time and then centrifuging or filtering; Euglena alga body dry powder added to an aqueous solvent and shaken to uniformly disperse, and then centrifuged or filtered. Method, etc.
It is also possible to heat the aqueous solvent after the addition of Euglena to facilitate extraction.

Euglena water extraction can be carried out by a conventional method as shown below, but is not limited thereto. For example, the Euglena tissue and water are placed in a container and allowed to stand for a predetermined time with appropriate stirring or shaking, and the resulting extract can be used as a water extract as it is. In addition, for example, a supernatant obtained by centrifuging such an extract can be used as a water extract. Further, such an extract or supernatant can be concentrated and dried to remove water, and this can be used as a water extract. Water extraction may be performed by adding a small amount of alcohol, for example, 10% by mass or less, preferably ethanol, to water in order to increase extraction efficiency and shorten extraction time.
The extraction time in the case of performing water extraction is not particularly limited as long as it is a time during which the active ingredient is extracted, and can be appropriately set in the range of several seconds to several tens of hours according to the extraction temperature.

Extraction with hot water can be performed by a commonly used method as shown below, but is not limited thereto. Euglena is extracted by introducing it with water into a commonly used extractor and then heating. When extracting using boiling water or water in a supercritical state, it is necessary to use an extractor that can withstand the vapor pressure of water. The pressure at the time of extraction can be set to 1 to 5000 atmospheres, and is preferably set to 60 to 400 atmospheres.
When performing extraction under high temperature and high pressure, if the extraction time is too long, the active component may be decomposed or a chemical reaction may occur. Therefore, when extraction is performed under high temperature and high pressure, the extraction time is preferably short, for example, within 3 minutes, more preferably within 1 minute, and particularly preferably within 30 seconds.

The extracted Euglena extract can be used as it is as an active ingredient of the progenitor osteoblast proliferation agent, bone differentiation promoting agent, bone strengthening agent, and anti-osteoporosis agent according to the present embodiment, but the extract is further suitable. It is also possible to fractionate and use a fraction with high activity by a simple separation means (for example, partition extraction, gel filtration, silica gel chromatography, reversed phase or normal phase high performance liquid chromatography, etc.).
Further, the Euglena extract or a fraction thereof can be concentrated and dried to remove the aqueous solvent, and this can be used as the aqueous solvent extract.

<Progenitor osteoblast proliferation agent>
The progenitor osteoblast proliferating agent according to the present embodiment is a progenitor osteoblast proliferating agent containing Euglena alga or Euglena aqueous solvent extract as an active ingredient.
The progenitor osteoblast proliferating agent according to the present embodiment promotes the proliferation of progenitor osteoblasts that differentiate into osteoblasts.

<Bone differentiation promoter>
The bone differentiation promoting agent according to the present embodiment is a bone differentiation promoting agent containing Euglena alga or Euglena aqueous solvent extract as an active ingredient.
The bone differentiation promoting agent according to the present embodiment promotes bone differentiation in the process of osteoblast differentiation.
Specifically, the bone differentiation promoting agent according to the present embodiment promotes differentiation from precursor osteoblasts to osteoblasts.

<Bone density improving agent, bone strengthening agent>
“Bone strengthening agent” refers to an agent that strengthens bone. Bone strengthening can be evaluated by indices such as bone strength (bone density and bone mass) and bone quality (bone quality), but is not limited to these indices.
“Bone density improving agent (bone density improving agent)” refers to an agent that improves bone density, specifically, increases bone density. For example, the bone density is generally evaluated based on the bone density of L1-L4 corresponding to the lumbar spine, but is not limited to this index.
The bone density improving agent and the bone strengthening agent according to the present embodiment increase bone density and strengthen bone by the progenitor osteoblast proliferating action and bone differentiation promoting action by Euglena alga or Euglena aqueous solvent extract. Is.

<Anti-osteoporosis agent>
“Anti-osteoporosis agent” refers to an agent for preventing, improving or treating osteoporosis.
The anti-osteoporosis agent according to the present embodiment can prevent osteoporosis by the effect of improving bone density, progenitor osteoblast proliferation, bone differentiation promoting action, and bone strengthening action by Euglena alga or Euglena aqueous solvent extract. Is to improve, treat.

