JP2013129627A - Simple preparation method of phlorotannins and use of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of easily preparing phlorotannins in which various functions have been found to phlorotannins, if there is a simple preparing method, while that use advances further is expected, if the health of a diabetic patient or a person who has the risk can be recovered by an agent of a natural product origin of experienced food, those are very preferable.SOLUTION: The preparation method of phlorotannins includes: a process in which 2-10 pts.wt. of an ethanol-containing solvent is used for one pt.wt. of a dried product of brown algae raw material to be extracted, and an extract including phlorotannins is obtained; and a process in which 1.6-2.9 pts.wt. (desirably, 1.8-2.7 pts.wt., and further desirably, 2.0-2.5 pts.wt.) of activated carbon is added to 100 pts.wt. of the dried product of the brown algae raw material, and impurities are removed by adsorption. Moreover, a treatment medicinal of diabetes includes phlorotannins, fucoxanthin, magdisalicylate, and fucoidan.

Description

  The present invention relates to a simple method for preparing fluorotannins contained in brown algae polyphenol, and a composition containing the obtained fluorotannins. The compositions of the present invention are useful for the treatment of diabetes.

  Known fluorofluorotannins contained in brown algae polyphenols include phloroglucinol, equol, florofcofurecole A, diacor, 8,8'-bayecol, and the like. These have a structure in which phloroglucinol is polymerized. The functions of fluorotannins include suppression of oxidative damage (Non-patent Documents 1 and 2), inhibition of AGEs (Advanced Glycation End-products) formation (Non-patent Document 3), inhibition of acetylcholinesterase (Non-patent Document 4) ), Aldose reductase inhibitory activity (Non-patent Document 5) is known.

  On the other hand, it is known that the carotenoid fraction derived from brown algae can inhibit the complex formation of retinol-binding protein and transthyretin, and is useful for treatment of insulin-independent diabetes (patent document) 1). Further, a fraction having a molecular weight of 250 to 500 containing magnesium derived from brown algae is known to selectively inhibit COX-2 and to be useful as an immunostimulator or anti-inflammatory agent (Patent Document 2). ).

  On the other hand, diabetes is defined as a hyperglycemic state due to insufficient action of insulin. Diabetes is increasing at an extremely high rate regardless of whether it is a developed country or a developing country, and it is estimated that the number of diabetes patients in Japan is 8.9 million. Most of these diabetics are classified as type 2 diabetes, which is caused by insulin resistance and / or insulin secretion failure. Complications to be noted in diabetes are typical complications, and typical examples include increased risk of nephropathy and retinopathy due to AGEs, and neuropathy. Diabetes has few subjective symptoms in the early stages. However, it can progress slowly but reliably and cause serious complications. It is said that improving habits and keeping blood hemoglobin A1C (HbA1c) levels below 6.5 is important to avoid complications.

JP2007-314451 JP2008-120738

Hwang et al., Int. J. Cancer, 119, 2742-2749, 2006. Zhang et al., Eur. J. Pharm., 591, 114-123, 2008. Okada et al., J. Nat. Prod., 67, 103-105, 2004) Yoon Na Y. et al., Fisheries Sci., 74, 200-207, 2008. H. Nakamura et al., Natural Medicines, 51 (2), 162-169, 1997.

Fluorotannins have been found to have various functions, and if there is a simple preparation method, it is expected that the use will further advance.
On the other hand, it goes without saying that it would be very desirable to restore the health of diabetic patients or those who are at risk by using an agent containing an active ingredient derived from a natural product with abundant dietary experience.

  The inventors have been engaged in research on brown algae for many years, and paid attention to various functions of fluorotannins, which are polyphenols of brown algae. Most conventional drugs that promote insulin secretion target the β cells of the islets, but the α cells of the islets secrete acetylcholine as a substance that primes the functions of the β cells. Fluorotannins have acetylcholinesterase and aldose reductase inhibitory activity, as well as antioxidant activity and AGEs formation inhibitory activity, etc., and if used in the treatment of diabetes, they can exert an effect by an unprecedented mechanism There is expected.

