JP5339664B2 - AMPK activator - Google Patents

Description

  The present invention relates to an AMPK (AMP-activated protein kinase) activator useful as a substitute for leptin and adiponectin.

  AMPK functions as a “metabolic sensor” that promotes metabolism and promotes ATP synthesis under the circumstances where intracellular ATP levels decrease such as exercise.

  AMPK is known to affect mitochondrial fatty acid oxidation through regulation of acetyl CoA carboxylase (ACC) activity. Carnitine palmitoyltransferase (CPT-1), which takes in long-chain fatty acids into mitochondria, is a rate-limiting enzyme for fatty acid oxidation in mitochondria and is strongly inhibited by malonyl-CoA, the product of ACC. Therefore, it is considered that AMPK phosphorylates ACC (Ser79) to suppress the activity of ACC and reduce the amount of malonyl CoA, thereby increasing the activity of CPT-1 and promoting fatty acid oxidation. As described above, AMPK is not only activated in the presence of intracellular energy, but is thought to have an important effect on the energy metabolism or nutrient metabolism of the living body.

Recent studies have reported that AMPK is also activated by adipocyte-derived hormones such as leptin (Non-patent Document 1) and adiponectin (Non-patent Document 2), and these proteins are administered medically. Therefore, treatment of various diseases has been attempted.
Nature, Vol.415, pp339-343, 2002 Nature, Vol.423, pp762-769, 2003

However, it is impossible for the general public to use leptin or adiponectin. In view of such a situation, if a compound having an AMPK activating action can be found, the action of leptin or adiponectin can be easily mimicked, and it is considered useful as a substitute for them.
Accordingly, an object of the present invention is to provide an AMPK activator that is excellent in safety and useful as a substitute for leptin and adiponectin.
Furthermore, activation of AMPK enhances energy production, so if the catechins of the present invention are ingested in situations that require energy, such as during exercise, or in daily life, the production of energy is promoted. Thus, it is possible to improve athletic ability and reduce fatigue.

Therefore, the present inventor has searched for an AMPK activator that is safe even when ingested for a long time and has few side effects, discovered a completely new function of AMPK activation action for catechins having a long dietary experience, and has found leptin and adiponectin. It was found to be useful as an alternative substance.
That is, this invention provides the AMPK activator which uses catechin as an active ingredient.

  The AMPK activator of the present invention is useful as a substitute for leptin and adiponectin because it is highly safe and can be taken for a long time.

  In the present invention, catechins is a general term for catechin, gallocatechin, catechin gallate, gallocatechin gallate, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, or a mixture thereof. Catechins and tea extracts rich in catechins are effective in the present invention.

  The catechins that are the components used in the present invention include tea leaves made from tea leaves obtained from the genus Camellia, for example, C. sinensis, C. assamica, and bamboo seeds, or hybrids thereof. Can be extracted with the addition of an extraction aid. The tea leaves produced include (1) green teas such as sencha, bancha, gyokuro, tencha, and kettle; (2) iron kannon, color species, golden katsura, wushuiwa tea, etc. (3) Fermented teas such as Darjeeling, Uba, and Keyman called black tea are included. About the method of extracting tea, it can carry out by conventional methods, such as stirring extraction. Moreover, you may add organic acids or organic acid salts, such as sodium ascorbate, to the water at the time of extraction beforehand. 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. Further, instead of extracting directly from tea leaves, a concentrate or purified product of tea extract may be blended.

  The concentrate of tea extract here is a concentrate obtained by concentrating an extract extracted from tea leaves with hot water or a water-soluble organic solvent. The purified tea extract is purified using a solvent or a column. Is. Examples thereof include those prepared by the methods exemplified in detail in JP-A-59-219384, JP-A-4-20589, JP-A-5-260907, JP-A-5-306279, and the like. Commercially available products include Tokyo Food Techno Co., Ltd. “Polyphenone”, ITO EN “Theafranc”, Taiyo Kagaku Co., Ltd. “Sunphenon”, Suntory Co., Ltd. “Sunwoolon”, and the like. In addition, catechins are also used in other raw materials such as grapes, processed products made from grapes such as wine and juice, cocoa beans, processed products made from these raw materials, and chemical synthetic products. it can. Examples of the form of the concentrate of tea extract and the purified product of tea extract include various forms such as solids, aqueous solutions, and slurries. Examples of the solution for dissolving and diluting the concentrate of tea extract or the purified product of tea extract include water, carbonated water, generally extracted tea extracts and the like.

  As catechins, a concentrate of tea extract or a purified product of tea extract is generally used, but it is particularly preferable to use a concentrate of green tea extract or a purified product of green tea extract.

  The AMPK activator of the present invention can be administered to humans and animals as a pharmaceutical. When used as a pharmaceutical, for example, oral solid preparations such as tablets and granules, and oral liquid preparations such as oral liquids and syrups can be used. In addition, the AMPK activator of the present invention can be blended in various pharmaceuticals, foods and drinks, pet foods and the like.