<Locomotive syndrome preventive agent, locomotive syndrome treatment agent>
The agent for improving bone density and bone strengthening agent according to the present embodiment contains a Euglena alga or Euglena aqueous solvent extract as an active ingredient, and a locomotive syndrome preventive or locomotive syndrome therapeutic agent for preventing or treating locomotive syndrome Can also be used.
“Treatment of locomotive syndrome” means that the symptoms of locomotive syndrome are reduced compared to the state before ingesting the Euglena aqueous solvent extract, which is an active ingredient of the bone density improving agent and bone strengthening agent of the present embodiment. That means.

<Application>
The bone density improving agent, progenitor osteoblast proliferation agent, bone differentiation promoting agent, bone strengthening agent, and anti-osteoporosis agent according to the embodiment are administered to a patient who has received a definite diagnosis of bone density reduction and / or osteoporosis.
Further, the bone density improving agent, progenitor osteoblast proliferating agent, bone differentiation promoting agent, bone strengthening agent and anti-osteoporosis agent of the present embodiment are configured as a pharmaceutical composition, health food, etc., and have manifested subjective symptoms of osteoporosis. It is administered prophylactically to persons with osteoporosis reserve, such as those who are apt to decrease bone density, those who are likely to suffer from osteoporosis genetically or considering living conditions.

  Euglena alga or Euglena aqueous solvent extract can be ingested as a food and has no side effects, and therefore can be administered even before receiving a definite diagnosis of bone density reduction or osteoporosis. In addition, before receiving a definitive diagnosis, any time after receiving a definitive diagnosis, for example, bone strength (bone density and bone mass) is a predetermined standard (bone density is 70% of the average value of young adults (YAM)) Or -2.5SD or less), or when switching to another bone strengthening agent, etc.

In general, it is known that osteoporosis is caused by various factors such as diet, lack of exercise, aging, genetic factors and the like.
Therefore, the osteoporosis of the bone density improving agent, progenitor osteoblast proliferating agent, bone differentiation promoting agent, bone strengthening agent, and anti-osteoporosis agent of the present embodiment is affected by factors such as dietary habits, lack of exercise, aging, and genetic factors. Can be continuously administered for a long period of time to people in an environment where there is a high possibility of suffering from, for example, a person in a family with a disordered diet, a person suffering from osteoporosis in a family, and the like.

  Furthermore, a subject to which a bone density improving agent, a precursor osteoblast proliferating agent, a bone differentiation promoting agent, a bone strengthening agent, and an anti-osteoporosis agent containing the Euglena alga body or Euglena aqueous solvent extract according to this embodiment as an active ingredient is It is not limited to those with the above symptoms or conditions, or animals other than humans.

For example, for postmenopausal women who have been reported to have a high prevalence of osteoporosis, bone density improving agent, progenitor osteoblast proliferating agent, bone differentiation agent containing Euglena alga or Euglena aqueous solvent extract as an active ingredient Accelerators, bone strengtheners and anti-osteoporosis agents can be administered or ingested.
In postmenopausal women, the decrease in female hormones due to menopause promotes bone resorption and induces osteoporosis. Therefore, Euglena alga body or Euglena aqueous solvent extract has an effect of improving bone density, precursor osteoblast. Osteoporosis can be prevented by strengthening bone or increasing bone density by cell proliferation, bone differentiation promoting and bone strengthening.

In addition, among humans under 20 years old whose bone strength is not sufficient, the Euglena algae or Euglena aqueous solvent extract is used as an active ingredient for humans whose bone strength (bone density and bone mass) is below a predetermined standard. The bone density improving agent, progenitor osteoblast proliferating agent, bone differentiation promoting agent, bone strengthening agent and anti-osteoporosis agent can be administered or ingested.
Humans under the age of 20 years, for example, humans of infancy and puberty age are in the process of increasing bone density, but the bone density improving action, precursor osteoblast provided by the Euglena alga or Euglena aqueous solvent extract is provided. Bone can be strengthened or bone density can be increased by cell proliferation action, bone differentiation promoting action, and bone strengthening action.