The present invention provides the following.
[1] A step of extracting 2 to 10 parts by weight of an ethanol-containing solvent with respect to 1 part by weight of a dried brown algae raw material to obtain an extract containing a fluorotannin
To the extract, 1.6 to 2.9 parts by weight (preferably 1.8 to 2.7 parts by weight, more preferably 2.0 to 2.5 parts by weight) of activated carbon is added to the equivalent of 100 parts by weight of the dried brown algae material to adsorb impurities. Removing;
A method for producing fluorotannins, comprising:
[2] The fluorotannins contain at least one substance selected from the group consisting of phloroglucinol, equol, florofcofurecole A, diacor, and 8,8′-bayecol, [1] The manufacturing method as described in.
[3] A therapeutic agent for diabetes comprising phlorotannins, fucoxanthin, magdisalicylate and fucoidan obtained by the production method of [1] or [2].
[4] The agent according to [3] for inoculating phlorotannins at 0.1 to 500 mg / day, preferably 50 to 100 mg / day.

  In the present invention, fluorotannins include at least one selected from the group consisting of the following substances, unless otherwise specified.

  The term “brown algae” as used herein refers to algae classified as Phaeophyceae unless otherwise specified. Brown algae includes the order of Chordariales, Laminariales, Dictyosiphonales, Dictyotales, Fucales, Ralfsiales, Scytosiphonales and Midi Includes algae belonging to Ectocarpales, Sphacelariales, Desmarestiales, Cutleriales, Sporochnales, Syringodermatales, and also from the genus Cleidae, Davilia, Mozuku, Algae belonging to the genus, Lessonia, Kajime, Macrocystis, Hibamata, or Ascophilum are included. Scientific name: Undaria pinnatifida is a seaweed brown algae of the order of the brown algae, specifically, wakame (Undaria pinnatifida) in the Chigaiso family, Durvillea antarctica (Durvillea antarctica), Mozuku ( Cladoshiphon caredoniae), Matsuma spp. (Nemacystis decipiens), Lessonia sp. Lessonia nigrescens, Kasime sp. Ecklonia cava, Macrocystis sp. evanescens), Fucus vesiculosus, and Ascophyllum genus Ascophyllum nodosum. Especially as a raw material of the fluoro tannins used for this invention, a wakame can be used suitably.

  As the raw material brown algae, the collected ones themselves may be used, but it is preferable to dry them before use in the extraction step. This is because the solid content per weight is higher in the dried product than in the raw material, and as a result, the extraction efficiency is increased.

  The method for preparing fluorotannins of the present invention is a step of extracting 2 to 10 parts by weight of an ethanol-containing solvent with respect to 1 part by weight of the dried brown algae raw material to obtain an extract containing fluorotannins; To the extract, 1.6 to 2.9 parts by weight (preferably 1.8 to 2.7 parts by weight, more preferably 2.0 to 2.5 parts by weight) of activated carbon is added to the equivalent of 100 parts by weight of the dried brown algae material to adsorb impurities. Removing. Conventionally, fluorotannins were prepared by extracting a necessary fraction from an alga body with an ethanol solution, and then using an organic solvent such as hexane, dichloromethane, ethyl acetate, and butanol. The use of such organic solvents may not be appropriate in producing oral compositions. According to the method of the present invention, fluorotannins can be more easily prepared using only a solvent acceptable as a medicine or food.

  In the present invention, “corresponding to a dried product (1 part by weight)” refers to the basis of the weight of the dried product, unless otherwise specified. For example, even when the raw brown algae contains a large amount of moisture as it is collected from nature, the standard is the weight of the dried product (sometimes referred to as “solid amount”). In the present invention, the ratio of components and the like is based on weight unless otherwise specified.

  In the present invention, the term “ethanol-containing solvent” refers to a solvent comprising 80% or more of ethanol and water, unless otherwise specified. Fluorotannins dissolve in 80% or more of ethanol, methyl alcohol, and ethyl acetate, but do not dissolve in water.

In the present invention, the extraction step using an ethanol-containing solvent of 2 to 10 parts by weight, preferably 3 to 8 parts by weight, more preferably 4 to 6 parts by weight, with respect to 1 part by weight of the dried brown algae raw material. Is implemented.

  In the production method of the present invention, the extract containing fluorotannins obtained by extraction using an ethanol-containing solvent is subjected to an activated carbon treatment step. The activated carbon that can be used in the production method of the present invention is not limited to raw materials, production methods, and shapes (for example, may be in the form of fibers, honeycombs, columns, crushed shapes, granules, and powders). Activated carbon used in the field of production or food production can be applied.