  In addition, when preparing an oral solid preparation, an excipient, if necessary, a binder, a disintegrant, a lubricant, a coloring agent, a corrigent, a flavoring agent and the like are added to catechins, followed by a conventional method. Thus, tablets, coated tablets, granules, powders, capsules and the like can be produced. Moreover, when preparing a liquid preparation for oral use, a liquid preparation, a syrup, an elixir, etc. can be manufactured by a conventional method by adding a corrigent, a buffer, a stabilizer, a corrigent and the like.

  The amount of catechins in each of the above preparations varies depending on the form of use, but in the case of pharmaceuticals such as oral solid preparations such as tablets, granules and capsules, oral liquid preparations such as oral liquids and syrups, etc. Is usually 0.01 to 95% by weight, more preferably 5 to 95% by weight, and particularly preferably 10 to 95% by weight. When blended in foods and drinks, pet foods, etc., 0.01 to 5% by weight, more preferably 0.05 to 5% by weight, and particularly preferably 0.1 to 1% by weight.

  The dose (effective intake) of the AMPK activator of the present invention is preferably 100 to 3000 mg / 60 kg body weight per day, particularly 250 to 2000 mg / 60 kg body weight, more preferably 250 to 1000 mg / 60 kg body weight. preferable.

Test Example 1 (AMPK activation effect by catechins)
Activation of AMPK was evaluated using a mouse liver cell line (Hepa1-6) and phosphorylation of AMPK and ACC (acetyl-CoA carboxylase) as an index.
A mouse liver cell line (Hepa1-6) is seeded in a 25 cm ² flask and cultured in DMEM (+ 10% FBS, + antibacterial agent) at 37 ° C. for 1-2 days. When subconfluent, the culture solution is removed, washed with PBS (−), replaced with DMEM (−FBS), and further cultured for 1 day. After removing the culture solution, DMEM (-FBS) containing a predetermined concentration of catechins or tea extract is added and cultured for 60 minutes. Thereafter, the culture solution was removed, washed with PBS (−), and then cell lysate (10 mM Tris (pH 7.4), 50 mM NaCl, 30 mM sodium pyrophosphate, 0.5% Triton X-100, protease inhibitor cocktail (SIGMA P2714) 200 μL of phosphatase inhibitor cocktail-1 (SIGMA P2850) and phosphatase inhibitor cocktail-2 (SIGMA P5726)) was added, and the cell lysate was recovered with a cell scraper. The collected cell lysate was well homogenized by passing a 23G syringe with a needle three times, and then left on ice for 30 minutes. After centrifugation at 15000 rpm for 15 minutes at 4 ° C., the supernatant protein was used in the following experiment.

  After measuring the concentration of the supernatant protein, the protein concentration between the samples was adjusted to be constant. Add one-fourth volume of SDS buffer (250 mM Tris, 12.5% SDS, 20% glycerol), then add 2-mercaptoethanol and bromophenol blue, heat denaturation at 95 ° C, and rapid cooling at 4 ° C. Samples for electrophoresis were prepared.

A certain amount (about 20-40 μg) of the sample for electrophoresis is subjected to SDS-PAGE (4 or 12% gel), transferred to a membrane, anti-phospho-AMPKα (Thr72) antibody (manufactured by Cell Signaling), anti -phospho-AMPKβ (Ser108) antibody (Cell Signaling) or anti-phospho-ACC (Ser79) antibody (Cell Signaling) primary antibody, anti-rabbit-HRP antibody (Amersham) secondary antibody, phospho-AMPKα, phospho-AMPKβ and phospho-ACC were detected using phototope-HRP Western Detection System (manufactured by Cell Signaling) as a detection reagent. The degree of activation of AMPK is expressed as a relative value relative to the intensity of the detected band (pixels) by image analysis (EDAS 290 image analysis system: KODAK), with the control (catechin-free group) being 100. It was. In addition, the catechin preparation used the thing by SIGMA company or Wako Pure Chemical Industries Ltd. Tea extract 90% manufactured by ITO EN Co., Ltd. was used as the tea extract (tea catechin).

  From the results of Tables 1 to 3, it is clear that catechins have an excellent AMPK activating effect, in particular, gallocatechin gallate, epigallocatechin gallate, gallocatechin or epigallocatechin has an excellent AMPK activating effect. It was. Moreover, it became clear that the tea extract containing catechins also exhibits an excellent AMPK activation effect.

Test Example 2 (Catechins' ability to improve exercise ability and fatigue relief):
Mice (BALB / c strain, male, 6 weeks old) were preliminarily raised for 1 week, and then the limit swimming time was measured twice. The mouse was allowed to swim at a flow rate of 7 L / min in the simple pool, and the time when it became impossible to swim against the flow was defined as the limit swimming time, and that time was recorded. There were 10 animals per group so that there was no difference between the groups in the limit swimming time. They were raised using food prepared with the formulation shown in Table 4. While rearing for 10 weeks, the limit swimming time of each group was measured once a week. Table 5 shows the limit swimming time of mice at 10 weeks after the start of the test.

  From the results in Table 5, it can be seen that by taking epigallocatechin gallate (EGCG), exercise ability is improved and fatigue is less likely.

Claims (2)

An AMPK activator comprising as an active ingredient a catechin selected from epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate and epigallocatechin gallate .   The AMPK activator according to claim 1, wherein the catechin is a tea extract.