Here, “infanthood” is a period of less than one year from the age of birth, and “infanthood” is a period of one to six years of age.
The term “puberty” refers to the period from when secondary sexual characteristics begin, to maturity, until height development finally stops. For men, it is generally between 12 and 17 years old, but for women, according to the definition of the Japan Obstetrics and Gynecology Association, “beginning with the development of sexual function (breast development, pubic growth, etc.) The period until the completion of the secondary sexual characteristics and the menstrual cycle are almost smooth. In the case of current Japanese, the average is between 8 and 9 years old and 17 and 18 years old. " According to WHO (World Health Organization), it is a concept having a range depending on gender and individual differences as defined as 10 to 19 years old, but it is a period of 8 to 19 years old.

In addition, for humans over 30 years old, a bone density improving agent, progenitor osteoblast proliferating agent, bone differentiation promoting agent, bone strengthening agent and anti-osteoporosis agent containing Euglena alga or Euglena aqueous solvent extract as an active ingredient Can be administered.
Humans 30 years or older can suppress the decrease in bone density by improving bone density, progenitor osteoblast proliferation, promoting bone differentiation, and strengthening bone.

  Further, the bone density improving agent, progenitor osteoblast proliferating agent, bone differentiation promoting agent, bone strengthening agent and anti-osteoporosis agent containing the Euglena alga body or Euglena aqueous solvent extract according to the present embodiment as an active ingredient are pharmacologically. Can be used as a composition such as a pharmaceutical composition, a cosmetic composition, and a food composition.

(Pharmaceutical composition)
The bone density improving agent, progenitor osteoblast proliferating agent, bone differentiation promoting agent, bone strengthening agent and anti-osteoporosis agent of the present embodiment can be used as a pharmaceutical composition.
In the pharmaceutical field, the bone density improving agent, progenitor osteoblast proliferating agent, bone differentiation promoting agent, bone strengthening agent and anti-osteoporosis agent of the present embodiment are used to improve bone density, progenitor osteoblast proliferating activity, bone A drug having such an effect by blending a pharmaceutically acceptable carrier or additive together with an Euglena alga or Euglena aqueous solvent extract in an amount capable of effectively exerting differentiation promoting action, bone strengthening action, and anti-osteoporosis action. A composition is provided. The pharmaceutical composition may be a pharmaceutical or a quasi drug.
The pharmaceutical composition may be applied internally or externally. Therefore, the pharmaceutical composition is used in a pharmaceutical form such as an internal preparation, intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection and / or intraperitoneal injection, transmucosal application agent, transdermal application agent, and the like. be able to.
The dosage form of the pharmaceutical composition can be appropriately set depending on the form of application, for example, solid preparations such as tablets, granules, capsules, powders, powders, liquid preparations such as liquids and suspensions, Semi-solid preparations such as ointments or gels are mentioned.

In the pharmaceutical composition according to this embodiment, one or more pharmaceutically acceptable additives can be freely selected and contained.
For example, when the pharmaceutical composition according to the present embodiment is applied to an oral preparation, for example, an excipient, a binder, a disintegrant, a surfactant, a preservative, a coloring agent, a corrigent, a fragrance, a stabilizer, an antiseptic, for example. All additives that can be usually used in the field of pharmaceutical preparations, such as agents and antioxidants, can be contained. In addition, a sustained-release preparation can be obtained using a drug delivery system (DDS).

  In addition to Euglena alga or Euglena aqueous solvent extract, the pharmaceutical composition according to this embodiment includes calcium, magnesium, vitamin D, vitamin K, soy isoflavone, β-crypt, which are known to have bone strengthening action. One or more substances such as xanthine and milk basic protein (MBP) can be added.