  In the production method of the present invention, the activated carbon comes into contact with the extract, but the amount of necessary activated carbon can be determined relative to 100 parts by weight of the dried material of the brown algae raw material. Usually, the amount of activated carbon is 1.6 to 2.9 parts by weight, preferably 1.8 to 2.7 parts by weight, and more preferably 2.0 to 2.5 parts by weight with respect to 100 parts by weight of the dried alga raw material.

In the present invention, pigments and other contaminants can be efficiently removed from the ethanol-containing solvent extract of brown algae by using a specific amount of activated carbon.
The present invention provides an agent (composition) containing phlorotannins. Fluorotannins inhibit oxidative damage (Non-Patent Documents 1 and 2), AGEs (Advanced Glycation End-products) formation inhibition (Non-Patent Document 3), acetylcholinesterase inhibition (Non-Patent Document 4) ), Aldose reductase inhibitory activity (Non-Patent Document 5) is known, and is therefore effective in treating diabetes and reducing the risk of developing diabetic complications.

The agent of the present invention contains fucotandan, fucoxanthin or analogs thereof, and magdisalicylate in addition to fluorotannins as active ingredients.
When it is referred to as fucoidan in the present invention, a sulfated polysaccharide derived from brown algae composed of fucose, xylose, galactose, uronic acid, etc. (some raw material brown algae may contain mannose) unless otherwise specified Say. A substance having only fucose as a constituent sugar is referred to as “fucoidan”. A substance containing a sugar other than fucose is sometimes referred to as a “fucoidan-like polysaccharide”. The fucoidan herein includes both. As the raw material for fucoidan, natural or farmed Mozuku, especially Cladosiphon okamuranus (Cladoshiphon okamuranus Tokida), Cladoshiphon novae -caledoniae Kylin), or Cladoshiphon caledoniae Kylin it can be suitably used. It is preferable to use natural Cladoshiphon caledoniae Kylin from Tonga from the viewpoint of containing abundant minerals and not detecting harmful substances of heavy metals.

  In the present invention, fucoxanthin or an analog thereof is a fucoxanthin represented by the following formula (molecular weight: 658.88) unless otherwise specified.

Or the various stereoisomers (cis-trans isomer, optical isomer) are mentioned (patent document 1). Fucoxanthin is (3 S , 5 R , 6 S , 3 ' S , 5' R , 6 ' R ) -3'-acetoxy-5,6-epoxy-3,5'-dihydroxy-6', 7 ' -didehydro-5,6,7,8,5 ', 6'-hexahydro-β, β-caroten-8-one (IUPAC), or (3 S , 3' S , 5 R , 5 ' R , 6 S , 6 ' R ) -3'-(acetyloxy) -6 ', 7'-didehydro-5,6-epoxy-5,5', 6,6 ', 7,8-hexahydro-3,5'-dihydroxy- It may be expressed as 8-oxo-β, β-carotene (CAS). In the present invention, fucoxanthin or its analogs may be described by taking fucoxanthin as an example, but the description also applies to fucoxanthin analogs unless otherwise specified. In the present invention, the term “maggysalicylate” refers to a cyclooxygenase-2 (COX-2) inhibitor having a molecular weight of 250 to 500 and containing magnesium, derived from seaweeds belonging to brown algae, unless otherwise specified (Patent Documents above) 2). More specifically, magzisalicylate is contained in an extract of Davilia seaweed and selectively inhibits COX-2.

The agent of the present invention is effective in treating diabetes and reducing the risk of developing diabetic complications.
Whether or not the agent of the present invention is effective for the intended purpose can be confirmed by measuring blood glucose (GLU) and hemoglobin A1C (HbA1c) levels in the administered subject. The methods for measuring these values are well known to those skilled in the art.

  According to studies by the present inventors, the agent of the present invention has an action of lowering blood glucose level. Therefore, in addition to diabetes, the agent of the present invention can be used for the treatment of diseases or illnesses characterized by high blood glucose (GLU) and improved by lowering blood glucose level. The agent of the present invention can also improve blood hemoglobin A1C (HbA1c) levels. Therefore, in addition to diabetes, the agent of the present invention can be used for the treatment of diseases or illnesses characterized by high HbA1c level and improved by lowering HbA1c level.

