JP2009107995A - Differentiation inhibitor of osteoclast - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a differentiation inhibitor of osteoclast, having excellent activity for inhibiting differentiation of the osteoclast. <P>SOLUTION: The bone tissue is kept by a dynamic cycle of breaking (bone resorption) and formation (osteogenesis), and the bone resorption and the osteogenesis are carried out by the osteoclast and the osteoblast, respectively. When the activity balance of the osteoclast and the osteoblast is destroyed, the risk of osteoporosis increases. Epigallocatechin gallate of one kind of bone catechin has been found to have effects for ameliorating bone metabolism and to be an effective differentiation inhibitor of the osteoclast. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an osteoclast differentiation inhibitor.

Bone tissue is maintained by a dynamic cycle of destruction (bone resorption) and formation (bone formation), with osteoclasts and osteoblasts responsible for bone resorption and bone formation, respectively.
Osteoclasts are differentiated from mononuclear osteoclast precursor cells stimulated by RANKL (receptor activator of NF-κB ligand; RANKL) and M-CSF (macrophage colony stimulating factor) generated by osteoblasts, Then it is known to mature.

Under normal conditions, bone tissue maintains its shape and amount by maintaining a balance of activity between osteoclasts and osteoblasts.
If the above balance is lost due to the bone resorption rate becoming higher than the bone formation rate, many small holes occur in the bone, the bone density is lowered, the bone strength is weakened, and the risk of fragile fracture increases. Such a condition is osteoporosis. Osteoporosis is a symptom commonly seen in postmenopausal women and older men, and significantly reduces the quality of life (QOL) of the affected individuals, and is therefore a major problem for the modern aging society. It has become.

  As a method for preventing and treating osteoporosis, (1) bone formation promotion by ingestion of calcium and calcium absorption promoter, and (2) bone resorption suppression by ingestion of bone resorption inhibitor are known.

As calcium absorption promoters, various compounds such as twintose (Patent Document 1), fructooligosaccharide (Patent Document 2), casein phosphopeptide (Patent Document 3), calcium citrate malate (Patent Document 4) and the like are already known. ing. However, there is a problem that the promotion of calcium absorption by such foods is insufficient to prevent rapid bone loss accompanying menopause. Therefore, it is desirable to actively suppress bone resorption in order to prevent rapid bone loss due to menopause.
On the other hand, bisphosphonate preparations, selective estrogen receptor modulators (SERM), calcitonin preparations, and the like are already known as drugs that suppress bone resorption (Non-Patent Documents 1 to 3). Moreover, as a food ingredient having a bone resorption suppressing action, β-cryptoxanthin contained in curcumin contained in quercetin and turmeric contained in onions and the like, and Satsuma mandarin etc. are already known (Non-Patent Documents 4 to 6). .

  Catechins contained in green tea, black tea, cacao beans and the like are widely known to have a cholesterol elevation inhibitory action (Patent Document 5) and a blood sugar elevation inhibitory action (Patent Document 6). A polyphenol-rich extract selected from plants belonging to the family Rosaceae, Camelliaaceae or Mulberryaceae suppresses bone resorption (Patent Document 7), and epigallocatechin gallate which is a kind of catechins is immature osteoblasts It has already been known that a biphasic protective effect is exerted against cell death in the above to promote bone formation (Patent Document 8).

However, it has not been known so far that epigallocatechin gallate has a good osteoclast differentiation inhibitory action.
Japanese Patent Laid-Open No. 11-43438 JP 7-252156 A JP 7-308172 A JP-A-1-156985 JP-A-60-156614 JP-A-4-253918 JP-A-6-183985 JP 2004-161669 A Kushida K. Et al. Bone Miner. Metab. (2004) 22,462-468 Ohta H.:Clin.Calcium (2004) 14,73-80 Hosoi T.:CLINICAL CALCIUM (2004) 14 (11), 83-86 Wattel A. et al .: J. Cell. Biochem. (2004) 92,285-295 Bharti AC. Et al .: J. Immunol. (2004) 172,5940-5947 Uchiyama S. and Yamaguchi M .: Biochem. Pharmacol. (2004) 67,1297-1305

  The present invention relates to providing an osteoclast differentiation inhibitor having an excellent osteoclast differentiation inhibitory effect.

  The present inventor has examined compounds that improve bone metabolism. Surprisingly, epigallocatechin gallate has an excellent osteoclast differentiation inhibitory action and is useful as a pharmaceutical or food for osteoclast differentiation inhibition. I found out.

  That is, the present invention relates to an osteoclast differentiation inhibitor containing epigallocatechin gallate as an active ingredient.