(Food composition)
Further, the Euglena alga or Euglena aqueous solvent extract of the present embodiment can be used for foods.
The food composition for improving bone density, the food composition for proliferating osteoblasts, the food composition for promoting bone differentiation, the food composition for strengthening bone, and the food composition for anti-osteoporosis according to the present embodiment, Euglena algae or Euglena aqueous solvent extract that can effectively exert bone density improving action, progenitor osteoblast proliferation action, bone differentiation promoting action, bone strengthening action, and anti-osteoporosis action as a food material for various foods By blending, a food composition having the action can be provided.
That is, this invention can provide the food composition of the food displayed as the bone strengthening etc. in the field of food. Examples of the food composition include foods for specified health use, functional nutritional foods, functional labeling foods, hospital patient foods, and supplements in addition to general foods. It can also be used as a food additive.
Examples of the food composition include seasonings, processed meat products, processed agricultural products, beverages (soft drinks, alcoholic beverages, carbonated beverages, milk beverages, fruit juice beverages, tea, coffee, nutritional drinks, etc.), powdered beverages (powder) Juice, powdered soup, etc.), concentrated beverages, confectionery (candy (throat cake), cookies, biscuits, gum, gummi, chocolate, etc.), bread, cereal and the like. Moreover, in the case of food for specified health use, nutritional functional food, functional indication food, etc., it may be in the form of capsule, troche, syrup, granule, powder or the like.

Here, food for specified health use is a food containing health functional ingredients that affect physiological functions, etc., and can be used to indicate that it is suitable for specific health use with the permission of the Commissioner of the Consumer Affairs Agency. It is. In the present invention, as a specific health use, “helps maintain bone health by helping bone metabolism”, “helps bone health by maintaining bone component”, “maintenance of bone component” It is a food that is sold and labeled as “helpful”, “support bone maintenance by helping bone metabolism work”, and so on.
The nutritional functional food is a food used for supplementing nutritional components (vitamins and minerals) and displays the function of the nutritional components. In order to sell as a functional nutritional food, the amount of nutrients contained in the daily intake standard amount must be within the specified upper and lower limits. You also need to.
The functional labeling food is a food that displays the functionality based on the scientific basis at the responsibility of the operator. Information on safety and functionality grounds was reported to the Commissioner of the Consumer Affairs Agency before sales.

  In addition to Euglena algae or Euglena aqueous solvent extract, the food composition according to the present embodiment may be freely selected from one or more ingredients that can be used in normal food compositions. Is possible. For example, all additives that can be usually used in the food field, such as various seasonings, preservatives, emulsifiers, stabilizers, fragrances, colorants, preservatives, and pH adjusters can be contained.

  In addition to Euglena alga or Euglena aqueous solvent extract, the food composition according to this embodiment includes calcium, magnesium, vitamin D, vitamin K, soy isoflavone, β-crypt, which are known to have bone strengthening action. One or more substances such as xanthine and milk basic protein (MBP) can be added.

<Dosage and administration>
Examples of usage of the bone density improving agent, progenitor osteoblast proliferating agent, bone differentiation promoting agent, bone strengthening agent, and anti-osteoporosis agent of the present embodiment include, for example, powders, capsules, tablets, granules, liquids, syrups, etc. Oral administration is good.
The dosage and administration form of the bone density improving agent, progenitor osteoblast proliferating agent, bone differentiation promoting agent, bone strengthening agent, and anti-osteoporosis agent of the present embodiment are appropriately selected depending on the subject, the disease state, its progress, and other conditions. do it. For example, as a Euglena aqueous solvent extract, when Euglena water extract is selected and administered orally for the purpose of obtaining bone strengthening action for humans (adults), or when Euglena alga is orally administered, In general, Euglena water extract obtained by using Euglena as a raw material at a dry weight of 1 to 5000 mg per day, preferably 100 to 3000 mg, more preferably about 500 to 2000 mg per day (about 1 to 2 times a day) And evening) and should be administered continuously at a rate of 5 or more per week.