  The agent of the present invention is surprisingly effective for diabetic patients whose hemoglobin A1c and fasting blood glucose level hardly improve even with various drugs. When the fasting blood glucose level is high, the effect of insulin does not appear, and as a result, the high value of hemoglobin A1c continues. It has been found that if this condition persists, the risk of developing complications (renal disorder, retinopathy and neuropathy) increases. Renal disorder, retinopathy and neuropathy are also said to be the three major complications of diabetes. In addition to the treatment / prevention of diabetes, the agent of the present invention can reduce the risk of developing three major complications of diabetes or prevent the three major complications. An agent for reducing the risk of developing complications of diabetes is provided for the first time by the present application.

  Fucoidan, fucoxanthin, and magdisalicylate, all contained in the agent of the present invention, are components mainly involved in improving blood glucose levels in the treatment of diabetes. In addition, phlorotannins have an AGEs formation inhibitory action and an acetylcholinesterase inhibitory action (Non-patent Documents 3 and 4). However, the present invention is the first attempt on the prevention of diabetic complications by components having an inhibitory effect on AGEs formation and the improvement of blood glucose level by β-cell activation via β-cell priming by islet α-cells by inhibiting acetylcholinesterase. Will. In addition, the present invention provides for the first time a therapeutic agent for diabetes including a reduction in the risk of developing three major complications by a combination of fucoidan, fucoxanthin, magdisalicylate, and fluorotannins.

  As used herein, “treatment” for a disease or condition, except for special cases, treats, prevents, delays or stops progression, or maintains a good condition for the disease or condition. The treatment includes coping treatment that suppresses symptoms and fundamental treatment.

  The acute toxicity of phlorotannins is not observed in a single dose acute toxicity study at 2,000 mg / kg body weight. Moreover, the fluorotannins of the present invention were stable when heated at 100 ° C. for 15 minutes, and were stable in the range of pH 3.0 to 8.5.

  The phlorotannins dose can be, for example, preferably from 0.1 to 500 mg, more preferably from 100 to 200 mg, still more preferably from 30 to 150 mg per adult day. Smaller amounts may be effective for prevention and maintenance purposes. The daily dose may be administered in a single dose, or may be divided into multiple doses (eg, twice or three times a day).

  The component ratio of the agent of the present invention can be appropriately determined as long as the desired effect can be obtained. As a guideline, about 2.5 to 20 times as much fucoidan as that of fluorotannins and about 2.5 to 20 times of that of magdisalisylate Double phlorotannins can be used. Moreover, about 2 to 10 times as much fluorotannins as that of magnesialicylate can be used.

  The agent of the present invention can be administered orally. A person skilled in the art can make the pharmaceutical composition of the present invention into various preparation forms according to the administration route. For example, the low molecular weight compound of the present invention may be made into a dry powder by solution, freeze drying, spray drying or the like, or prepared in a paste form. More specifically, powders, granules, capsules, tablets, pills, liquids, and injections can be used. Oral preparations are one of the preferred examples.

  The agent (composition) of the present invention is a food or pharmaceutically acceptable additive such as an excipient, binder, stabilizer, preservative, disintegrant, lubricant, coating agent, solubilizer, buffering agent. Can be used. In addition, the low molecular weight compound of the present invention or the pharmaceutical composition of the present invention can be used in combination with other components effective for the treatment of the target disease or condition.

1． Preparation of fluorotannins
To 1 kg of micronized dry wakame, 5 L of 95% (v / v) ethanol was added and left at room temperature for 24 hours with stirring. The extract is clarified by filtration, 1 to 30 g of activated carbon (trade name: activated carbon, reagent grade, manufacturer: Wako Pure Chemical Industries, Ltd., shape: powder) is added, stirred and mixed, and then the activated carbon is removed by filtration. did. Ethanol was removed by concentration under reduced pressure (evaporator) to obtain a dried product.

  The ethanol-eluted fraction of wakame contains carotenoids such as chlorophyll and fucoxanthin, some fats and oils, in addition to fluorotannins. As a method for easily and efficiently removing these, activated carbon, which is recognized as a food processing aid, was used, and the amount used was examined.

  Each component was confirmed by a thin layer chromatogram. Since A and B have characteristic colors, they were visually confirmed, and C was confirmed using vanillin-sulfuric acid as a color solution after developing by thin layer chromatography. When the amount of the activated carbon used was 0.5% or less of the raw material algae, the obtained extract had a strong seaweed odor and a blackish brown color, which was not suitable as a food raw material. In addition, at 1.5% or less, the green color of the chlorophylls was removed, but carotenoids such as fucoxanthin remained and were orange-red. Fucoxanthin was easily oxidized and turned blue under acidity, and its storage / stability was poor, which also seemed unsuitable as a food ingredient. In the use of 2.0 to 2.5%, chlorophylls and carotenoids were removed, and the loss of fluorotannins was 5% or less. However, at 3.0% or more, most of the fluorotannins were adsorbed on the activated carbon.