  ADVANTAGE OF THE INVENTION According to this invention, foodstuffs, a pharmaceutical, etc. which have the outstanding osteoclast differentiation inhibitory effect can be provided, without increasing the compounding quantity as the whole catechin.

  In the present invention, “suppression of osteoclast differentiation” refers to an action of suppressing or inhibiting excessive differentiation of osteoclast precursor cells into osteoclasts. Since tartrate-resistant acid phosphatase (TRAP) is strongly expressed in osteoclasts and TRAP activity represents osteoclast activity, osteoclast differentiation inhibition can be grasped as inhibition of TRAP activity.

  Epigallocatechin gallate used in the present invention can be produced by extraction, separation, purification or chemical synthesis from, for example, green tea or other tea leaves or other plants containing epigallocatechin gallate. Commercially available products can also be used. Examples of such commercially available products include Teabigo (DSM Nutritional Product Co., Ltd.), “PF-EGCg90” (manufactured by Pharma Foods Laboratories, Inc.), Sanphenon EGCg (Taiyo Kagaku), Polyphenon. E (Mitsui Agriculture and Forestry).

Extraction from the above green tea or other tea leaves is carried out by adding water or hot water or an extraction aid to tea leaves made from tea leaves obtained from Camellia genus such as C. sinensis , C. assamica , or hybrids thereof. It can be carried out by stirring extraction and the like. Moreover, you may use together the method of extracting in so-called non-oxidative atmosphere, ventilating inert gas, such as boiling deaeration and nitrogen gas, and removing dissolved oxygen. Examples of the extraction aid include organic acids such as sodium ascorbate or organic acid salts thereof.
The tea leaves produced above are: (1) Green teas such as Sencha, Bancha, Gyokuro, Tencha, Kamasencha; (2) Iron kannon, color types, golden katsura, wushuiwa tea, etc. (3) Fermented teas such as Darjeeling, Uba, and Keyman called black tea are included.

  Concentration of the green tea extract or other tea leaf extract can be performed by concentrating the extract, and purification of epigallocatechin gallate from the tea extract is performed using a solvent or a column. Can be done.

In addition, extracts such as green tea contain catechins such as gallocatechin, epigallocatechin, catechin, epicatechin, gallocatechin gallate, epicatechin gallate and catechin gallate in addition to epigallocatechin gallate.
As shown in a comparative example described later, purified epigallocatechin gallate has an osteoclast differentiation inhibitory action superior to such an extract. Therefore, when used as an osteoclast differentiation inhibitor, the proportion of epigallocatechin gallate in the catechins is preferably 50 mass% to 99.99 mass%, more preferably 70 mass% to 99.99 mass%, 80 mass% to 99.99 mass% is still more preferable. That is, it is preferable to use isolated epigallocatechin gallate substantially free of gallocatechin, epigallocatechin, catechin, epicatechin, gallocatechin gallate, epicatechin gallate and catechin gallate.

  As shown in Examples described later, epigallocatechin gallate can be an osteoclast differentiation inhibitor having an action of suppressing osteoclast differentiation induced by RANKL and M-CSF. Therefore, the osteoclast differentiation inhibitor of the present invention is a study for preventing or improving various disorders such as osteoporosis, fracture associated with osteoporosis, pain, spinal deformity, etc. It can be used as a reagent or as a food or pharmaceutical or quasi drug for humans or animals, or it can be used to produce them.

  When the osteoclast differentiation inhibitor of the present invention is used as a pharmaceutical, it can be administered in any dosage form. Administration forms include oral and parenteral administration. Examples of the dosage form of the preparation for oral administration include tablets, pills, capsules, powders, granules, syrups, elixirs, liquids and the like. Examples of parenteral administration include injection, infusion, transdermal, transmucosal, nasal, inhalation, suppository, and bolus.

  In such a preparation, the epigallocatechin gallate of the present invention may be used in combination with a pharmaceutically acceptable carrier. Such carriers include, for example, excipients, binders, disintegrants, lubricants, diluents, osmotic pressure adjusters, pH adjusters, emulsifiers, preservatives, stabilizers, antioxidants, colorants, ultraviolet light Examples include absorbents, humectants, thickeners, brighteners, activity enhancers, anti-inflammatory agents, bactericides, flavoring agents, and flavoring agents.

  The content of epigallocatechin gallate in the preparation is usually 0.01% to 95% by weight of the total weight of the preparation, preferably 5% to 95% by weight, and preferably 10% to 95% by weight. Is more preferable.

  The dosage or intake of the formulation may vary according to the patient's condition, weight, sex, age, or other factors. The daily dose per adult is usually 20 to 1200 mg / body weight 60 kg / day, preferably 50 to 800 mg / body weight 60 kg / day, and more preferably 50 to 400 mg / body weight 60 kg / day. The formulation can be administered according to any dosage regimen.