<Manufacturing method of bone density improving agent, progenitor osteoblast proliferation agent, bone differentiation promoting agent, bone strengthening agent and anti-osteoporosis agent>
The bone density improving agent, progenitor osteoblast proliferation agent, bone differentiation promoting agent, bone strengthening agent and anti-osteoporosis agent of the present embodiment are produced by the following method.
First, a dispersion preparation step is performed in which an aqueous solvent is added to Euglena and stirred and / or shaken to obtain a dispersion. In this dispersion preparation step, Euglena powder (a dried product of Euglena alga) may be used as Euglena.
Subsequently, the dispersion liquid is centrifuged, and an extraction step is performed to obtain a supernatant as an aqueous solvent extract of Euglena.

In addition, after the dispersion preparation step and before the extraction step, the heating step may be performed by heating the dispersion using heating means such as a high-pressure steam sterilizer.
Specifically, first, a dispersion preparation step is performed in which an aqueous solvent is added to Euglena and stirred and / or shaken to obtain a dispersion.
Next, a heating step is performed in which the dispersion is heated using a heating means and is heated and extracted.
Furthermore, the dispersion liquid is centrifuged, and an extraction step is performed in which the supernatant is obtained as an aqueous solvent extract of Euglena.

  In the heating step, the set temperature of the heating means is room temperature or higher, for example, 20 ° C or higher and 50 ° C or lower, 50 ° C or higher and 80 ° C or lower, 80 ° C or higher and 100 ° C or lower, 100 ° C or higher and 120 ° C or lower, 120 ° C or higher and 150 ° C or lower, It can be set to 150 ° C. or higher and 200 ° C. or lower.

In the heating step, when the Euglena aqueous solvent dispersion is placed in a sealed container, the set temperature of the heating means matches the temperature of the Euglena aqueous solvent dispersion.
In the heating step, when the Euglena aqueous solvent dispersion is placed in an open container, the temperature of the Euglena aqueous solvent dispersion is determined according to the atmospheric pressure around the open container. It becomes about 100 ° C.

  In the heating step, when the Euglena aqueous solvent dispersion is placed in an open container, the heating step and the Euglena aqueous solvent extract concentration step are performed simultaneously.

In addition, instead of centrifuging the dispersion, an aqueous solvent extract of Euglena may be obtained by a general separation method. For example, an aqueous solvent extract of Euglena may be obtained from the filtrate obtained by filtering the dispersion.
Furthermore, a dispersion obtained by adding an aqueous solvent and shaking without performing centrifugation or filtration can be directly used as an Euglena aqueous solvent extract.

The obtained Euglena aqueous solvent extract is further fractionated to a highly active fraction by an appropriate separation means (for example, partition extraction, gel filtration, silica gel chromatography, reversed phase or normal phase high performance liquid chromatography, etc.). A fractionation step obtained in this manner may be performed.
Further, a concentration step of concentrating the obtained Euglena aqueous solvent extract or fraction and / or a drying step of evaporating and drying the aqueous solvent may be performed.

  The aqueous solvent extract of Euglena obtained as described above can be used as the bone density improving agent, precursor osteoblast proliferating agent, bone differentiation promoting agent, bone strengthening agent, and anti-osteoporosis agent of the present embodiment.

EXAMPLES Hereinafter, although this invention is demonstrated concretely based on a specific Example, this invention is not limited to these.
In the following examples, Euglena powder is used as a raw material, and extraction is performed using water, hot water, and ethanol as an aqueous solvent, and the influence of Euglena aqueous solvent extract on proliferation and bone differentiation of progenitor osteoblasts is examined. Went. We also examined the effects of oral intake of Euglena alga on human bones.

<Example 1>
Euglena water extract was prepared using Euglena gracilis powder (Euglena algae, Euglena Co., Ltd.) as a raw material and water as an aqueous solvent.
1 g of ultrapure water was added to 0.5 g of Euglena powder (Euglena Gracilis, Lot. EU-16189, manufactured by Euglena Co., Ltd.), shaken and dispersed uniformly, and then extracted at room temperature (25 ° C.) for 10 minutes. The supernatant obtained by centrifugation (3000 rpm, 3 minutes, 25 ° C.) was collected and filtered through a 0.45 μm sterilizing filter to prepare a Euglena water extract.