  Therefore, the manufacturing method of the fluoroaltannin derived from brown algae was established as follows. After extraction with ethanol having a final concentration of 85% (v / v) or more, 2.0 to 2.5% activated carbon is added to the clarified liquid with respect to the original alga body (corresponding to the dried product) to remove impurities.

By this method, 1 kg of finely powdered dried wakame was processed to obtain about 5 g of fluorotannins.
Each component of this fraction was examined based on the method of Nagayama et al. (Patent No. 146146: antibacterial agent mainly composed of phlorotannins, Patent No. 4105535: antiviral substance). As a result, the main components were diacor and flurofucophore A, and phloroglucinol, equol and 8,8'-bayecol were also included.
The following table summarizes the main characteristics of the fluoroaltanines of the brown algal polyphenols obtained by this method.

Fluorotannins prepared by this method were used in the following experiments.
2. Human volunteer test It is known that acetylcholinesterase inhibitors stimulate insulin secretion by stimulating muscarinic receptors (R. Rodriguez-Diaz et. Al., Alpha cells secrete acetylcholine as a nonneuronal paracrine signal priming beta cell) function in humans. Nature Medicine, 17, 888-892, 2011). Complications are most noticeable in diabetes, and typical examples include increased risk of nephropathy and retinopathy due to AGEs (Advanced Glycation End-products), and neuropathy. Neuropathy is particularly serious, and this is caused by an increase in the intracellular concentration of polyol (mainly sorbitol) caused by aldose reductase, which causes cell damage. The aldose reductase inhibitory effect of phlorotannins has been found by Hiroshi Nakamura et al. (Natural Medicines, 51 (2), 162-169, 1997) Yes.

  On the other hand, since phlotannins have not been shown to improve insulin resistance directly, fucoxanthin (diabetes treatment; JP 2007-314451) and phlorotannin have been shown to improve insulin resistance. We prepared a mixture of varieties in fucoidan (table below). Specifically, 250 mg of the powder mixture shown in the table below was filled into capsules, and 4 capsules per day (powder mixture 1 g / day) were ingested. No additives such as a carrier were added to the capsule.

  1 g per day was ingested, and hemoglobin A1c and fasting blood glucose level were measured every month. Volunteers were helping patients with type 2 diabetes who had little improvement in hemoglobin A1c and fasting blood glucose levels with various drugs.

  The background of each volunteer is shown in the table below.

  The results are shown in the table below.

At daily intakes of 0.5 g and 1 g of the test food, 1 g more effectively decreased both the blood glucose level and hemoglobin A1c.
From these results, the anti-diabetic action of the test food containing fucoidan, fucoxanthin, fluorotannins and magdisalicylate is clear. Furthermore, for volunteers B and C, both blood glucose levels and hemoglobin A1c levels that did not improve even with hypoglycemic drugs improved. It can be said that the improvement effect of the test food was also observed in diabetic patients having drug resistance. From the impressions of each volunteer, not only did diabetes not completely cure, but there was a growing awareness that it could be improved, and there was a willingness to actively improve lifestyle.

Claims (4)

Extracting 2 to 10 parts by weight of an ethanol-containing solvent with respect to 1 part by weight of the dried brown algae raw material to obtain an extract containing fluorotannins;
To the extract, 1.6 to 2.9 parts by weight (preferably 1.8 to 2.7 parts by weight, more preferably 2.0 to 2.5 parts by weight) of activated carbon is added to the equivalent of 100 parts by weight of the dried brown algae material to adsorb impurities. Removing;
A method for producing fluorotannins, comprising: The fluorotannins according to claim 1, comprising at least one substance selected from the group consisting of phloroglucinol, equol, florofcofurecol A, diecol, and 8,8'-bayecol. Production method. A therapeutic agent for diabetes comprising phlorotannins, fucoxanthin, magdisalicylate and fucoidan obtained by the production method of claim 1 or 2. The agent according to claim 3, for inoculating phlorotannins at 0.1 to 500 mg / day, preferably 50 to 100 mg / day.