  When the osteoclast differentiation inhibitor of the present invention is used as a food, in addition to general foods, it is intended for the prevention or improvement of bone resorbable diseases, beauty foods, foods for the sick, functional nutrition foods, or specified health use It can be set as functional foods, such as food. The food can be solid, semi-solid or liquid. Examples of foods include breads, noodles, confectionery, jelly, dairy products, frozen foods, frozen foods, other processed foods, beverages, soups, seasonings, nutritional supplements, and the like, and their raw materials. . The food may be in tablet form, pill form, capsule form, liquid form, syrup form, powder form, granule form and the like.

  When the osteoclast differentiation inhibitor of the present invention is used as a food, epigallocatechin gallate may be used in combination with other food materials, and if necessary, it contains food additives. Also good. Examples of food additives include solvents, oils, softeners, emulsifiers, preservatives, stabilizers, antioxidants, colorants, UV absorbers, humectants, thickeners, brighteners, sweeteners, flavorings, etc. Can be mentioned.

  In addition, the content of epigallocatechin gallate in the food is usually 0.0025% by mass to 5% by mass of the total mass of the food, preferably 0.0125% by mass to 5% by mass, and 0.025% by mass. -1 mass% is more preferable.

  The pharmaceutical or food product may be prepared in combination with at least one calcium intake or absorption enhancer or bone metabolism improving agent. Examples of calcium intake or absorption promoters include calcium salts such as calcium carbonate, twinose, fructooligosaccharides, casein phosphopeptides, calcium citrate malate, vitamin D, glucose-1-phosphate, and the like. Examples of the bone metabolism improving agent include vitamin D, vitamin K, soybean isoflavone, collagen, MBP (milk basic protein) and the like.

  EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.

  Epigallocatechin gallate (Wako Pure Chemical) and green tea extract Theafuran 90S (Itoen) were used as test substances, respectively, and osteoclast differentiation inhibitory activity was measured.

Example 1 Measurement of osteoclast differentiation inhibitory activity

RAW264.7 cells were seeded on a 48-well plate at a concentration of 3000 cells / well and cultured in MEM medium (Dainippon Sumitomo Pharma) containing 10% FCS. On the next day, the medium was replaced with a medium containing a test substance and cultured for 1 day. Then, osteoclast differentiation was induced with 50 ng / ml sRANKL (PEPROTECH EC) and 50 ng / ml M-CSF (R & D SYSTEMS) in the presence of the test substance. . The test substance differentiation-inhibiting activity was determined by the positive control (100% differentiation) of the TRAP (tartrate-resistant acid phosphatase) activity of the well to which only the solvent of the test substance was added, and the negative control (0%) of the well that was not induced to differentiate. It was expressed as a percentage as differentiation).
The TRAP activity was measured by the following method. The culture solution was removed from the plate with an aspirator, and 0.4 ml of 3.7% formalin solution was added and left at room temperature for 10 minutes or more to fix the cells. Thereafter, the formalin solution was removed with an aspirator, 0.4 ml of ethanol: acetone = 1: 1 was added, and the mixture was allowed to stand for about 30 seconds. This was removed with an aspirator and allowed to air dry. To an air-dried plate, 0.4 ml of a TRAP activity measurement solution (5.5 mM p-Nitrophenylphosphate, 50 mM sodium tartrate, 50 mM citrate buffer (pH 4.5)) was added and shaken at 37 ° C. for 1 to 2 hours. . 100 μl of the reaction solution in each well was placed in a 96-well plate to which 20 μl of 0.1N NaOH had been added in advance, and the absorbance (405 nm) was measured. p-Nitrophenylphosphate, sodium tartrate, citric acid monohydrate, and sodium hydroxide were all manufactured by Wako Pure Chemical. The results are shown in FIG.

  FIG. 1 shows that epigallocatechin gallate has an excellent TRAP activity inhibitory action and has an osteoclast differentiation inhibitory action. Further, at any final concentration, TRAP activity when epigallocatechin gallate was used was theafuran 90S (epigallocatechin gallate: 34.2% (Ikeda I et al. J Nutr. 2005 Feb; 135 (2) : 155-9)) is lower than TRAP activity, and epigallocatechin gallate has a higher inhibitory effect on osteoclast differentiation than theafuran 90S.

  From the results shown in FIG. 1, it can be seen that epigallocatechin gallate has a TRAP activity inhibitory action superior to that of theafuran which is a mixture, and is useful as an osteoclast differentiation inhibitor.

It is a figure which shows the osteoclast differentiation inhibitory activity of an epigallocatechin gallate.

Claims (1)

  An osteoclast differentiation inhibitor containing epigallocatechin gallate as an active ingredient.

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