<Example 2>
Euglena hot water extract was prepared using hot water as an aqueous solvent using Euglena gracilis powder (Euglena algae, Euglena Co., Ltd.) as a raw material.
Euglena powder (Euglena Gracilis, Lot. EU-16189, manufactured by Euglena Co., Ltd.) 0.5 g was added with 1 ml of ultrapure water, shaken and dispersed uniformly, and then heated and extracted using a dry heat sterilizer (100 (C, 10 minutes). The supernatant obtained by centrifugation (3000 rpm, 3 minutes, 25 ° C.) was collected and filtered through a 0.45 μm sterilizing filter to prepare a Euglena hot water extract.

<Example 3>
Euglena ethanol extract was prepared using Euglena gracilis powder (Euglena alga, manufactured by Euglena Co., Ltd.) as a raw material and ethanol (EtOH) as an aqueous solvent.
1 ml of ethanol was added to 0.5 g of Euglena powder (Euglena Gracilis, Lot. EU-16189, manufactured by Euglena Co., Ltd.), shaken and dispersed uniformly, and then extracted at room temperature (25 ° C.) for 10 minutes. The supernatant obtained by centrifugation (3000 rpm, 3 minutes, 25 ° C.) was collected and filtered through a 0.45 μm sterilizing filter to prepare an Euglena ethanol extract.

<Test 1 Progenitor osteoblast proliferation test>
(Test method)
MC3T3-E1 cells (progenitor osteoblasts) were extracted with the extracts of Examples 1 to 3 in MEM-α (0.1% FBS, 1% Penicillin-Streptomycin Solution, no ascorbic acid) medium at a final concentration of 5 μg / mL. Alternatively, the cell proliferation rate was measured using Cell Counting Kit 24 hours later.
As a control, the cell proliferation rate was measured in the same procedure using MEM-α (0.1% FBS, 1% Penicillin-Streptomycin Solution, no ascorbic acid) without addition of an extract.

(Result of Test 1)
The results are shown in FIGS.
By adding the extracts of Examples 1 to 3, the cell growth rate was significantly higher than the control. That is, it was found that an aqueous solvent extract of Euglena exhibited cell proliferation activity against progenitor osteoblasts.
In any of the extracts, the cell growth rate was significantly improved as the extract concentration increased.

<Test 2 Bone differentiation (calcification) acceleration test>
(Test method)
After making MC3T3-E1 cells (progenitor osteoblasts) confluent, each extract of Examples 1 to 3 was added to a bone differentiation induction medium supplemented with 50 μg per 1 mL of the medium. After culturing for 21 days, calcium was stained using the Von Kossa method, and image analysis was performed using Image J.
As a control, analysis was performed in the same procedure using a bone differentiation induction medium to which no extract was added.

(Result of Test 2)
The results are shown in FIGS.
Addition of each extract of Examples 1 to 3 showed significantly higher calcium deposition than the control. That is, it was found that an aqueous solvent extract of Euglena exhibited an action of promoting differentiation (calcification) of progenitor osteoblasts.

<Summary of Tests 1 and 2>
From the results of Tests 1 and 2, it was revealed that the Euglena aqueous solvent extract has proliferative activity of progenitor osteoblasts and bone differentiation promoting action.
Therefore, it was found that the Euglena aqueous solvent extract can be used as a progenitor osteoblast proliferation agent, a bone differentiation promoter, a bone strengthening agent, and an anti-osteoporosis agent.

<Test 3 Examination of the effect of Euglena intake on human bones>
In Test 3, the effect of oral intake of Euglena gracilis powder (Euglena algae, Euglena Co., Ltd.) on human bone was examined.

(Test method)
We examined the effects of continuous intake of Euglena gracilis powder or placebo food on bone mineral density in postmenopausal women aged 50 or older and younger than 70 years who have no health problems. Specifically, 4 capsules (1 g) was taken 3 times a day for a total of 3 g of Euglena gracilis powder or placebo food.

  The test was carried out in a double-blind comparative test, and the bone density before intake and 24 weeks after intake was compared using the DEXA (Dual-Energy X-ray Absorptometry) method.

(Result of test 3)
As shown in FIG. 8, when the value of bone density of L1-L4 corresponding to the lumbar vertebrae was ingested Euglena gracilis powder, the amount of change after 24 weeks of ingestion (half year later) was +0.00603. When the placebo food was ingested, the change amount after 24 weeks (half year) was −0.00390, and the Euglena intake statistically significantly suppressed the decrease in bone density (t test, p <0.05).

  From the results of Test 3, it was shown that Euglena gracilis powder (Euglena alga) has an action of suppressing a decrease in bone density. From the results of Test 1 and Test 2, the effect of suppressing the decrease in bone density is presumed to be based on Euglena's progenitor osteoblast proliferation action and bone differentiation promoting action.

  Therefore, it was shown that Euglena alga or Euglena aqueous solvent extract can be used for uses such as bone strengthening, anti-osteoporosis, progenitor osteoblast proliferation, and bone differentiation promotion. At this time, Euglena algae or an Euglena aqueous solvent extract can be used to suppress a decrease in bone density, improve bone density, or increase (improve) bone density.

Claims (18)

  A food composition for improving bone density, comprising Euglena alga or Euglena aqueous solvent extract as an active ingredient.   The said Euglena aqueous solvent extract is at least 1 sort (s) selected from the group containing the Euglena water extract, the Euglena hot water extract, and the Euglena ethanol extract, The bone density improvement use of Claim 1 characterized by the above-mentioned. Food composition.   A bone density improving agent comprising Euglena alga or Euglena aqueous solvent extract as an active ingredient.   A food composition for progenitor osteoblast proliferation comprising Euglena alga or Euglena aqueous solvent extract as an active ingredient.   A food composition for promoting bone differentiation, comprising Euglena alga or Euglena aqueous solvent extract as an active ingredient.   A food composition for bone strengthening comprising Euglena alga or Euglena aqueous solvent extract as an active ingredient.   A food composition for anti-osteoporosis, comprising Euglena alga or Euglena aqueous solvent extract as an active ingredient.   A progenitor osteoblast proliferating agent comprising Euglena alga or Euglena aqueous solvent extract as an active ingredient.   A bone differentiation promoting agent comprising Euglena alga or Euglena aqueous solvent extract as an active ingredient.   A bone strengthening agent comprising Euglena alga or Euglena aqueous solvent extract as an active ingredient.   An anti-osteoporosis agent comprising Euglena alga or Euglena aqueous solvent extract as an active ingredient. A dispersion preparation step in which an aqueous solvent is added to Euglena and shaken to obtain a dispersion;
And a step of centrifuging the dispersion to obtain a supernatant as a Euglena aqueous solvent extract.   The method for producing a bone density improving agent according to claim 10, wherein a heating step of heating the dispersion is performed after the dispersion preparation step and before the extraction step.   The said aqueous solvent is at least 1 sort (s) selected from the group containing water, a hot water, and ethanol, The manufacturing method of the bone density improving agent of Claim 10 or 11 characterized by the above-mentioned. A dispersion preparation step in which an aqueous solvent is added to Euglena and shaken to obtain a dispersion;
An extraction step of centrifuging the dispersion to obtain a supernatant as an Euglena aqueous solvent extract. A dispersion preparation step in which an aqueous solvent is added to Euglena and shaken to obtain a dispersion;
And a step of centrifuging the dispersion to obtain a supernatant as an Euglena aqueous solvent extract. A dispersion preparation step in which an aqueous solvent is added to Euglena and shaken to obtain a dispersion;
And a step of centrifuging the dispersion to obtain a supernatant as an Euglena aqueous solvent extract. A dispersion preparation step in which an aqueous solvent is added to Euglena and shaken to obtain a dispersion;
And a step of centrifuging the dispersion to obtain a supernatant as an Euglena aqueous solvent extract. A method for producing an anti-osteoporosis agent, comprising: