TWI778047B - Production method of saccharolytic enzyme inhibitor - Google Patents

Abstract

The present invention provides a method for producing a natural product-derived saccharolytic enzyme inhibitor with a long edible history and high safety. A saccharolytic enzyme inhibitor is obtained by using an earthworm or its ground product or a dry powder of the earthworm or its ground product as a raw material. The saccharolytic enzyme inhibitor is obtained by performing the extraction process of obtaining the earthworm extract and the separation process of obtaining the component with a molecular mass of less than 3 kDa derived from the earthworm powder extract. One of the predetermined saccharolytic enzymes to be inhibited is α-glucosidase.

Description

Production method of saccharolytic enzyme inhibitor

The present invention relates to a method for producing a saccharolytic enzyme inhibitor having an activity-inhibiting effect on a specific saccharolytic enzyme.

硫酸分子內鹽結構，且顯示與市售的α-葡萄糖苷酶抑制劑阿卡波糖相同程度的活性(非專利文獻18)。又，專利文獻1及非專利文獻17中，作為顯示抑制效果之成分而分別舉出小麥及芸豆的蛋白質(分子質量3,500Da以上)，專利文獻10中作為顯示抑制效果之成分舉出分解馬鈴薯的蛋白質而得到之肽。 　　[0013] 蚯蚓為被分類為環節動物門寡毛綱之繩狀生物，主要生活在土壤中並攝食有機物，且藉由消化吸收和排洩而發揮分解土壤中的有機物之作用。於該方面，蚯蚓促進土壤的耕犁，並且將植物能夠利用之有機物帶至土壤中來，對農業而言係必不可少之存在。蚯蚓位於食物鏈的底層，在自然界中，被魚類、兩棲類、爬行類、鳥類、哺乳類等廣範圍的生物捕食。又，亦被用作魚或雞的飼料，亦存在人類於各地食用的記錄。在東方，以解熱、鎮痛、利尿、促進血流等目的而作為中藥亦被長期利用。如此關於蚯蚓，有具有足夠的安全性之充分的食用歷史，且亦生產有使用了蚯蚓乾燥粉末之健康食品(專利文獻11、專利文獻12)。 　　[0014] 目前，從蚯蚓的體腔液或粉碎液及由該等製造之蚯蚓乾燥粉末中，除了上述澱粉酶活性(非專利文獻8)以外，還發現纖維素酶活性(非專利文獻20)、脂肪酶活性(非專利文獻21)、尿激酶樣活性或組織纖溶酶原激活物(t-PA)樣活性等複數種蛋白酶活性(非專利文獻22、非專利文獻23)。進而，從蚯蚓粉碎液的丙酮沉澱組分發現有彈性蛋白酶抑制活性、基質金屬蛋白酶抑制活性及酪胺酸酶抑制活性(非專利文獻24)，從蚯蚓乾燥粉末萃取液的小於10kDa的低分子組分發現有二肽基肽酶IV抑制活性(專利文獻13)，同樣地從5kDa以下的低分子組分發現有血管收縮素轉化酶抑制活性(專利文獻14)。本發明人等最近示出蚯蚓萃取液的3kDa以下的組分含有促進胰蛋白酶、胰凝乳蛋白酶、脂肪酶等酶活性之物質(日本專利申請2017-076108；酶活性促進劑的製造方法及酶活性促進劑的含有物)。然而，目前為止沒有與蚯蚓成分中的α-澱粉酶抑制活性或α-葡萄糖苷酶抑制活性有關的報告。 (先前技術文獻) (專利文獻) 　　[0015] 　　專利文獻1：日本特開2014-51473號公報 　　專利文獻2：日本特開2005-220110號公報 　　專利文獻3：日本特開2010-222277號公報 　　專利文獻4：日本特開2000-319192號公報 　　專利文獻5：日本特開2002-10753號公報 　　專利文獻6：日本特開2004-352649號公報 　　專利文獻7：日本特開2006-104100號公報 　　專利文獻8：日本專利第4403457號公報 　　專利文獻9：國際公開第2008/090999號公報 　　專利文獻10：日本特開平10-292000號公報 　　專利文獻11：日本專利第5548931號公報 　　專利文獻12：日本特開2015-48353號公報 　　專利文獻13：日本專利第5901092號公報 　　專利文獻14：日本特開2015-168631號公報 (非專利文獻) 　　[0016] 　　非專利文獻1：厚生勞働省、平成24年國民健康·營養調查結果的概要 　　非專利文獻2：厚生勞働省、平成26年(2014年)患者調查的概況 　　非專利文獻3：厚生勞働省、平成25年度 國民醫療費的概況 　　非專利文獻4：今堀和友、山川民夫(監修)：生物化學詞典，第4版，Tokyo Kagaku Dojin，東京，142-143頁(胰島素)、2007年 　　非專利文獻5：今堀和友、山川民夫(監修)：生物化學詞典，第4版，Tokyo Kagaku Dojin，東京、931頁(糖尿病)、2007年 　　非專利文獻6：今堀和友，山川民夫(監修)：生物化學詞典，第4版，Tokyo Kagaku Dojin，東京、66-67頁(澱粉酶)、2007年 　　非專利文獻7：The Amylase Research Society of Japan.(日本澱粉酶研究學會)：Enzyme chemistry and molecular biology of amylases and related enzymes.(澱粉酶和相關酶的酶化學和分子生物學)(Yamamoto, T., ed.), CRC Press, Florida, USA, pp. 196-197 (1995) 　　非專利文獻8：Ueda, M., et al.: Purification and characterization of novel raw-starch-digesting and cold-adapted alpha-amylases from Eisenia foetida. Comp. Biochem. 　　Physiol. B. Biochem. Mol. Biol. 150, 125-130 (2008) 　　非專利文獻9：廣海啟太郎：澱粉酶的作用方式和亞位點結構.澱粉科學，21、190-203 (1974) 　　非專利文獻10：Ramasubbu, N., et al.: Human salivary α-amylase Trp58 situated at subsite -2 is critical for enzyme activity 　　Eur. J. Biochem. 271, 2517-2529 (2004). 　　非專利文獻11：今堀和友、山川民夫(監修)：生物化學詞典，第4版，Tokyo Kagaku Dojin，東京、404頁(α-葡萄糖苷酶)，2007年 　　非專利文獻12：今堀和友、山川民夫(監修)：生物化學詞典，第4版，Tokyo Kagaku Dojin，東京，934頁(糖轉運體)，2007年 　　非專利文獻13：Voet, D., Voet, J. G., and Pratt, C. W.(著)、田宮信雄、村松正實、八木達彥、遠藤斗志也(譯)：沃特基本生物化學，第1版，Tokyo Kagaku Dojin，東京，452-453頁，2004年 　　非專利文獻14：秋山徹、河府和義：抑制劑手冊、YODOSHA CO., LTD.，東京，404-419頁，2006年 　　非專利文獻15：Wako Pure Chemical Industries, LTD.：α-葡萄糖苷酶抑制劑. 試劑首頁，2017年，(http://www.wako-chem.co.jp) 　　非專利文獻16：Dabhi, A. S., Bhatt, N. R., and Shah, M. J.: Voglibose: An alpha glucosidase inhibitor. J. Clin. Diagn. Res. 7, 3023-3027 (2013) 　　非專利文獻17：吉川秀樹、桑島千榮、小垂真：芸豆中的α-澱粉酶抑制劑活性和其性質.Kyoto Koka Women’s University研究簡報，47，227-237(2009) 　　非專利文獻18：吉川雅之：藥用植物的糖尿病預防成分. 化學和生物、40、172-178、(2002) 　　非專利文獻19：村尾澤夫、大山邦夫、村井英繼、後藤章、松井良博、福原健一、宮田茂一、住田光夫、荒井基夫：由放線菌精算之澱粉酶抑制劑. 澱粉科學、26、157-164、(1979) 　　非專利文獻20：Ueda, M., et al.: A novel cold-adapted cellulose complex from Eisenia foetida: characterization of a multienzyme complex with carboxymethylcellulase, beta-glucosidase, beta-1, 3 glucanase, and beta-xylosidase. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 157, 26-32 (2010) 　　非專利文獻21：Nakajima, N., et al.: An isozyme of earthworm serine proteases acts on hydrolysis of triacylglycerol. Biosci Biotechnol. Biochem. 69, 2009-2011 (2005) 　　非專利文獻22：Nakajima, N., et al.: Characterization of potent fibrinolytic enzymes 　　in earthworm, Lumbricus rubellus. Biosci. Biotechnol. Biochem. 57, 1726-1730 (1993) 　　非專利文獻23：Phan, T. T., et al.: Purification and characterization of novel fibrinolytic proteases as potential antithrombotic agents from earthworm Perionyx excavates. AMB Express 1, 1-11 (2011) 　　非專利文獻24：Azmi, N., et al.: Anti-elastase, anti-tyrosinase and matrix metalloproteinase-1 inhibitory activity of earthworm extracts as potential new anti-agingagent. Asian. Pac. J. Trop. Biomed. 4, S348-S352 (2014) 　　非專利文獻25：廣海啓太郎：新·入門酶化學，改訂第2版(西澤一俊、志村憲助、編著)，Nankodo Co., LTD.，東京，21-93頁，1995年 　　非專利文獻26：井上國世：第一次酶化學(井上國世、編著)、CMC Publishing CO., LTD.，東京，241-365頁，2016年 　　非專利文獻27：SIGMA Quality Control Test Procedure: Enzymatic assay of α-glucosidase. Sigma (1996) 　　非專利文獻28：濱岡直裕、中川良二、比良徹、八卷幸二：小豆子葉部對α-葡萄糖苷酶活性及GLP-1分泌的影響.Nippon shokuhin kagaku kogaku kaishi, 60, 43-47 (2013)According to the survey of the Japanese Ministry of Health, Labour and Welfare, in recent years, the number of Japanese adult men and women who are strongly suspected of having diabetes has risen to about 9.5 million, and the number of people who cannot rule out the possibility of diabetes has also risen to about 11 million (non-diabetic). Patent Document 1). According to reports, the actual number of patients receiving treatment is about 3.17 million, and the annual medical expenses are about 1.2 trillion yen, which is a big problem in terms of national health and medical finance (Non-Patent Document 2, Non-Patent Document 3). [0003] Diabetes is a disease caused by insufficient insulin production or a decrease in the function of insulin, and the blood sugar level rises, and symptoms such as sugar also appear in the urine. Insulin is a hormone produced and secreted by β-cells of the pancreas. In addition to the function of promoting the uptake of glucose from the blood in the whole body organs and lowering blood sugar, it also promotes the synthesis of glucose to glycogen in the liver or muscle, and inhibits the synthesis of glucose in the liver. It has many important roles such as the decomposition of glycogen into glucose (Non-Patent Document 4). It is known that in diabetic patients, since the hyperglycemia state caused by insufficient insulin continues, damage accumulates in the cells of blood vessels and develops arteriosclerosis, which in turn causes diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, etc. Serious complications (Non-Patent Document 5). The main cause of diabetes is overeating, lack of exercise, obesity, stress, genetic factors, etc., and is roughly divided into type I diabetes and type II diabetes. Type I diabetes is absolutely insufficiency of insulin due to pancreatic β-cell disorder, and requires insulin injections during treatment. On the other hand, type II diabetes is relatively insufficiency of insulin due to a decrease in insulin production or insulin resistance, so diet therapy, exercise therapy, or drug therapy is performed in the treatment. 95% of diabetic patients in Japan are type II diabetes (Non-Patent Document 5). [0005] Saccharolytic enzymes play an important role in the digestion and absorption of sugars ingested in the diet. For example, starch is first partially cleaved in the oral cavity by salivary α-amylase (hereinafter, referred to as salivary amylase), and then in the small intestine by pancreatic α-amylase (hereinafter, referred to as pancreatic amylase) ) to be decomposed to maltose, and then decomposed to glucose by maltase. The product glucose is taken up by small intestinal epithelial cells via a sugar transporter, and then transported into blood vessels (Non-Patent Document 6). Amylase is a general term for enzymes that hydrolyze starch in a broad sense, and is classified into endo-type enzymes that cut sugar chains at non-specific sites and exo-type enzymes that cut a certain number of glucose units from non-reducing ends of sugar chains. Dicer. α-Amylase (enzyme number EC 3.2.1.1) is an endo-type enzyme that hydrolyzes only α1→4 glucosidic bonds of sugar chains, and salivary amylase and pancreatic amylase are also known as isozymes in humans (non-patent literature). 6). This isozyme has a very high identity in the DNA sequence, is more than 90% identical in the amino acid sequence, and has very similar properties as an enzyme (Non-Patent Document 7). In fact, the same isoenzymes are also commonly found in human serum or urine. In addition, α-amylase has been found in animals, plants, microorganisms, and a wide range of organisms, and is also reported to be present in earthworms (Non-Patent Document 8). About the mode of action of saccharolytic enzymes mainly composed of α-amylase, in addition to the distinction of endo-type and exo-type, it is classified from the following multiple viewpoints, that is, based on the type of glucosidic bond to be cut classification, classification based on the type of the final product, classification based on the relationship between the degree of polymerization of the linear matrix and the decomposition rate, classification based on the difference in the cutting position of the matrix oligosaccharide, and the like. The difference in these complex modes of action is derived from the three-dimensional structure of each enzyme, but by assuming that there is a sugar-bonding site called a subsite in the structure including the active site before and after the cleavage of the glucosidic bond Theoretical explanation was given in advance by considering the structural unit (Non-Patent Document 9). Then, according to X-ray crystal structure analysis, the functions of many enzymes were identified by correlating with subsite structures. For example, according to reports, human salivary α-amylase consists of 496 amino acids, 3 regions Formation, subsites from -4 to +3 are present in the deep cracked structure of region A (sugar chain that becomes the matrix is cut between -1 and +1); subsite -2 is important in developing activity tryptophan residues (Non-Patent Document 10). Exo-amylases separate a specific number of glucose units from the non-reducing ends of starch or amylose. Exo-amylases can be further classified according to the chain length of the isolated glucose units. The enzyme that separates glucose units one by one is called glucoamylase (enzyme number EC3.2.1.3); the enzyme that separates 2 glucose units (that is, with maltose units) at a time is called beta-amylase (EC3.2.1. 2); The enzyme that separates 3 glucose units (that is, with maltotriose units) at a time is called exoisomaltotriose hydrolase (EC3.2.1.95); 4 glucose units are separated at a time ( That is, the enzyme in maltotetraose units) is called exoisomaltotetraose hydrolase (EC 3.2.1.60); the enzyme that separates 6 glucose units (i.e., in maltohexaose units) at a time The enzyme is called exoisomaltohexahydrolase (EC 3.2.1.98). In addition, among the endo-amylases, in addition to α-amylase (EC3.2.1.1) that only hydrolyzes α1→4 glucosidic bonds, there are also pullulanases (EC3) that selectively hydrolyze α1→6 glucosidic bonds. .2.1.41) or isoamylase (EC3.2.1.68) (Non-Patent Document 6). Maltase, i.e. α-glucosidase, is a general term for exoglycosidase that hydrolyzes the α-D-glucosidic bond present at the non-reducing end of sugar chain, but in a narrow sense refers to α-glucosidase ( EC 3.2.1.20), and maltose, amylose and their oligosaccharides were used as substrates (Non-Patent Document 11). In addition, α-glucosidase in a broad sense includes sucrase that decomposes sucrose into glucose and fructose, lactase that decomposes lactose into glucose and galactose, cleaves between isomaltose or low molecular α1→6 glucosidic bonds Enzymes such as isomaltase. Phenyl-α-D-glucoside or p-nitrophenyl α-D-glucoside is also hydrolyzed, and the enzymatic activity is measured using this in many cases (Non-Patent Document 11). As described above, both endo-amylase and exo-amylase are enzymes that hydrolyze the α-D-glucosidic bond to be the target, and although the target selection system is different, the basic mechanism of action is considered to be the same. On the other hand, sugar transporter is the general term for the protein that crosses biofilm and carries out the transport of sugar, and is known to have glucose transporter (GLUT) family and sodium-glucose co-transporter (SGLT) family according to its transport mode . The former is a transporter of the diffusion-promoting system that takes up glucose in accordance with the concentration gradient of glucose, and the latter is a transporter of the kinetic transport system that takes up glucose against the glucose concentration gradient while transporting sodium using the intracellular and intracellular concentration gradients of sodium ions (Non-patent literature). 12). In the small intestine, SGLT1, which is present in villi cells, plays a major role in the uptake of glucose into cells, and GLUT5 contributes to the uptake of fructose. In addition, GLUT2, which is present in a large amount despite its low affinity for glucose, contributes to the transfer of glucose into the blood (Non-Patent Document 13). [0011] Currently, inhibitors of the above-mentioned saccharolytic enzymes or inhibitors of sugar transporters have been developed and utilized as therapeutic drugs for diabetes. Various α-glucosidase inhibitors are used as diabetes treatment drugs for suppressing the rise in blood sugar levels after meals. 1-deoxynojirimycin is an inhibitor of α-glucosidase and β-glucosidase, and the corresponding _IC50 values are 12.6 μM and 47 μM, respectively (Non-Patent Document 14). In addition, acarbose, miglitol, and voglibose are widely used. Acarbose has a linear tetrasaccharide-like structure and exhibits inhibitory effect on α-amylase together with α-glucosidase. Voglibose shares a monosaccharide-like structure with miglitol. These have a stronger inhibitory effect on α-glucosidase than acarbose (Non-Patent Document 15, Non-Patent Document 16). Regarding voglibose, it is described that the inhibitory effect of porcine intestinal maltase and sucrase is 20 times and 30 times stronger than that of acarbose, respectively, and it is 270 times and 190 times stronger than that of rat intestinal maltase and sucrase, respectively. times the inhibitory effect. On the other hand, the inhibitory effect on pancreatic amylase in pigs and rats was 1/3000 that of acarbose. Voglibose is more selective for α-glucosidase than α-amylase, but has _IC50 values of 10 μM to 1 nM for α-glucosidase (Non-Patent Document 14, Non-Patent Document 15, Non-Patent Document 16). These pharmaceuticals inhibit glucose uptake in the small intestine by inhibiting α-glucosidase activity, but do not directly lower blood glucose levels. In addition, according to reports, when the decomposition of sugar is excessively inhibited, side effects such as abdominal distension and symptoms of hypoglycemia are caused. On the other hand, as a sugar transporter, canagliflozin (canagliflozin ( Canagliflozin) or Ipragliflozin, etc. These are reported to reduce the amount of glucose reabsorbed from the urine, but since they act on the kidneys, it is necessary to pay attention to their drug dynamics. In addition, they also need to pay attention to side effects such as polyuria or hypoglycemia. [0012] In addition, under these circumstances, various attempts have been made to search for inhibitory components for α-amylase or α-glucosidase that can be orally ingested from natural products such as vegetables, seaweed, plants, and microorganisms. For example, from wheat (Patent Document 1), buckwheat (Patent Document 2), tea (Patent Document 3), Grifola frondosa (Patent Document 4), olive leaf (Patent Document 5), kidney beans (Non-Patent Document 17), etc. α-amylase inhibitory effect, and from walnut (patent document 6), seaweed (patent document 7), plum (patent document 8), shochu moromi (patent document 9), mulberry leaf (non-patent document 18), five layer dragon Genus plants (Non-Patent Document 18) and the like have found an α-glucosidase inhibitory effect, and a plurality of those having an inhibitory effect on both α-amylase and α-glucosidase have been reported. A number of inhibitor-related contents have also been reported from compounds derived from microorganisms. As examples, those containing oligosaccharides as components and those containing proteins as components, such as nojirimycin as an α-glucosidase inhibitor or S-AI as an α-amylase inhibitor (Non-Patent Documents) 19). Among the ingredients showing these inhibitory effects, there are those whose properties or structures are known. For example, the laver of Patent Document 7 includes a structure containing a sulfated sugar, that is, Porphyran, the tea of Patent Document 3, or Porphyran. The olive leaves of Patent Document 5 contain flavonoids such as flavonoids and flavonols or polyphenol compounds containing their glycosides, the mulberry leaves contain 1-deoxynojirimycin, and the phyllostachys plants contain salacinol and Kotalanol. Required for Salacinol and Kotalanol to develop activity

It has an intramolecular salt structure of sulfuric acid and exhibits the same activity as acarbose, a commercially available α-glucosidase inhibitor (Non-Patent Document 18). In addition, in Patent Document 1 and Non-Patent Document 17, proteins of wheat and kidney bean (molecular mass 3,500 Da or more) are listed as components exhibiting an inhibitory effect, respectively, and in Patent Document 10, as a component exhibiting an inhibitory effect, decomposed potato is exemplified. peptides derived from proteins. [0013] Earthworms are rope-like organisms classified as Annelid phylum Oligochaetes, mainly live in soil and feed on organic matter, and play the role of decomposing organic matter in soil by digestion, absorption and excretion. In this respect, earthworms are essential to agriculture by facilitating the ploughing of the soil and bringing organic matter that can be used by plants into the soil. Earthworms are located at the bottom of the food chain. In nature, they are preyed on by a wide range of organisms such as fish, amphibians, reptiles, birds, and mammals. In addition, it is also used as feed for fish or chickens, and there are records of human consumption in various places. In the East, it has also been used for a long time as a traditional Chinese medicine for the purposes of antipyretic, analgesic, diuretic, and promoting blood flow. As described above, earthworms have a sufficient history of consumption with sufficient safety, and health foods using dry earthworm powders are also produced (Patent Document 11, Patent Document 12). At present, in addition to the above-mentioned amylase activity (non-patent document 8), cellulase activity (non-patent document 20), cellulase activity (non-patent document 20), Multiple protease activities such as lipase activity (Non-Patent Document 21), urokinase-like activity or tissue plasminogen activator (t-PA)-like activity (Non-Patent Document 22, Non-Patent Document 23). Furthermore, elastase inhibitory activity, matrix metalloproteinase inhibitory activity, and tyrosinase inhibitory activity were found from the acetone-precipitated fraction of the earthworm pulverized liquid (Non-Patent Document 24), and the low molecular weight group less than 10 kDa in the earthworm dry powder extract A dipeptidyl peptidase IV inhibitory activity has been distributed (Patent Document 13), and similarly angiotensin-converting enzyme inhibitory activity has been found from a low molecular weight fraction of 5 kDa or less (Patent Document 14). The present inventors have recently shown that the fraction of 3 kDa or less in the earthworm extract contains substances that promote enzyme activities such as trypsin, chymotrypsin, and lipase (Japanese Patent Application No. 2017-076108; Production method and enzyme of enzyme activity accelerator). active accelerator content). However, so far, there has been no report on the α-amylase inhibitory activity or α-glucosidase inhibitory activity in earthworm components. (Prior Art Document) (Patent Document) [0015] Patent Document 1: Japanese Patent Application Laid-Open No. 2014-51473 Patent Document 2: Japanese Patent Application Laid-Open No. 2005-220110 Patent Document 3: Japanese Patent Application Laid-Open No. 2010-222277 4: Japanese Patent Laid-Open No. 2000-319192 Patent Document 5: Japanese Patent Laid-Open No. 2002-10753 Patent Document 6: Japanese Patent Laid-Open No. 2004-352649 Patent Document 7: Japanese Patent Laid-Open No. 2006-104100 Patent Document 8: Japanese Patent No. 4403457 Patent Document 9: International Publication No. 2008/090999 Patent Document 10: Japanese Patent Laid-Open No. 10-292000 Patent Document 11: Japanese Patent No. 5548931 Patent Document 12: Japanese Patent Laid-Open No. 2015-48353 Patent Document 13: Japanese Patent No. 5901092 Patent Document 14: Japanese Patent Laid-Open No. 2015-168631 (Non-Patent Document) [0016] Non-Patent Document 1: Ministry of Health, Labour and Welfare, 2009 National Health and Nutrition Survey Results Outline of Non-Patent Document 2: Ministry of Health, Labour and Welfare, overview of patient survey in 2014 (2014) Non-patent document 3: Ministry of Health, Labour and Welfare, overview of national medical expenses in 2015 (Supervised): Dictionary of Biochemistry, 4th Edition, Tokyo Kagaku Dojin, Tokyo, pp. 142-143 (Insulin), 2007 Non-Patent Document 5: Kazuto Imabori, Tomo Yamakawa (Supervisor): Dictionary of Biochemistry, 4th Edition , Tokyo Kagaku Dojin, Tokyo, pp. 931 (Diabetes), 2007 Non-Patent Document 6: Kazuto Imabori, Tomoyama Yamakawa (Supervisor): Dictionary of Biochemistry, 4th Edition, Tokyo Kagaku Dojin, Tokyo, pp. 66-67 (starch Enzyme), 2007 Non-Patent Document 7: The Amylase Research Society of Japan. (Japan Amylase Research Society): Enzyme chemistry and molecular biology of amylases and related enzymes. (Enzyme chemistry and molecular biology of amylase and related enzymes) (Yamamoto, T., ed.), CRC Press, Florida, USA, pp. 196-197 (1995) Non-Patent Literature 8: Ueda, M., et al.: Purification and characterization of novel raw-starch-digesting and cold-adapted a lpha-amylases from Eisenia foetida. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 150, 125-130 (2008) Non-patent document 9: Ketaro Hirokai: Mode of action and subsite structure of amylase. Starch Science, 21, 190-203 (1974) Non-Patent Literature 10: Ramasubbu, N., et al.: Human salivary α-amylase Trp58 situated at subsite -2 is critical for enzyme activity Eur. J. Biochem. 271, 2517 -2529 (2004). Non-patent document 11: Kazuto Imabori, Tomoyama Yamakawa (supervising): Dictionary of Biochemistry, 4th edition, Tokyo Kagaku Dojin, Tokyo, p. 404 (α-glucosidase), 2007 Non-patent document 12: Kazuyoshi Imabori, Tomoyama Yamakawa (supervising): Dictionary of Biochemistry, 4th Edition, Tokyo Kagaku Dojin, Tokyo, p. 934 (sugar transporter), 2007 Non-patent literature 13: Voet, D., Voet, JG, and Pratt, CW (author), Nobuo Tamiya, Masami Muramatsu, Tatsuhiko Yagi, Toshiya Endo (translated): Water Basic Biochemistry, 1st Edition, Tokyo Kagaku Dojin, Tokyo, pp. 452-453, 2004 Non-patent Document 14: Toru Akiyama, Kazuyoshi Kawafu: Handbook of Inhibitors, YODOSHA CO., LTD., Tokyo, pp. 404-419, 2006 Non-patent Document 15: Wako Pure Chemical Industries, LTD.: α-Glucosidase Inhibitor . Reagent top page, 2017, (http://www.wako-chem.co.jp) Non-patent literature 16: Dabhi, AS, Bhatt, NR, and Shah, MJ: Voglibose: An alpha glucosidase inhibitor. J. Clin . Diagn. Res. 7, 3023-3027 (2013) Non-Patent Document 17: Hideki Yoshikawa, Chiei Kushima, and Makoto Kotari: α-amylase inhibitor activity in kidney beans and its properties. Kyoto Koka Women's University Research Brief, 47, 227-237 (2009) Non-Patent Document 18: Yoshikawa Masaru: Diabetes-preventing ingredients of medicinal plants. Chemistry and Biology, 40, 172-178, (2002) Non-Patent Document 19: Murao Zeo, Oyama Kunio, Murai Eiji, Goto Akira, Matsui Ryohiro, Fukuhara Kenichi, Miyata Shigeru, Sumita Mitsuo, Arai Keifu: Amylase Inhibitors Calculated from Actinomycetes. Starch Science, 26, 157-164, (1979) Non-Patent Literature 20: Ueda, M., et al.: A novel cold-adapted cellulose complex from Eisenia foetida: characterization of a multienzyme complex with carboxymethylcellulase, beta-glucosidase, beta -1, 3 glucanase, and beta-xylosidase. Comp. Biochem. Physiol. B. Biochem. Mol. 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(Problem to be solved by the present invention) The main problem of the present invention is to provide a food with a long history and safety that has the activity-inhibiting effect of saccharolytic enzymes, especially the activity-inhibiting effect of α-amylase and α-glucosidase The method for producing a natural product-derived saccharolytic enzyme inhibitor with high property can also provide a food, a pharmaceutical, and the like that are effective in preventing and improving diabetes and obesity. (Means for Solving the Problems) [0018] The present inventors have completed the present invention as a result of intensive research in order to solve the above-mentioned problems. That is, the first feature of the present invention is a method for producing a saccharolytic enzyme inhibitor having an activity-inhibiting effect on a specific saccharolytic enzyme, wherein one of the specific saccharolytic enzymes is α-glucosidase, The following process is performed to obtain the aforementioned saccharolytic enzyme inhibitor, that is, an extraction process, using earthworm or its pulverized product or dry powder of earthworm or its pulverized product as a raw material and adding water to obtain an earthworm extract; and a separation process to obtain an extract derived from The molecular mass of the earthworm extract is less than 3kDa, that is, the component of the extract is less than 3kDa. A second characteristic configuration of the present invention is characterized in that one of the specific saccharolytic enzymes is α-amylase. According to this constitution, earthworm has a long history of eating, and is a natural raw material with high safety, so it can be expected to have a saccharolytic enzyme activity by inhibiting the small intestine, especially α-amylase activity or α-amylase activity. - The effect of inhibiting the digestion and absorption of sugar by glucosidase activity, and the application of effective pharmaceuticals, health foods, supplements, etc. for the prevention and improvement of lifestyle diseases such as diabetes, obesity, and metabolic syndrome. In addition, the target is not limited to human beings, and it is also considered to be used as pharmaceuticals or to improve pet mammals such as dogs and cats, and livestock such as cattle, horses, and pigs, for which diseases similar to lifestyle diseases have become a problem in recent years as in human beings. Application of pet food for health purpose. The earthworms used as raw materials can be easily reproduced, and there are also advantages in terms of productivity. Also, according to this constitution, by obtaining the separation process of the component with a molecular mass less than 3kDa in the earthworm extract, that is, the component with a molecular mass less than 3kDa in the extract, it is possible to remove the macromolecule whose molecular mass is more than 3kDa in the earthworm extract. quality components. As described above, according to reports, earthworms themselves contain an enzyme component having α-amylase activity, so the separation process is also important for the purpose of excluding earthworm-derived α-amylase from the saccharolytic enzyme inhibitor of the present invention meaning. Further, by applying a concentration operation such as freeze-drying to the obtained component whose molecular mass is less than 3 kDa, that is, the extract is less than 3 kDa, the α-amylase activity inhibitory effect or α-amylase activity contained in the component can be obtained at a high concentration. Saccharolytic enzyme inhibitor with α-glucosidase activity inhibitory effect. The freeze-dried powder is also excellent in storage stability and workability. The third feature of the present invention is characterized in that at least one selected from the group consisting of freeze-thaw treatment, acid treatment, heat treatment, and organic solvent treatment is performed on the components of the extract recovered by the aforementioned separation process that are less than 3 kDa. The aforementioned saccharolytic enzyme inhibitor was obtained. [0022] According to this configuration, by performing the above freezing and thawing process, the components that are not resistant to freezing and thawing can be modified and removed. In addition, the saccharolytic enzyme inhibitor of the present invention can be expected to be stably stored for a long period of time even during freezing. Moreover, by performing the above-mentioned acid treatment, components that are not resistant to acid can be modified and removed. Moreover, the heat-labile component can be reformed and removed by performing the above-mentioned heat treatment. Furthermore, the sterilization effect by heat can also be expected. Moreover, by performing the above-mentioned organic solvent treatment, the components transferred to the organic layer and the components transferred to the interface can be removed. After each treatment, a separation operation such as centrifugation is performed, so that the modified components can be easily removed. Furthermore, these treatments can be carried out in combination rather than individually, so that a saccharolytic enzyme inhibitor with higher purity can be obtained. (Effect of the Invention) [0023] According to the present invention, it is possible to provide a method for producing a natural product-derived saccharolytic enzyme inhibitor with a long edible history and high safety. In addition, it can be provided as a food, a pharmaceutical, and the like effective for the prevention and improvement of diabetes and obesity.

[0043] 如表1所示，於使用了基質試液的原液之條件下，以最終濃度成為40mg/mL之方式添加了萃取液小於3kDa的組分粉末時，α-澱粉酶活性抑制率為約35%，但於用緩衝液對基質試液進行稀釋而將相對濃度設為0.50之條件下，α-澱粉酶活性抑制率成為約38%。進而，於將基質試液的相對濃度設為0.1之條件下抑制率為約43%，將相對濃度設為0.05之條件下抑制率為約49%。進一步對基質試液進行稀釋，將相對濃度設為0.03時，伴隨酶反應之405nm的吸光度明顯小，且很難追蹤其變化。如以上所示，酶反應液中所含有之α-澱粉酶濃度和萃取液小於3kDa的組分粉末濃度一定，但隨著基質濃度減少而抑制率上升。又，相反地，隨著基質濃度的增大而抑制率減少。由此推斷，本發明的糖分解酶抑制劑具有藉由與基質針對酶活性部位的鍵結競爭而抑制α-澱粉酶活性之類型的抑制方式。 　　[0044] 進而，利用酶反應速度論(非專利文獻25、非專利文獻26)對本發明的基於糖分解酶抑制劑的α-澱粉酶抑制的性質進行了分析。將基質試液的相對濃度標繪於橫軸，將藉由圖4的各資料求出之反應速度(v)標繪於縱軸而製作了米氏曲線(圖5)。進而，製作了將基質試液的相對濃度的倒數(1/v)標繪於橫軸，將反應速度的倒數標繪於縱軸而成的賴威佛-柏克(Lineweaver-Burk)曲線(圖6)及將基質試液的相對濃度標繪於橫軸，將基質試液的相對濃度除以反應速度而得之值標繪於縱軸而成之哈尼斯-伍爾夫(Hanes-Woolf)曲線(圖7)。從各自的曲線得到基於最小二乘法的近似直線，藉此求出了米氏常數K_m (單位：M)及最大速度V_max (單位：M/s)的酶反應速度論參數。又，分子活性(轉變數)k_cat (單位：1/s)以V_max /[E]₀ 表現。在此，[E]₀ (單位：M)為反應液中的酶初始濃度。特異性常數k_cat /K_m (單位：1/(Ms))係從k_cat 與K_m 計算。此外，基質試液中所含有之GalG2CNP的莫耳濃度雖不明確，但於標記該等的參數之基礎上，將基質試液原液的GalG2CNP的莫耳濃度表示為常數[S_S ]。 　　[0045] 基於抑制劑的酶抑制大體分類為競爭型與混合型(非專利文獻26)。競爭型抑制藉由抑制劑與酶上與基質鍵結之部位(基質鍵結部位)鍵結，且基質與抑制劑相互競爭爭奪基質鍵結部位而引起。於抑制劑的存在下，酶與基質的鍵結受到抑制劑干擾，因此酶與基質之間的親和力降低(亦即K_m 增大)，且抑制酶-基質複合物(ES複合物)的形成。但是，當基質濃度充分大時，實質上能夠無視抑制劑對酶-基質之間的鍵結的干擾，所得到之反應速度(亦即最大速度V_max )成為與不存在抑制劑時得到之最大速度V_max 相同的值。 　　[0046] 混合型抑制中，抑制劑與和基質鍵結部位不同的部位(抑制劑鍵結部位)鍵結，藉此針對基質鍵結部位的基質鍵結或酶反應速度常數受到影響。混合型抑制的特殊情況亦即非競爭抑制中，認為基質與抑制劑對酶的相互作用彼此獨立，並且於酶-抑制劑複合物(EI)中失活。該情況下，活性亦可以由游離的酶(E)帶來，因此與不存在抑制劑的情況相比，存在抑制劑之情況下，最大速度V_max 雖降低，但K_m 未發現發生變化。通常的混合型抑制中，基質與抑制劑對酶的相互作用相互影響，因此與不存在抑制劑的情況相比，抑制劑存在的情況下，可發現於酶與基質的親和力及酶活性這兩方面發生變化。 　　[0047] 酶抑制劑不僅是僅與酶的基質鍵結部位鍵結之競爭型抑制或僅與抑制劑鍵結部位鍵結之混合型抑制，亦可考慮於這兩個鍵結部位鍵結之情況。該情況下，認為有可能依賴於抑制劑針對基質鍵結部位或抑制劑鍵結部位的親和力的程度，而顯現競爭型抑制(亦即於抑制劑存在下V_max 及k_cat 未發生變化，但K_m 增大)和混合型抑制(於抑制劑存在下V_max 及k_cat 減少，K_m 增大)這兩者的性質。 　　[0048] 圖5的米氏(Michaelis)曲線中，各曲線表示飽和曲線，得知當以最終濃度成為40mg/mL之方式添加了萃取液小於3kDa的組分粉末時，反應速度(v)亦即酶活性降低。認為本發明的基於糖分解酶抑制劑的α-澱粉酶活性抑制的方式只要是競爭型，則最大反應速度V_max 於萃取液小於3kDa的組分粉末的添加、非添加條件下變相同。但是，即使於賦予最大基質濃度之基質試液的原液之條件下，反應速度亦未達到最大值。這表明為了達到最大反應速度，需要進一步高濃度的基質，但至少僅從圖5很難明確判斷是競爭型抑制還是混合型抑制。 　　[0049] 因此，製作賴威佛-柏克曲線，並求出了酶反應速度論參數(圖6)。最大反應速度於萃取液小於3kDa的組分粉末的最終濃度為0mg/mL、40mg/mL之任意條件下均成為大致相同的值。亦即，表示於基質濃度充分過剩的條件下賦予(預測)之最大速度V_max 與萃取液小於3kDa的組分粉末的有無添加無關而未發生變化。另一方面，藉由添加萃取液小於3kDa的組分粉末(最終濃度40mg/mL)，K_m 值大致增大2倍，由此表示α-澱粉酶與基質的親和性顯著降低。該等結果強烈表明本發明的基於糖分解酶抑制劑的α-澱粉酶活性抑制的方式有可能係競爭型。 　　[0050] 利用相同的資料製作哈尼斯-伍爾夫曲線，並求出了酶反應速度論參數(圖7)。藉由添加最終濃度40 mg/mL的萃取液小於3kDa的組分粉末，顯示最大反應速度及α-澱粉酶與基質的親和性這兩者均降低。亦即，K_m 值大致增大1.5倍，V_max (及k_cat )值減少了21%。該等結果係表明基於萃取液小於3kDa的組分粉末中的酶抑制劑的α-澱粉酶活性抑制為混合型抑制方式者。但是，與對K_m 值的影響相比，對V_max (及k_cat )值的影響很小，但不可無視競爭型抑制的可能性。 　　[0051] 作為該等圖6與圖7的結果的差異的理由，認為如下，亦即當為競爭型抑制時，若相對於抑制劑濃度增加基質濃度則抑制效果會減少，但賴威佛-柏克曲線於其特性上，權重施加到基質濃度低的區域的測定資料，相對於此，哈尼斯-伍爾夫曲線具有權重施加到基質濃度高的區域的測定結果的性質。無法從圖6的曲線中讀取之混合型抑制的特徵有可能反應在圖7的曲線中。 　　[0052] 依據以上內容，作為本發明的基於糖分解酶抑制劑的α-澱粉酶活性的抑制機制，會引發以下疑問。 　　第一，所觀察到的抑制是由單一抑制物質引起，還是兩種以上的抑制物質引起。 　　如果設為由單一物質引起抑制，則該物質是與α-澱粉酶的基質鍵結部位鍵結而引起競爭型抑制，還是與抑制劑鍵結部位鍵結而引起混合型抑制，或者與這兩個鍵結部位鍵結而顯示競爭型與混合型的抑制型。 　　進而，抑制並不是由單一抑制劑引起的，而是由抑制型不同的兩種以上的抑制劑引起者，該等與酶的基質鍵結部位或抑制劑鍵結部位鍵結，其效果有可能引起基質的鍵結親和性降低(亦即K_m 的增大)或分子觸媒活性降低(亦即V_max 及k_cat 的減少)。 　　於抑制物質的種類、是否為競爭型抑制還是混合型抑制的判別、酶的基質鍵結部位及抑制劑鍵結部位的辨識方面，上述研究之酶反應速度論的方法有限。將來需要推進抑制物質的有效成分的純化，並進行使用了酶與抑制劑的複合物的X射線晶體分析或NMR等之高精度的結構分析，藉此分析抑制劑是與基質鍵結部位鍵結，還是與和其不同的抑制劑鍵結部位鍵結，或者是與這兩個部位鍵結，並確定抑制方式。 　　[0053] [比較試驗例3] 　　用緩衝液稀釋上述α-澱粉酶活性測定試驗中所使用之基質試液(原液)而製備了濃度不同之基質試液稀釋液。具體而言，將基質試液原液的濃度設為相對濃度1而分別製備了相對濃度0.5及相對濃度0.25的基質試液稀釋液。又，實施例1中所得到之萃取液小於3kDa的組分粉末以成為80、40、20、10、5mg/mL之方式溶解於緩衝液中，並按濃度製備了萃取液小於3kDa的組分粉末的試料溶液。與前述α-澱粉酶抑制效果測定試驗的情況相同，將預先於37℃下進行了保溫之各濃度的試料溶液75μL和緩衝液33.5μL於微孔板的孔內進行混合，對其添加0.5μg/mL的α-澱粉酶溶液5μL，並於37℃下進行了5分鐘的保溫。接著，藉由添加預先於37℃下進行了保溫之各濃度的基質試液36.5μL而作為反應液開始進行反應，並使用酶標儀以1分鐘的間隔經15分鐘測定了37℃下的405nm的吸光度(A₄₀₅ )。此外，將各測定各進行了3次。 　　[0054] 關於各測定結果，藉由上述方法進行分析，按基質試液的相對濃度，將萃取液小於3kDa的組分粉末的最終濃度標繪於橫軸(x軸)，將反應速度的倒數標繪於縱軸(y軸)之迪克遜曲線(Dixon plot)(圖8)。從各曲線得到基於最小二乘法的回歸直線，並藉由該回歸方程，分別求出了基質濃度不同之回歸直線的交點。迪克遜曲線中，改變了該交點的x坐標的符號之值表示抑制物質常數(inhibitor constant)K_i (非專利文獻25、非專利文獻26)。從而，本發明的基於糖分解酶抑制劑的α-澱粉酶活性抑制的抑制物質常數(K_i )顯示為39±2mg/mL。在此，若假設萃取液小於3kDa的組分粉末中所含有之物質的平均分子量為3,000，則推定K_i 以莫耳濃度計為13mM。又，若假設該抑制物質的平均分子量為1,500，則推定K_i 為26mM。但是，認為萃取液小於3kDa的組分粉末中所含有之所有的物質並不都是抑制劑成分，因此認為實質上的K_i 係成為進一步小的值者。 　　[0055] ＜α-葡萄糖苷酶活性測定試驗＞ 　　關於α-葡萄糖苷酶活性，參閱非專利文獻27及非專利文獻28的方法，藉由α-葡萄糖苷酶，將基質對硝基苯基-α-D-吡喃葡萄糖苷(PNPG)水解成對硝基苯基(PNP)和D-葡萄糖(G)，並測定源自所產生之對硝基苯基之400nm的吸光度變化量來進行了評價。 　　[0056] 關於α-葡萄糖苷酶，將源自大鼠腸丙酮粉末大鼠者(產品編號I1630，SIGMA，批號：SLBN7104V)用作酶源。使0.5g的丙酮粉末懸浮於10mL的50mM磷酸鉀緩衝液(pH7.0)中，一邊將容器浸在冰水中一邊使用均質機(NS-51，Microtec Co., LTD.製，千葉縣船橋市)，以旋轉調整器的刻度20計粉碎了3分鐘。將粉碎液於4℃、15,000xg離心分離15分鐘而得到之上清液作為酶液。關於酶液的蛋白質量，藉由布拉德福德法以牛血清白蛋白(BSA)為標準進行了定量。所求出之酶液的蛋白質濃度為4.5mg/mL。 　　將基質對硝基苯α-D-吡喃葡萄糖苷(簡稱為PNPG；產品編號25032-91, Nacalai Tesque Inc.製，批號：M6E7187)於50mM磷酸鉀緩衝液(pH7.0)溶解成3mM者作為PNPG基質液。 　　[0057] 將酶液12.5μL與預先於37℃下進行了保溫之50mM磷酸鉀緩衝液(pH7.0)87.5μL添加到96微孔板，並於37℃下保溫5分鐘。對其添加預先於37℃下進行了保溫之PNPG基質液50μL而開始進行反應，並使用酶標儀以1分鐘的間隔經15分鐘測定了37℃下的400nm的吸光度(A₄₀₀ )。另一方面，作為對照試驗，替代酶液而添加不含有α-葡萄糖苷酶之50mM磷酸鉀緩衝液(pH7.0)並以同樣的方式進行測定，且將其作為空白。從添加了酶液時的測定值減去空白值而求出每一測定時間的400nm的吸光度變化(ΔA₄₀₀ )，確認該吸光度變化隨著反應時間而直線增加，並依該直線的斜率而求出了每1分鐘的吸光度變化量(ΔA₄₀₀ /min)。在此，生成物PNP的400nm中的分子吸光係數ε₄₀₀ 係18,300M^-1 cm^-1 (非專利文獻27)，並且，PNP的pK_a 係7.15，測定時的pH係7.0，因此依漢-哈二氏方程式(Henderson-Hasselbalch equation)(非專利文獻26)，測定條件下的ε₄₀₀ 成為7,585M^-1 cm^-1 。又，從所使用之微孔板的光路長以反應液量150μL計為0.458cm，從該等值和ΔA₄₀₀ /min求出每1分鐘所生成之PNP量，且將其作為反應速度亦即酶活性。此時，於不含有萃取液小於3kDa的組分粉末之條件下求出之α-葡萄糖苷酶活性為3.4×10^-6 M/min。 　　[0058] ＜α-葡萄糖苷酶抑制效果測定試驗＞ 　　圖9表示以各種濃度將於實施例1中得到之萃取液小於3kDa的組分粉末添加到反應液時觀察到之α-澱粉酶活性、生成物量(400nm的吸光度)的經時變化。圖例的EB表示酶的空白，DB表示酶及萃取液小於3kDa的組分粉末的雙空白。此外，EB以最終濃度成為10mg/mL的方式含有萃取液小於3kDa的組分粉末。 　　[0059] 關於萃取液小於3kDa的組分的α-澱粉酶抑制效果，具體而言藉由以下方法進行了測定。萃取液小於3kDa的組分的試料溶液以成為80、40、20、10、4、2 mg/mL之方式將於實施例1中得到之萃取液小於3kDa的組分粉末溶解於50mM磷酸鉀緩衝液(pH7.0)而進行了製備。於微孔板的孔中將預先於37℃下進行了保溫之各濃度的試料溶液75μL與pH7.0的50mM磷酸鉀緩衝液12.5μL進行混合，對其添加酶液12.5μL並於37℃進行了5分鐘的保溫。接著，藉由添加預先於37℃下進行了保溫之PNPG基質液50μL而作為反應液開始進行反應，使用酶標儀以1分鐘間隔經15分鐘測定了37℃下的400nm的吸光度(A₄₀₀ )。此外，將各測定各進行了3次。 　　[0060] 將測定結果示於圖9。得知隨著添加到反應液之萃取液小於3kDa的組分的濃度的增大，表示400nm的吸光度變化(ΔA₄₀₀ )之直線的斜率減少。 　　另一方面，不含有α-葡萄糖苷酶，且以最終濃度成為10mg/mL之方式含有萃取液小於3kDa的組分之條件的反應液中，未觀察到PNPG分解活性(圖9的EB)。從該等結果顯示，於實施例1中得到之萃取液小於3kDa的組分中含有依賴濃度而抑制α-葡萄糖苷酶活性之成分，並且該成分完全不具有α-葡萄糖苷酶樣活性。 　　進而，藉由上述方法對圖9的測定結果進行了分析。藉由α-葡萄糖苷酶從每1分鐘生成之PNP量求出之不含有萃取液小於3kDa的組分粉末時的酶反應速度(v_o )為3.4×10^-6 M/min。同樣地，計算添加某一濃度的萃取液小於3kDa的組分粉末時的酶反應速度(v_i )，將基於萃取液小於3kDa的組分粉末的抑制率(單位：%)定義為[1-(v_i /v_o )]×100，從而求出了存在各種濃度的萃取液小於3kDa的組分粉末時的抑制率。製作了將萃取液小於3kDa的組分粉末的最終濃度標繪於橫軸，將抑制率標繪於縱軸之圖10。藉由圖10可知，增加添加到反應液的萃取液小於3kDa的組分粉末的最終濃度時，抑制率雖增加但顯示飽和曲線行為。在此，將抑制50%的α-葡萄糖苷酶活性之萃取液小於3kDa的組分粉末的最終濃度設為IC₅₀ 時，IC₅₀ 為7.4mg/mL。通常，當酶與抑制劑鍵結而形成酶-抑制劑複合物時，其平衡常數相當於抑制物質常數K_i ，其值大致相當於IC₅₀ 。從而，能夠將萃取液小於3kDa的組分粉末相對於α-葡萄糖苷酶的抑制物質常數K_i 視為7.4mg/mL。當使用了濃度相同的萃取液小於3kDa的組分粉末時的α-澱粉酶活性抑制率為約10%。又，如上所述，萃取液小於3kDa的組分粉末相對於α-澱粉酶的抑制物質常數K_i 為39±2mg/mL。從以上內容顯示，與α-澱粉酶活性相比本發明的糖分解酶抑制劑對α-葡萄糖苷酶活性發揮高5.3倍的抑制效果。 　　[0061] 接著，對向於實施例1中得到之萃取液小於3kDa的組分粉末進一步進行了各種處理製程之實施例進行說明。 　　[0062] [實施例2] 　　製備了以成為80mg/mL的方式將於實施例1中得到之萃取液小於3kDa的組分粉末溶解於DIAMONDCOLOR•AMY-L DIRECT的緩衝液(KTAM-103，α-澱粉酶抑制試驗用)或50mM磷酸鉀緩衝液(pH7.0，α-葡萄糖苷酶抑制試驗用)中之試料溶液。使用-80℃的超低溫冷凍機(MDF-U384, SANYO Electric Co., LTD.製，大阪府守口市)對該溶液進行冷凍處理，保持一晚之後，將容器浸漬於自來水中而將其融化。將該溶液作為“冷凍融化處理溶液”(實施例2)。 　　[0063] [實施例3] 　　以成為80mg/mL的方式將於實施例1中得到之萃取液小於3kDa的組分粉末溶解在10mM的HCl(pH2.0)中。於室溫下將該溶液保持3小時之後，添加1M的NaOH而進行了中和。進而進行冷凍處理之後，使用冷凍乾燥機將其製成再度冷凍乾燥處理粉末。以成為溶解在10mM的HCl時相同的體積的方式向該再冷凍乾燥處理粉末添加DIAMONDCOLOR•AMY-L DIRECT的緩衝液(KTAM-103、α-澱粉酶抑制試驗用)或50mM磷酸鉀緩衝液(pH7.0、α-葡萄糖苷酶抑制試驗用)而製備了試料溶液。將該溶液作為“酸處理溶液”(實施例3)。 　　[0064] [實施例4] 　　製備了以成為80mg/mL的方式將於實施例1中得到之萃取液小於3kDa的組分粉末溶解在DIAMONDCOLOR•AMY-L DIRECT的緩衝液(KTAM-103，α-澱粉酶抑制試驗用)或50mM磷酸鉀緩衝液(pH7.0，α-葡萄糖苷酶抑制試驗用)之試料溶液。使用鋁塊恆溫槽(DTU-1BN，Tietech Co., LTD.製，東京)，於100℃下對該溶液進行了10分鐘或30分鐘加熱處理。 　　在此，於溶解在α-葡萄糖苷酶抑制試驗用亦即50mM磷酸鉀緩衝液(pH7.0)中之80mg/mL的萃取液小於3kDa的組分粉末中，加熱處理後視覺辨認到沉澱物，因此以15,000xg離心分離5分鐘而去除沉澱物，並收集了上清液。 　　將如以上藉由加熱處理所收集之萃取液小於3kDa的組分粉末的溶液冷卻至室溫而成者作為“加熱處理溶液”(實施例4)。 　　[0065] [實施例5] 　　製備了以成為80mg/mL的方式將於實施例1中得到之萃取液小於3kDa的組分粉末溶解在DIAMONDCOLOR•AMY-L DIRECT的緩衝液(KTAM-103，α-澱粉酶抑制試驗用)或50mM磷酸鉀緩衝液(pH7.0，α-葡萄糖苷酶抑制試驗用)之試料溶液。將該溶液放入玻璃樣品瓶，並添加等量的己烷(特級試劑，含有正己烷97%，Nacalai Tesque Inc.製、京都市)後加蓋，使用旋渦混合器激烈攪拌了5分鐘。以10,000xg離心分離5分鐘而充分分層之後，去除己烷層，並小心地回收水層。將該水層的溶液作為“己烷萃取溶液”(實施例5)。此外，亦製備了對不含有萃取液小於3kDa的組分粉末之DIAMONDCOLOR•AMY-L DIRECT的緩衝液(KTAM-103、α-澱粉酶抑制試驗用)或50mM磷酸鉀緩衝液(pH7.0、α-葡萄糖苷酶抑制試驗用)，添加等量的己烷並進行了相同的萃取操作者，將該等分別作為對照組。 　　[0066] [針對實施例2～5的α-澱粉酶抑制效果測定試驗] 　　使用於上述實施例2～5中得到之各試料溶液，並藉由與前述α-澱粉酶抑制效果測定試驗相同的方法進行了測定。將預先於37℃下進行了保溫之各試料溶液75μL與緩衝液33.5μL於微孔板的孔內進行混合，對其添加0.5μg/mL的α-澱粉酶溶液5μL，且於37℃下保溫了5分鐘。接著，藉由添加預先於37℃下進行了保溫之基質試液36.5μL而作為反應液開始進行反應，使用酶標儀以1分鐘間隔經15分鐘測定了37℃下的405nm的吸光度。此外，將各測定各進行了3次。藉由上述方法分析結果，且分別計算了對未含有萃取液小於3kDa的組分粉末時的α-澱粉酶活使用了各試料溶液時的抑制率(表2)。此外，為了進行比較，將實施例1的以最終濃度成為40mg/mL的方式添加了萃取液小於3kDa的組分粉末時之α-澱粉酶活性抑制率亦組合示於表2中。 　　[0067]

[0068] 首先，關於實施例2的冷凍融化處理溶液進行詳細說明。與未添加萃取液小於3kDa的組分粉末之情況相比，使用了於-80℃下進行冷凍，保持一晚之後融化之冷凍融化處理溶液之情況下，亦抑制了35%的α-澱粉酶活性。這表示如下內容：與使用了未處理的萃取液小於3kDa的組分粉末時效果相同，因此顯示α-澱粉酶活性抑制效果之成分對實施例2的冷凍融化處理具有穩定性及耐冷凍融化性。又，由此能夠期待使不耐低溫之成分改質而去除之效果或能夠防止微生物的繁殖並長期保存之可能性。 　　[0069] 接著，關於實施例3的酸處理溶液，與未添加萃取液小於3kDa的組分粉末之情況相比，使用了以pH2保持3小時之後進行了中和之酸處理溶液之情況下，亦抑制了34%的α-澱粉酶活性。該結果顯示α-澱粉酶活性抑制效果之成分對實施例3的酸處理具有穩定性及耐酸性。又，由此能夠期待使不耐酸之成分改質而去除之效果或基於酸的殺菌效果。進而口服攝取時，亦能夠期待耐胃酸而到達小腸並抑制α-澱粉酶之可能性。 　　[0070] 進而，關於實施例4的加熱處理溶液，與未添加萃取液小於3kDa的組分粉末之情況相比，添加了於100℃下進行10分鐘及30分鐘加熱處理而成之加熱處理溶液之情況下，亦抑制了36%及40%的α-澱粉酶活性分別。這表示與添加了未實施加熱處理之萃取液小於3kDa的組分粉末時相同或其以上的抑制效果。從以上內容顯示，萃取液小於3kDa的組分粉末中的α-澱粉酶活性抑制劑成分相對於實施例4的加熱處理具有高穩定性及耐熱性。又，由此可期待藉由對萃取液小於3kDa的組分粉末進行加熱處理而使不耐熱之成分失活或者改質而去除之效果，進而能夠期待殺菌效果或殺滅病毒效果。 　　[0071] 又，關於實施例5的己烷萃取溶液，與未添加萃取液小於3kDa的組分粉末之情況相比，使用了進行將己烷添加到萃取液小於3kDa的組分粉末而攪拌、分層之萃取操作之後得到之水層之情況下，亦抑制了35%的α-澱粉酶活性。另一方面，使用水層對α-澱粉酶抑制進行了研究，該水層中替代萃取液小於3kDa的組分粉末而僅使用緩衝液進行了相同的萃取操作。進行萃取操作而得到之水層的抑制活性中未發現與未進行萃取操作之對照組(亦即未處理的緩衝液)的抑制活性之差。該等結果表示，顯示α-澱粉酶活性抑制效果之成分並不是源自藉由己烷萃取操作而有可能轉移到水層中之己烷者。又表示，萃取液小於3kDa的組分粉末中的α-澱粉酶抑制物質對己烷穩定，不轉移到己烷層或界面而保持於水層中，且為親水性高之物質。從以上結果能夠期待藉由使用己烷等有機溶劑之萃取操作，去除轉移到有機層之成分及轉移到界面之成分之效果。己烷為應用於生物物質的萃取製程中之具有最高的非極性和高疏水性之有機溶劑，通常使用高於己烷的非極性或疏水性之溶劑是不尋常的。從而，如上所述，該萃取液小於3kDa的組分粉末不會於己烷中削弱α-澱粉酶活性抑制效果或使該效果消失，因此能夠判斷除了己烷以外的有機溶劑中，亦係具有充分的穩定性和耐有機溶劑性者。 　　[0072] [針對實施例2～5的α-葡萄糖苷酶抑制效果測定試驗] 　　進而，使用於上述實施例2～5中得到之各試料溶液，並以與前述α-葡萄糖苷酶抑制效果測定試驗相同的方法進行了測定。將預先於37℃下進行了保溫之各濃度的試料溶液75μL與50mM磷酸鉀緩衝液(pH7.0)12.5μL於微孔板的孔內進行混合，對其添加酶液12.5μL，於37℃下保溫了5分鐘。接著，藉由添加預先於37℃下進行了保溫之PNPG基質液50μL而作為反應液開始進行反應，使用酶標儀以1分鐘間隔經15分鐘測定了37℃下的400nm的吸光度。此外，將各測定各進行了3次。藉由上述方法對結果進行分析，分別計算出對未含有萃取液小於3kDa的組分粉末時的α-葡萄糖苷酶活性使用了各試料溶液時的抑制率(表3)。此外，為了進行比較，將添加了實施例1的最終濃度40mg/mL的萃取液小於3kDa的組分粉末時之α-葡萄糖苷酶活性抑制率亦組合示於表3。 　　[0073]

[0074] 如表3所示，以最終濃度40mg/mL將實施例2的冷凍融化處理溶液或實施例3的酸處理溶液添加到反應液之情況下，α-葡萄糖苷酶活性抑制率亦與使用了實施例1的萃取液小於3kDa的組分粉末(未處理溶液)之情況相同。 　　從該等結果表示，顯示α-葡萄糖苷酶活性抑制效果之成分對實施例2的冷凍融化處理或實施例3的酸處理非常穩定，且具有高耐冷凍融化性、耐酸性。又，與使用了實施例1的萃取液小於3kDa的組分粉末(未處理溶液)之情況相比，將實施例4的加熱處理溶液添加到反應液之情況下，α-葡萄糖苷酶活性抑制率略微降低。進而，與使用了實施例1的萃取液小於3kDa的組分粉末(未處理溶液)之情況相比，將實施例5的己烷萃取溶液以最終濃度40mg/mL添加到反應液之情況下，亦發現α-葡萄糖苷酶活性抑制率略微降低。藉由對萃取液小於3kDa的組分粉末進行冷凍融化、酸性、加熱、己烷處理等前處理，與未進行該等之情況相比，亦觀察到抑制活性略微降低之例子，即使如此，該等前處理後的抑制效果亦充分大。從而，能夠判斷顯示α-葡萄糖苷酶活性抑制活性之成分為對實施例4的加熱處理或實施例5的己烷萃取處理具有高穩定性、耐熱性、耐有機溶劑性者。顯示α-葡萄糖苷酶活性抑制活性之成分中可觀察到之該等特性與顯示上述α-澱粉酶活性抑制效果之成分的特性相同，於保存性、加工性、應用性等方面亦可期待與上述相同的優異的效果。 　　[0075] 藉由以上結果，認為藉由對於實施例1中得到之萃取液小於3kDa的組分粉末，將冷凍融化處理、酸處理、加熱處理、有機溶劑處理單獨或組合複數種而執行，藉此能夠得到純度更高且具有α-澱粉酶活性抑制效果及α-葡萄糖苷酶活性抑制效果之糖分解酶抑制劑。 　　如此得到之源自蚯蚓的糖分解酶抑制劑能夠使用於醫藥品、功能性食品、添加劑、寵物食品等中。 　　[0076] [其他實施形態] 　　(1)上述實施形態中，成為原料之蚯蚓使用了紅蚯蚓(Eisenia fetida)，但亦可以使用以醫藥品、健康食品用途使用之其他種類的蚯蚓。 　　[0077] (2)上述實施形態中，使用了對蚯蚓的粉碎液進行靜水壓式高壓處理，對經離心分離之上清液進行冷凍乾燥而製備之蚯蚓乾燥粉末，但亦可以使用藉由其他方法製備之蚯蚓乾燥粉末。 　　[0078] (3)關於上述實施形態中的糖分解酶抑制劑的製造方法中所含有之各製程的處理條件能夠適當進行變更。 　　[0079] (4)上述實施形態中，例示了將蚯蚓乾燥粉末作為原料而製造糖分解酶抑制劑之情況，但亦可以將蚯蚓或其粉碎物作為原料而製造糖分解酶抑制劑。The present invention is described in detail. The saccharolytic enzyme inhibitor of the present invention is obtained by the following process, that is, an extraction process, in which water is added to dry earthworm powder prepared using a pulverized liquid obtained by crushing earthworms to be raw materials to obtain an earthworm extract; and a separation process , the earthworm extract obtained in the extraction process is subjected to ultrafiltration to obtain components with a molecular mass less than 3kDa, that is, components with a molecular weight less than 3kDa. The separation process for obtaining the fractions of less than 3 kDa in the extract is not limited to ultrafiltration, and known separation methods such as centrifugal separation and gel filtration can also be used. In the Examples to be described later, it was confirmed that the saccharolytic enzyme inhibitor containing the fraction of the extract as a main component having less than 3 kDa has the effect of inhibiting the α-amylase activity and the α-glucosidase activity. In the manufacturing method of the saccharolytic enzyme inhibitor of the present invention, the fraction of the extract obtained from the above-mentioned earthworm extract after the molecular mass separation is less than 3kDa is subjected to freeze-drying treatment to set the fraction of the extract less than 3kDa The freeze-dried powder is the component powder whose extract is less than 3kDa. By this process, the fraction of the extract with less than 3kDa can be concentrated, and a high concentration of saccharolytic enzyme inhibitor can be obtained. In addition, by making the extract liquid less than 3 kDa as a component powder, storage stability and workability can be improved. In addition, at least one treatment selected from the group consisting of freeze-thaw treatment, acid treatment, heat treatment, and organic solvent treatment can be performed on the fraction of the extracted liquid after the molecular mass separation is less than 3 kDa. The freeze-thaw treatment can be performed at a temperature of -80°C (extremely low temperature freezer) and -196°C (liquid nitrogen temperature). Regarding the acid treatment, the treatment can be performed using an acid up to pH 2. The heat treatment can be carried out under normal pressure and at a temperature of 100°C. Regarding the organic solvent treatment, an organic solvent such as hexane that can be separated into a water layer and an organic solvent layer can be used. These treatment processes can be carried out in combination regardless of their order, and the treated solution can be subjected to normal separation operations such as centrifugation or filtration, and a high-purity saccharolytic enzyme inhibitor can be obtained by removing solid matter . About the saccharolytic enzyme inhibitor of the present invention, it can be used as a medicine, health care product for the purpose of inhibiting enzymes, especially α-amylase activity or α-glucosidase activity, by setting it as a content containing it. food, supplements or pet food, etc. Embodiments of the present invention will be described below. However, the present invention is not limited to the following examples. <Method for producing dry earthworm powder> The dry earthworm powder used in the examples of the present invention was produced in accordance with the method of Patent Document 12. That is, after washing 30 kg of the living body of the cultured red earthworm (Eisenia fetida) as a raw material with tap water, it was immersed in a 5% (w/v) sodium bicarbonate aqueous solution for 1 hour, and the earthworm spit out the body cavity fluid to remove the body cavity fluid. The earthworms that have been washed again were ground, filled in plastic bags and sealed, and then treated with a hydrostatic pressure type high-pressure treatment device (SHP-100-50A, manufactured by SHINADA CO., LTD., Nagaoka City, Niigata Prefecture). Treatment was performed at 100 MPa and 60°C for 16 hours. Using a roller pump (RP-LVS, manufactured by FURUE SCIENCE CO., LTD., Shinjuku-ku, Tokyo) and a cylindrical ultracentrifuge (ASM160AP, manufactured by TOMOE ENGINEERING CO., LTD., Shinagawa-ku, Tokyo), The treatment was continuously centrifuged at 17,000 rpm. The obtained centrifugal supernatant was subjected to freeze-drying treatment using a vacuum freeze dryer (TF20-80TNNN, manufactured by TAKARA. Co. Ltd, Itabashi-ku, Tokyo), and then pulverized into powder. The powder was dried at 80° C. for 6 hours as the final dry earthworm powder. The recovery amount at this time was 4.0 kg, and the yield was 13% with respect to the initial live earthworm weight of 30 kg. [Example 1] 5 g of the dry earthworm powder obtained in the above preparation process was weighed in a beaker, and distilled water was added to make it 50 mL, thereby obtaining a 10% (w/v) suspension. After the suspension was stirred for 10 minutes using a stirrer, the precipitate was removed by centrifugation at 16,100×g for 10 minutes. This supernatant was used as earthworm extract. Next, the earthworm extract was subjected to ultrafiltration using Vivaspin 20 (3 kDa MWCO, manufactured by GE Healthcare, Little Chalfont, UK) to obtain a filtrate of low molecular weight components having a molecular mass of less than 3 kDa. The filtrate was used as the fraction with less than 3 kDa in the extract, and the powder lyophilized using a freeze dryer (FDU-1200, manufactured by EYELA, Tokyo) was used as the fraction with less than 3 kDa in the extract. At this time, 1.7 g of powder of the fraction of the extract of less than 3 kDa was obtained from 24 mL of the fraction of the extract of less than 3 kDa. Thus, the yield of fractional powders of less than 3 kDa in the extract from the initial 5 g of dry earthworm powder was 34%. That is, 1.3 kg of the fractional powder having an extract liquid of less than 3 kDa was obtained from an initial live weight of 30 kg of earthworms, and the yield was 4.4%. <Alpha-amylase activity assay test> Regarding the alpha-amylase activity, DIAMONDCOLOR.AMY-L DIRECT (KTAM-103 (buffer) and KTAM-113 (substrate test solution), TOYOBO CO., LTD., Osaka City, Osaka Prefecture) was measured. Hereinafter, the measurement principle of this product will be described in accordance with the manufacturer's instructions. That is, the α-amylase in the sample hydrolyzes the synthetic substrate α-2-chloro-4-nitrophenyl-galactosylmaltoside (GalG2CNP) to generate galactosylmaltoside (GalG2) and 2-chloro-4- Nitrophenyl (CNP). The α-amylase activity can be determined by measuring the increase in yellow absorbance per unit time by CNP at a wavelength of 405 nm. In addition, the molar absorption coefficient (ε ₄₀₅ ) at 405 nm of the CNP was 13,400 M ⁻¹ cm ⁻¹ . Regarding α-amylase, 20mM Tris-HCl (pH7.0) buffer was added to the powder of porcine pancreas-derived α-amylase (product number A3176, manufactured by SIGMA, lot number: SLBM2655V) to make the powder concentration After 10 mg/mL, inversion stirring was performed for 5 minutes, centrifugation was performed at 16,100×g for 10 minutes, and insoluble components were removed. For this supernatant, the protein concentration was quantified by the Bradford method using bovine serum albumin (BSA) as a standard. The protein concentration of this supernatant was 226 μg/mL. This supernatant was diluted to 0.5 μg/mL using the same buffer and used as a stock solution of α-amylase. 5 μL of the α-amylase solution of 0.5 μg/mL prepared by the above method and 108.5 μL of buffer solution previously incubated at 37 ° C were added to 96 microwell plates, and were carried out at 37 ° C for 5 minutes of insulation. The enzymatic reaction was started by adding 36.5 µL of the substrate solution previously incubated at 37°C, and the enzyme reaction was carried out for 1 minute using a microplate reader (xMark, manufactured by Bio-Rad Laboratories, Hercules, California, USA). The absorbance at 405 nm at 37°C was measured at intervals of 15 minutes. On the other hand, as a control experiment, a buffer solution not containing α-amylase was added in place of the α-amylase solution, and the measurement was performed in the same manner, and was used as a blank. For each measurement time, the blank value was subtracted from the measurement value when α-amylase was added, thereby confirming that the change in absorbance at 405 nm (ΔA ₄₀₅ ) at each measurement time increased linearly as the measurement time increased. From the slope of the straight line, the change in absorbance per minute (ΔA ₄₀₅ /min) was determined. The optical path length of the microplate used was 0.458 cm in the amount of 150 μL of the reaction solution. In addition, the molecular absorption coefficient ε ₄₀₅ of the product CNP was 13,400 M ^-1 cm ^-1 . From these equivalent values and ΔA ₄₀₅ /min, the amount of CNP produced per minute was determined, and this was taken as the reaction rate (ie, the enzyme activity). At this time, the α-amylase activity determined under the condition that the extract did not contain components less than 3 kDa was 6.5×10 ^-6 M/min. <Measurement test of α-amylase inhibitory effect> Fig. 1 is a diagram showing that the extract obtained in Example 1 was prepared in various concentrations to form component powders less than 3 kDa, and added to the α-amylase solution. Graph of the change with time of the observed product amount (absorbance at 405 nm). The α-amylase activity in each concentration was obtained from the slope of the straight line representing the time-dependent change in the production of the product. EB in the legend represents the blank of the enzyme, and DB represents the double blank of the enzyme and the component powder whose extract is less than 3 kDa. In addition, EB contains fractional powders with a final concentration of 10 mg/mL of less than 3 kDa in the extract. [0035] The α-amylase inhibitory effect of the fraction less than 3 kDa in the extract was specifically measured by the following method. The sample solution of the fraction whose extract was less than 3 kDa was prepared by dissolving the powder of the fraction whose extract was less than 3 kDa obtained in Example 1 in a buffer so as to be 80, 40, 20, 10, and 5 mg/mL. 75 μL of each concentration of the sample solution previously incubated at 37°C was mixed with 33.5 μL of buffer solution in the wells of the microplate, and 5 μL of 0.5 μg/mL α-amylase solution was added to it, and the reaction was carried out at 37°C. Keep warm for 5 minutes. Next, the reaction was started by adding 36.5 μL of the substrate test solution previously incubated at 37° C. as a reaction solution, and the absorbance at 405 nm at 37° C. was measured at 1-minute intervals for 15 minutes using a microplate reader. In addition, each measurement was performed three times. Measurement result is shown in Fig. 1. As a result of analyzing the measurement results of Fig. 1 by the above method, when the extract does not contain any component powder less than 3 kDa, the amount of CNP produced by α-amylase per minute, that is, the reaction rate, is calculated. is 6.5×10 ^-6 M/min. Furthermore, the α-amylase activity when each sample solution was used was relatively shown by making this value a relative value of 1 ( FIG. 2 ). According to FIG. 2 , it can be seen that the α-amylase activity is inhibited depending on the concentration of the component powder of which the extract solution obtained in Example 1 is less than 3 kDa is added to the enzyme reaction system. The α-amylase activity was reduced to 0.65 (65%) in relative activity when the fractional powder with a final concentration of 40 mg/mL extract less than 3 kDa was added. On the other hand, GalG2CNP-decomposing activity was not observed in the reaction solution under the condition that α-amylase was not contained, but the final concentration of 10 mg/mL of extract was less than 3 kDa in the reaction solution (EB in Fig. 1 ) . From these results, it is shown that the fraction of less than 3 kDa in the extract obtained in Example 1 contains a component that inhibits α-amylase activity in a concentration-dependent manner, and further, this component does not contain α-amylase-like activity at all. [Comparative Test Example 1] The preparation of the α-amylase stock solution in the above-mentioned α-amylase activity measurement test was changed based on the dilution of the buffer solution of the quantified α-amylase solution by the Bradford method. α-amylase stock solutions of 0.25 μg/mL and 0.1 μg/mL were prepared at the same rate. Using these α-amylase solutions at different concentrations, the α-amylase inhibitory effect based on fractions of less than 3 kDa in the extract was tested in the same manner as the aforementioned α-amylase inhibitory effect measurement test. The measurement was performed 3 times each. [0038] The measurement results were analyzed in the same manner as described above. The amount of CNP produced by α-amylase per minute, that is, the reaction rate, was measured when the extract did not contain component powder less than 3 kDa, and the concentration of the α-amylase stock solution was 0.25 μg/mL. 3.1×10 ^-6 M/min, and 1.3×10 ^-6 M/min at 0.1 μg/mL. Considering the results obtained when the above-mentioned 0.5 μg/mL α-amylase stock solution was used, it was found that the enzyme reaction rate changed in proportion to the enzyme concentration. This situation indicates that the enzymatic reaction follows Michaelis-Menten-type kinetics. Here, in the same manner as in FIG. 2 , at each concentration of the α-amylase stock solution, the reaction rate when the fraction powder less than 3 kDa in the extract is not contained is set as a relative value of 1, respectively, and the addition of The reaction speed of each concentration of extract liquid is less than 3kDa component powder. Furthermore, the calculated relative value was calculated by (1-relative value)×100, and this was taken as the inhibition rate (unit: %). Fig. 3 shows that the extract added to the reaction solution was less than The relationship between the final concentration of the 3 kDa component powder and the inhibition rate. [0039] As shown in FIG. 3 , the α-amylase activity inhibition rate became greater depending on the final concentration of the component powders of less than 3 kDa in the extract added to the reaction solution. Different enzyme concentrations will cause a slight deviation in the inhibition rate in the range where the final concentration of the component powder with the extract less than 3kDa is 10mg/mL or less, but when the final concentration of the component powder with the extract less than 3kDa is 20mg/mL or more No effect on inhibition rate. In addition, when the fractional powder of which the extract solution was less than 3 kDa was added at a final concentration of 40 mg/mL, the inhibition rate was 35% at any enzyme concentration, indicating that the saccharolytic enzyme inhibitor of the present invention did not inhibit sharply at a low dosage. α-amylase activity, but the possibility of stably acting and exerting an effect. [Comparative Test Example 2] The matrix test solution used in the above-mentioned α-amylase activity measurement test was diluted with a buffer solution to prepare the relative concentrations 0.75 and 0.5 when the stock solution of the matrix test solution was used as the relative concentration 1, respectively. , 0.25, 0.1, 0.05 of the matrix test solution dilution. The α-amylase activity measurement test was carried out by the same method as above using the substrate test solutions having different concentrations. In addition, the α-amylase stock solution of 0.5 microgram/mL was used as an enzyme liquid. In addition, using the matrix test solutions with different concentrations, and in the same manner as the aforementioned α-amylase inhibitory effect measurement test, the group in which the extract obtained in Example 1 was less than 3 kDa with the final concentration of 40 mg/mL was used. The α-amylase activity when the powder was added to the reaction solution was measured. Each measurement was performed three times. Measurement result is shown in Fig. 4. The graph (Fig. 4(a)) shows the result when the component powder whose extract solution is less than 3 kDa is not added, and the graph (Fig. 4(b)) shows the component powder whose extract solution is less than 3 kDa so that the final concentration is 40 mg/mL. Result when added to the reaction solution. The concentration of the matrix test solution is expressed as relative concentration. Here, regarding the relative concentration, the stock solution of the matrix test solution product is assumed to be a relative value of 1. Alpha-amylase activity can be calculated from the slope of the line observed at the respective substrate concentrations. In any case, the amount of change in absorbance at 405 nm (ΔA ₄₀₅ /min), that is, the enzyme activity, increases as the substrate concentration in the reaction solution increases. In addition, if the comparison is made at the same substrate concentration, ΔA ₄₀₅ /min is reduced and α-amylase activity is inhibited by adding the component powder of which the extract is less than 3 kDa at any substrate concentration. Here, the α-amylase activity inhibition rate was obtained when the fraction powder having an extract solution of less than 3 kDa was added so that the final concentration of the extract was 40 mg/mL at each relative substrate concentration by the above-mentioned analysis. (Table 1). [0042]

As shown in Table 1, under the condition of using the stock solution of the substrate test solution, when the final concentration becomes 40mg/mL, when the component powder of the extract solution less than 3kDa is added, the α-amylase activity inhibition rate is about 35%, but when the relative concentration was set to 0.50 by diluting the matrix test solution with a buffer, the α-amylase activity inhibition rate was about 38%. Furthermore, the inhibition rate was about 43% under the condition that the relative concentration of the matrix test solution was 0.1, and the inhibition rate was about 49% under the condition that the relative concentration was 0.05. When the substrate test solution was further diluted and the relative concentration was set to 0.03, the absorbance at 405 nm accompanying the enzymatic reaction was significantly small, and it was difficult to track the change. As shown above, the concentration of α-amylase contained in the enzyme reaction solution and the powder concentration of components less than 3 kDa in the extract are constant, but the inhibition rate increases as the substrate concentration decreases. Also, conversely, the inhibition rate decreased with increasing substrate concentration. From this, it is inferred that the saccharolytic enzyme inhibitor of the present invention has a type of inhibition mode that inhibits α-amylase activity by competing with the substrate for binding to the enzyme active site. Furthermore, the property of inhibiting α-amylase by the saccharolytic enzyme inhibitor of the present invention was analyzed using the enzyme reaction rate theory (Non-Patent Document 25, Non-Patent Document 26). The relative concentration of the matrix test solution was plotted on the horizontal axis, and the reaction velocity (v) obtained from each data in FIG. 4 was plotted on the vertical axis to create a Mie curve ( FIG. 5 ). Furthermore, a Lineweaver-Burk curve (Fig. 6) and plot the relative concentration of the matrix test solution on the horizontal axis, and plot the value obtained by dividing the relative concentration of the matrix test solution by the reaction speed on the vertical axis. The Hanes-Woolf curve ( Figure 7). An approximate straight line based on the least squares method was obtained from the respective curves, whereby the rate theory parameters of the enzyme reaction of the Michaelis constant K _m (unit: M) and the maximum velocity V _max (unit: M/s) were obtained. In addition, molecular activity (transition number) k _cat (unit: 1/s) is represented by V _max /[E] ₀ . Here, [E] ₀ (unit: M) is the initial enzyme concentration in the reaction solution. The specificity constant k _cat /K _m (unit: 1/(Ms)) is calculated from k _cat and K _m . In addition, although the molar concentration of GalG2CNP contained in the matrix test solution is not clear, the molar concentration of GalG2CNP in the matrix test solution stock solution is expressed as a constant [S _S ] on the basis of these parameters. [0045] The inhibitor-based enzyme inhibition is roughly classified into a competitive type and a mixed type (Non-Patent Document 26). Competitive inhibition is caused by the binding of the inhibitor to the site on the enzyme that binds to the substrate (substrate binding site), and the substrate and the inhibitor compete with each other for the substrate binding site. In the presence of the inhibitor, the binding of the enzyme to the substrate is disturbed by the inhibitor, so the affinity between the enzyme and the substrate decreases (that is, the _Km increases), and the formation of the enzyme-substrate complex (ES complex) is inhibited . However, when the substrate concentration is sufficiently large, the interference of the inhibitor on the bond between the enzyme and the substrate can be substantially ignored, and the obtained reaction rate (ie, the maximum rate V _max ) becomes the maximum obtained in the absence of the inhibitor The same value as the speed V _max . [0046] In the mixed-type inhibition, the inhibitor binds to a site (inhibitor binding site) different from the substrate binding site, whereby the substrate binding or enzyme reaction rate constant to the substrate binding site is affected. In a special case of mixed-type inhibition, ie non-competitive inhibition, it is believed that the interaction of the substrate and the inhibitor on the enzyme is independent of each other and is inactivated in the enzyme-inhibitor complex (EI). In this case, the activity can also be brought about by the free enzyme (E). Therefore, in the presence of the inhibitor, the maximum velocity V _max was decreased, but no change in K _m was observed compared with the absence of the inhibitor. In general mixed-type inhibition, the interaction between the substrate and the inhibitor on the enzyme interacts, so that in the presence of the inhibitor, the affinity of the enzyme and the substrate and the enzyme activity can be found in the presence of the inhibitor compared to the absence of the inhibitor. aspects have changed. The enzyme inhibitor is not only a competitive inhibition that binds only to the substrate binding site of the enzyme or a mixed-type inhibition that binds only to the inhibitor binding site, but also can be considered as a combination of these two binding sites. Happening. In this case, it is considered that there is a possibility that, depending on the degree of affinity of the inhibitor for the substrate-binding site or the inhibitor-binding site, competitive inhibition (that is, _Vmax and _kcat do not change in the presence of the inhibitor, but _Km increase) and mixed-type inhibition ( _Vmax and _kcat decrease, _Km increase in the presence of inhibitor). In the Michaelis curve of Fig. 5, each curve represents a saturation curve, and it is known that when the final concentration becomes 40mg/mL, when the component powder of the extract less than 3kDa is added, the reaction rate (v) is also That is, the enzyme activity is reduced. As long as the method of inhibiting α-amylase activity by the saccharolytic enzyme inhibitor of the present invention is a competitive type, the maximum reaction rate _Vmax is considered to be the same under addition and non-addition conditions of the fraction powder having an extract of less than 3 kDa. However, the reaction rate did not reach the maximum value even under the condition of giving the stock solution of the matrix test solution with the maximum matrix concentration. This suggests that in order to achieve the maximum reaction rate, a further high concentration of substrate is required, but it is difficult to clearly determine whether it is a competitive inhibition or a mixed inhibition, at least from Figure 5 alone. [0049] Therefore, the Rivever-Burke curve was made, and the enzymatic reaction rate theory parameters were obtained (Fig. 6). The maximum reaction rate becomes approximately the same value under any condition that the final concentration of the component powder of which the extract solution is less than 3 kDa is 0 mg/mL and 40 mg/mL. That is, it shows that the maximum velocity _Vmax imparted (predicted) under the condition that the substrate concentration is sufficiently excessive does not change regardless of the presence or absence of addition of the component powder of which the extract solution is less than 3 kDa. On the other hand, by adding the component powder (final concentration: 40 mg/mL) of less than 3 kDa in the extract, the K _m value was approximately increased by a factor of 2, indicating that the affinity of α-amylase to the substrate was significantly reduced. These results strongly suggest that the α-amylase activity inhibition mode based on the saccharolytic enzyme inhibitor of the present invention is likely to be a competitive type. Utilize identical data to make Hannis-Wolff curve, and obtained the rate theory parameter of enzyme reaction (Fig. 7). Both the maximum reaction rate and the affinity of the alpha-amylase to the substrate were shown to be reduced by adding fractional powders of less than 3 kDa in the extract at a final concentration of 40 mg/mL. That is, the value of K _m is approximately increased by a factor of 1.5, and the value of V _max (and k _cat ) is decreased by 21%. These results indicate that the inhibition of alpha-amylase activity based on the enzyme inhibitor in the fraction powder of the extract is less than 3 kDa is a mixed type of inhibition. However, the effect on V _max (and k _cat ) value is small compared to the effect on K _m value, but the possibility of competitive inhibition cannot be ignored. As the reason for the difference between the results of Fig. 6 and Fig. 7, it is considered as follows, that is, in the case of competitive inhibition, if the substrate concentration is increased relative to the inhibitor concentration, the inhibitory effect will be reduced, but Rewei Fu- In contrast to the characteristic of the Burke curve that weights are applied to the measurement data in the region where the substrate concentration is low, the Hannes-Wulff curve has the property of weighting the measurement results in the region where the substrate concentration is high. It is possible that features of mixed inhibition that cannot be read from the curves of FIG. 6 are reflected in the curves of FIG. 7 . [0052] Based on the above, the following questions may be raised as the inhibition mechanism of the α-amylase activity by the saccharolytic enzyme inhibitor of the present invention. First, whether the observed inhibition was caused by a single inhibitory substance or by more than two inhibitory substances. If it is assumed that the inhibition is caused by a single substance, whether the substance binds to the substrate binding site of α-amylase to cause competitive inhibition, binds to the inhibitor binding site and causes mixed inhibition, or combines with both Each binding site is bound to show competitive and mixed inhibition. Furthermore, the inhibition is not caused by a single inhibitor, but is caused by two or more inhibitors with different inhibitory types, and these are bound to the substrate-binding site of the enzyme or the inhibitor-binding site, and the effect may be possible. This results in a decrease in the binding affinity of the substrate (ie, an increase in _Km ) or a decrease in the molecular catalyst activity (ie, a decrease in _Vmax and _kcat ). In terms of the types of inhibitory substances, the discrimination of competitive inhibition or mixed inhibition, and the identification of enzyme substrate binding sites and inhibitor binding sites, the above-mentioned methods of enzyme reaction rate theory are limited. In the future, it is necessary to advance the purification of the active ingredient of the inhibitor, and to perform high-precision structural analysis such as X-ray crystallography or NMR using the complex of the enzyme and the inhibitor to analyze whether the inhibitor is bonded to the substrate bonding site. , or it is bound to a different inhibitor binding site than it, or it is bound to both sites, and the inhibition mode is determined. [Comparative Test Example 3] The substrate test solution (stock solution) used in the above-mentioned α-amylase activity measurement test was diluted with a buffer to prepare substrate test solution dilutions with different concentrations. Specifically, matrix test solution dilutions with a relative concentration of 0.5 and a relative concentration of 0.25 were prepared by setting the concentration of the matrix test solution stock solution as a relative concentration of 1. In addition, the powders of the components whose extracts were less than 3 kDa obtained in Example 1 were dissolved in the buffer solution in a manner of 80, 40, 20, 10, and 5 mg/mL, and the components whose extracts were less than 3 kDa were prepared according to the concentration. Powder sample solution. As in the case of the aforementioned α-amylase inhibitory effect measurement test, 75 μL of the sample solution of each concentration and 33.5 μL of the buffer solution previously incubated at 37° C. were mixed in the wells of the microplate, and 0.5 μg was added thereto. 5 μL/mL of α-amylase solution and incubated at 37°C for 5 minutes. Next, the reaction was started by adding 36.5 μL of the substrate test solution of each concentration previously incubated at 37° C. as a reaction solution, and the 405 nm at 37° C. was measured for 15 minutes at 1-minute intervals using a microplate reader. Absorbance (A ₄₀₅ ). In addition, each measurement was performed three times. About each measurement result, analyze by the above-mentioned method, according to the relative concentration of the matrix test solution, the final concentration of the component powder of which the extract is less than 3kDa is plotted on the horizontal axis (x axis), and the reciprocal of the reaction rate is plotted. Dixon plot (Figure 8) plotted on the vertical axis (y-axis). A regression line based on the least squares method was obtained from each curve, and the intersection points of the regression lines with different substrate concentrations were obtained from the regression equation. In the Dixon curve, the value in which the sign of the x-coordinate of the intersection is changed represents the inhibitor constant K _i (Non-Patent Document 25, Non-Patent Document 26). Thus, the inhibitory substance constant (K _i ) of α-amylase activity inhibition by the saccharolytic enzyme inhibitor of the present invention was shown to be 39±2 mg/mL. Here, assuming that the average molecular weight of the substance contained in the component powder of which the extract solution is less than 3 kDa is 3,000, K _i is estimated to be 13 mM in molar concentration. Furthermore, assuming that the average molecular weight of the inhibitory substance is 1,500, K _i is estimated to be 26 mM. However, it is considered that not all substances contained in the fraction powder having an extract liquid of less than 3 kDa are inhibitor components, and therefore it is considered that the substantial K _i is a further smaller value. <Alpha-glucosidase activity measurement test> Regarding the alpha-glucosidase activity, refer to the methods of Non-Patent Document 27 and Non-Patent Document 28, by α-glucosidase, the substrate p-nitrophenyl- α-D-glucopyranoside (PNPG) was hydrolyzed into p-nitrophenyl (PNP) and D-glucose (G), and the amount of change in absorbance at 400 nm derived from the generated p-nitrophenyl was measured. Evaluation. Regarding α-glucosidase, the one derived from rat intestinal acetone powder rat (product number I1630, SIGMA, lot number: SLBN7104V) was used as the enzyme source. 0.5 g of acetone powder was suspended in 10 mL of 50 mM potassium phosphate buffer (pH 7.0), and a homogenizer (NS-51, manufactured by Microtec Co., LTD., Funabashi City, Chiba Prefecture) was used while immersing the container in ice water. ), pulverized for 3 minutes on the scale of 20 on the rotary adjuster. The pulverized liquid was centrifuged at 4° C. and 15,000×g for 15 minutes, and the supernatant liquid was obtained as an enzyme liquid. The protein amount of the enzyme solution was quantified by the Bradford method using bovine serum albumin (BSA) as a standard. The protein concentration of the obtained enzyme solution was 4.5 mg/mL. The substrate p-nitrophenyl α-D-glucopyranoside (abbreviated as PNPG; product number 25032-91, manufactured by Nacalai Tesque Inc., batch number: M6E7187) was dissolved in 50 mM potassium phosphate buffer (pH 7.0) to 3 mM as PNPG matrix fluid. [0057] 12.5 μL of the enzyme solution and 87.5 μL of 50 mM potassium phosphate buffer (pH 7.0) previously incubated at 37° C. were added to a 96-well plate, and incubated at 37° C. for 5 minutes. The reaction was started by adding 50 µL of the PNPG substrate solution previously incubated at 37°C, and the absorbance (A ₄₀₀ ) at 400 nm at 37° C. was measured at 1-minute intervals for 15 minutes using a microplate reader. On the other hand, as a control test, a 50 mM potassium phosphate buffer (pH 7.0) not containing α-glucosidase was added in place of the enzyme solution, and the measurement was performed in the same manner, and this was used as a blank. The change in absorbance at 400 nm per measurement time (ΔA ₄₀₀ ) was obtained by subtracting the blank value from the measured value when the enzyme solution was added. It was confirmed that the change in absorbance increased linearly with the reaction time, and was obtained from the slope of the straight line. The change in absorbance per minute (ΔA ₄₀₀ /min) is shown. Here, the molecular absorption coefficient ε ₄₀₀ at 400 nm of the product PNP is 18,300 M ^-1 cm ^-1 (Non-Patent Document 27), and the pK _a of PNP is 7.15, and the pH at the time of measurement is 7.0, so according to Han- According to the Henderson-Hasselbalch equation (Non-Patent Document 26), ε ₄₀₀ under the measurement conditions was 7,585 M ^-1 cm ^-1 . In addition, the optical path length of the microplate used was calculated as 0.458 cm in the amount of 150 μL of the reaction solution, and the amount of PNP generated per minute was obtained from the equivalent value and ΔA ₄₀₀ /min, and this was taken as the reaction rate, that is, enzymatic activity. At this time, the α-glucosidase activity determined under the condition that the fraction powder of less than 3 kDa in the extract was not contained was 3.4×10 ^-6 M/min. <Measurement test of α-glucosidase inhibitory effect> FIG. 9 shows the α-amylase activity observed when the extract obtained in Example 1 is less than 3 kDa component powder at various concentrations to the reaction solution, Time-dependent change in product amount (absorbance at 400 nm). EB in the legend represents the blank of the enzyme, and DB represents the double blank of the enzyme and the component powder whose extract is less than 3 kDa. In addition, EB contains the component powder whose extract liquid is less than 3 kDa so that the final concentration may be 10 mg/mL. The α-amylase inhibitory effect of the fraction less than 3 kDa in the extract was specifically measured by the following method. The sample solution of the component whose extract solution is less than 3kDa is dissolved in 50mM potassium phosphate buffer in the form of 80, 40, 20, 10, 4, and 2 mg/mL. The powder of the component whose extract solution is less than 3kDa obtained in Example 1 solution (pH 7.0) and prepared. 75 μL of each concentration of the sample solution previously incubated at 37° C. was mixed with 12.5 μL of 50 mM potassium phosphate buffer pH 7.0 in the wells of the microplate, and 12.5 μL of the enzyme solution was added thereto, and the assay was carried out at 37° C. Keep warm for 5 minutes. Next, the reaction was started by adding 50 μL of the PNPG base solution previously incubated at 37° C. as a reaction solution, and the absorbance at 400 nm (A ₄₀₀ ) at 37° C. was measured at 1-minute intervals for 15 minutes using a microplate reader. . In addition, each measurement was performed three times. [0060] The measurement results are shown in Figure 9. It was found that the slope of the straight line representing the change in absorbance at 400 nm (ΔA ₄₀₀ ) decreased as the concentration of the component less than 3 kDa in the extract added to the reaction solution increased. On the other hand, no PNPG-decomposing activity was observed in the reaction solution containing no α-glucosidase and containing fractions of less than 3 kDa in the extract at a final concentration of 10 mg/mL (EB in FIG. 9 ). From these results, it was shown that the fraction less than 3 kDa of the extract obtained in Example 1 contained a component that inhibited α-glucosidase activity in a concentration-dependent manner, and this component had no α-glucosidase-like activity at all. Furthermore, the measurement result of FIG. 9 was analyzed by the above-mentioned method. The enzymatic reaction rate (v _o ) obtained from the amount of PNP produced per minute by α-glucosidase when the fraction powder of less than 3 kDa in the extract was not contained was 3.4×10 ^-6 M/min. Similarly, to calculate the enzyme reaction rate (vi ) when adding a certain concentration of the component powder whose extract solution is less than _3kDa , the inhibition rate (unit: %) based on the component powder whose extract solution is less than 3kDa is defined as [1- (v _i / _vo )]×100, the inhibition rate was obtained in the presence of component powders having various concentrations of the extract solution of less than 3 kDa. Figure 10 was created in which the final concentration of the component powders of which the extract was less than 3 kDa was plotted on the horizontal axis and the inhibition rate was plotted on the vertical axis. As can be seen from FIG. 10 , when the final concentration of the component powder of less than 3 kDa in the extract solution added to the reaction solution was increased, the inhibition rate increased but showed a saturation curve behavior. Here, IC ₅₀ was 7.4 mg/mL when the final concentration of the fraction powder whose α-glucosidase activity was inhibited by 50% was less than ₃ kDa in the extract. Generally, when an enzyme binds to an inhibitor to form an enzyme-inhibitor complex, its equilibrium constant corresponds to the inhibitory substance constant K _i , and its value roughly corresponds to IC ₅₀ . Therefore, the inhibitory substance constant K _i of the fraction powder having an extract solution of less than 3 kDa with respect to α-glucosidase can be regarded as 7.4 mg/mL. The α-amylase activity inhibition rate was about 10% when the same concentration of the extract was less than 3kDa component powder. In addition, as described above, the inhibitory substance constant K _i with respect to α-amylase of the fraction powder of which the extract solution is less than 3 kDa was 39±2 mg/mL. From the above, it was revealed that the saccharolytic enzyme inhibitor of the present invention exerts a 5.3-fold higher inhibitory effect on α-glucosidase activity than α-amylase activity. [0061] Next, an example in which various processing procedures were further performed on the component powder of the extract obtained in Example 1 and less than 3kDa was further described. [Example 2] A buffer solution (KTAM-103, α, KTAM-103, α) was prepared by dissolving the component powder of the extract obtained in Example 1 less than 3 kDa in DIAMONDCOLOR·AMY-L DIRECT so as to be 80 mg/mL. - sample solution in amylase inhibition assay) or 50 mM potassium phosphate buffer (pH 7.0, alpha-glucosidase inhibition assay). The solution was frozen using a -80°C ultra-low temperature freezer (MDF-U384, manufactured by SANYO Electric Co., LTD., Moriguchi-shi, Osaka Prefecture) and kept overnight, and then the container was immersed in tap water to melt it. This solution was referred to as "freeze-thaw treatment solution" (Example 2). [Example 3] The powder of the fraction of the extract obtained in Example 1 less than 3 kDa was dissolved in 10 mM HCl (pH 2.0) so as to be 80 mg/mL. After the solution was kept at room temperature for 3 hours, 1 M NaOH was added for neutralization. After further freezing treatment, it was made into a freeze-drying treatment powder again using a freeze-drying machine. DIAMONDCOLOR·AMY-L DIRECT buffer (KTAM-103, for α-amylase inhibition test) or 50 mM potassium phosphate buffer ( pH 7.0, for α-glucosidase inhibition test), a sample solution was prepared. This solution was referred to as "acid treatment solution" (Example 3). [Example 4] The component powder of which the extract solution obtained in Example 1 was less than 3 kDa was dissolved in DIAMONDCOLOR·AMY-L DIRECT buffer solution (KTAM-103, α) so as to be 80 mg/mL. - sample solution for amylase inhibition test) or 50 mM potassium phosphate buffer (pH 7.0, for α-glucosidase inhibition test). This solution was heat-treated at 100° C. for 10 minutes or 30 minutes using an aluminum block thermostat (DTU-1BN, manufactured by Tietech Co., LTD., Tokyo). Here, in the fraction powder having an extract solution of less than 3 kDa at 80 mg/mL dissolved in 50 mM potassium phosphate buffer (pH 7.0) for the α-glucosidase inhibition test, a precipitate was visually recognized after heat treatment , so the precipitate was removed by centrifugation at 15,000 xg for 5 minutes, and the supernatant was collected. The solution of the component powder whose extract liquid was less than 3 kDa collected by the above heat treatment was cooled to room temperature as a "heat treatment solution" (Example 4). [Example 5] The component powder of which the extract solution obtained in Example 1 was less than 3 kDa was dissolved in DIAMONDCOLOR·AMY-L DIRECT buffer solution (KTAM-103, α) so as to be 80 mg/mL. - sample solution for amylase inhibition test) or 50 mM potassium phosphate buffer (pH 7.0, for α-glucosidase inhibition test). This solution was put into a glass sample bottle, an equal amount of hexane (special grade reagent, containing 97% of n-hexane, manufactured by Nacalai Tesque Inc., Kyoto City) was added, and the solution was capped, and vigorously stirred with a vortex mixer for 5 minutes. After sufficient separation by centrifugation at 10,000 xg for 5 minutes, the hexane layer was removed, and the aqueous layer was carefully recovered. The solution of the aqueous layer was referred to as "hexane extraction solution" (Example 5). In addition, DIAMONDCOLOR·AMY-L DIRECT buffer (KTAM-103, α-amylase inhibition test) or 50 mM potassium phosphate buffer (pH 7.0, α-glucosidase inhibition test), those who added the same amount of hexane and performed the same extraction operation were used as control groups, respectively. [Measurement test of α-amylase inhibitory effect for Examples 2 to 5] Using each of the sample solutions obtained in the above-mentioned Examples 2 to 5, the same method as the above-mentioned α-amylase inhibitory effect measurement test was carried out. method was determined. Mix 75 μL of each sample solution previously incubated at 37°C with 33.5 μL of buffer in the wells of the microplate, add 5 μL of 0.5 μg/mL α-amylase solution, and incubate at 37°C 5 minutes. Next, the reaction was started by adding 36.5 μL of the substrate test solution previously incubated at 37° C. as a reaction solution, and the absorbance at 405 nm at 37° C. was measured at 1-minute intervals for 15 minutes using a microplate reader. In addition, each measurement was performed three times. The results were analyzed by the above-mentioned method, and the inhibition rate when each sample solution was used for the α-amylase activity when the fraction powder containing less than 3 kDa in the extract solution was not contained was calculated (Table 2). In addition, for comparison, the α-amylase activity inhibition rate of Example 1 when the final concentration of the component powder of less than 3 kDa in the extract is added so that the final concentration is 40 mg/mL is also shown in Table 2 together. [0067]

First, the freeze-thaw treatment solution of Example 2 will be described in detail. Compared with the case where the component powder of less than 3kDa in the extract was not added, the α-amylase was also inhibited by 35% in the case of using the freeze-thaw treatment solution which was frozen at -80°C and thawed after being kept overnight. active. This means that the effect is the same as when the untreated extract is less than 3 kDa component powder, and therefore the component showing the α-amylase activity inhibitory effect has stability and freeze-thaw resistance to the freeze-thaw treatment of Example 2 . In addition, it is possible to expect the effect of modifying and removing components that are not resistant to low temperature, or the possibility of preventing the growth of microorganisms and storing them for a long period of time. Next, with regard to the acid treatment solution of Example 3, compared with the case where the component powder having an extract liquid of less than 3 kDa was not added, when the acid treatment solution neutralized after being kept at pH 2 for 3 hours was used, Alpha-amylase activity was also inhibited by 34%. This result shows that the component with the α-amylase activity inhibitory effect has stability and acid resistance to the acid treatment of Example 3. In addition, it is possible to expect an effect of modifying and removing components that are not resistant to acid or a bactericidal effect by an acid. Furthermore, in the case of oral ingestion, it is expected that it is resistant to gastric acid, reaches the small intestine, and inhibits α-amylase. Furthermore, with regard to the heat treatment solution of Example 4, compared with the case where the component powder of less than 3 kDa in the extract was not added, a heat treatment solution obtained by heat treatment at 100° C. for 10 minutes and 30 minutes was added In this case, the α-amylase activity was also inhibited by 36% and 40%, respectively. This represents the same or higher inhibitory effect as when the component powder of less than 3 kDa in the extract without heat treatment was added. From the above, it was shown that the α-amylase activity inhibitor component in the component powder whose extract solution is less than 3 kDa has high stability and heat resistance compared to the heat treatment in Example 4. Furthermore, it is possible to expect the effect of inactivating or modifying and removing heat-labile components by heat-treating the fraction powder of which the extract is less than 3 kDa, and further expecting a bactericidal effect or a virus-killing effect. In addition, with regard to the hexane extraction solution of Example 5, compared with the case where the component powder of which the extract liquid was less than 3 kDa was not added, hexane was added to the component powder of which the extract liquid was less than 3 kDa and stirred, The alpha-amylase activity was also inhibited by 35% in the case of the aqueous layer obtained after the layered extraction operation. On the other hand, alpha-amylase inhibition was investigated using an aqueous layer in which the same extraction operation was performed using buffer only in place of the powders of the fractions less than 3 kDa in the extract. In the inhibitory activity of the aqueous layer obtained by the extraction operation, no difference was found between the inhibitory activity of the control group (that is, the untreated buffer solution) that was not subjected to the extraction operation. These results indicate that the component showing the α-amylase activity inhibitory effect is not derived from hexane that is likely to be transferred to the aqueous layer by the hexane extraction operation. It is also indicated that the α-amylase inhibitory substance in the component powder of which the extract is less than 3 kDa is stable to hexane, does not transfer to the hexane layer or the interface but remains in the water layer, and is a highly hydrophilic substance. From the above results, the effect of removing the components transferred to the organic layer and the components transferred to the interface by the extraction operation using an organic solvent such as hexane can be expected. Hexane is the most non-polar and highly hydrophobic organic solvent used in the extraction process of biomass, and it is unusual to use solvents that are generally more non-polar or hydrophobic than hexane. Therefore, as described above, the fraction powder of the extract liquid less than 3kDa will not weaken the α-amylase activity inhibitory effect or make the effect disappear in hexane, so it can be judged that organic solvents other than hexane also have Sufficient stability and resistance to organic solvents. [Measurement test of α-glucosidase inhibitory effect for Examples 2 to 5] Further, each sample solution obtained in the above-mentioned Examples 2 to 5 was used to measure the α-glucosidase inhibitory effect with the above-mentioned α-glucosidase. The test was determined in the same way. 75 μL of each concentration of the sample solution previously incubated at 37°C was mixed with 12.5 μL of 50 mM potassium phosphate buffer (pH 7.0) in the well of the microplate, 12.5 μL of enzyme solution was added to it, and the mixture was heated at 37°C. Insulated for 5 minutes. Next, the reaction was started by adding 50 μL of the PNPG substrate solution previously incubated at 37° C. as a reaction solution, and the absorbance at 400 nm at 37° C. was measured at 1-minute intervals for 15 minutes using a microplate reader. In addition, each measurement was performed three times. The results were analyzed by the above-mentioned method, and the inhibition rate when each sample solution was used for the α-glucosidase activity when the fraction powder containing less than 3 kDa in the extract was not contained was calculated (Table 3). In addition, for comparison, the α-glucosidase activity inhibition rate when the fraction powder having a final concentration of 40 mg/mL of the extract of Example 1 of less than 3 kDa was added is also shown in Table 3 in combination. [0073]

As shown in Table 3, when adding the freeze-thaw treatment solution of Example 2 or the acid treatment solution of Example 3 to the reaction solution with a final concentration of 40 mg/mL, the α-glucosidase activity inhibition rate was also the same as The same applies to the use of the component powder (untreated solution) of which the extract of Example 1 is less than 3 kDa. These results show that the component showing the α-glucosidase activity inhibitory effect is very stable to the freeze-thaw treatment of Example 2 or the acid treatment of Example 3, and has high freeze-thaw resistance and acid resistance. In addition, when the heat-treated solution of Example 4 was added to the reaction solution, the α-glucosidase activity was inhibited compared with the case of using the fraction powder (untreated solution) of which the extract solution of Example 1 was less than 3 kDa. rate decreased slightly. Furthermore, when the hexane extraction solution of Example 5 was added to the reaction solution at a final concentration of 40 mg/mL, compared with the case of using the component powder (untreated solution) of which the extract solution of Example 1 was less than 3 kDa, A slight decrease in the inhibition rate of alpha-glucosidase activity was also found. By performing pretreatments such as freeze-thaw, acidity, heating, hexane treatment, etc. to the component powder whose extract solution is less than 3kDa, it is also observed that the inhibitory activity is slightly reduced compared with the case where it is not carried out. Even so, the The inhibitory effect after the pretreatment is also sufficiently large. Therefore, it can be judged that the component showing the α-glucosidase activity inhibitory activity has high stability, heat resistance, and organic solvent resistance to the heat treatment of Example 4 or the hexane extraction treatment of Example 5. These properties observed in the component exhibiting α-glucosidase activity inhibitory activity are the same as those of the component exhibiting the above-mentioned α-amylase activity inhibitory effect, and can also be expected in terms of storage stability, processability, and applicability. The same excellent effect as above. From the above results, it is considered that by performing freeze-thaw treatment, acid treatment, heat treatment, organic solvent treatment alone or in combination with the component powder of the extract obtained in Example 1 that is less than 3kDa, by This makes it possible to obtain a saccharolytic enzyme inhibitor having higher purity and an α-amylase activity inhibitory effect and an α-glucosidase activity inhibitory effect. The earthworm-derived saccharolytic enzyme inhibitor thus obtained can be used in pharmaceuticals, functional foods, additives, pet foods, and the like. [Other Embodiments] (1) In the above-described embodiment, the earthworms used as raw materials are red earthworms (Eisenia fetida), but other types of earthworms used for pharmaceuticals and health food applications may also be used. (2) In the above-mentioned embodiment, the earthworm dry powder prepared by the hydrostatic pressure type high-pressure treatment to the pulverizing liquid of earthworm is used, and the earthworm dry powder prepared by lyophilization is carried out to the centrifuged supernatant liquid, but also can be used by Earthworm dry powder prepared by other methods. (3) The processing conditions of each process included in the method for producing a saccharolytic enzyme inhibitor in the above-described embodiment can be appropriately changed. (4) In the above-mentioned embodiment, the case where the saccharolytic enzyme inhibitor is produced by using the dry powder of earthworms as a raw material is exemplified, but the saccharolytic enzyme inhibitor may be produced by using earthworms or their pulverized materials as raw materials.

1 is a graph showing the amount of the product (405 nm of the observed product when the extract obtained in Example 1 is less than 3 kDa in powders of components prepared in various concentrations and added to the α-amylase solution. A graph of the change in absorbance over time. Figure 2 is the relative value of the α-amylase activity when the component powder with the extract solution obtained in Example 1 less than 3kDa is added to the α-amylase solution at each concentration (without adding the component powder with the extract solution less than 3kDa relative value of α-amylase activity when compared with α-amylase). Fig. 3 is a graph showing the inhibition rate of α-amylase activity when the component powders of which the extract solution obtained in Example 1 is less than 3 kDa is added to the reaction solution at each concentration according to the concentration of α-amylase added to the reaction solution . Figure 4 is a graph showing the effect of changes in the concentration of the substrate test solution added to the reaction solution on the α-amylase activity. Fig. 5 shows that under the condition that the component powder of less than 3 kDa in the extract obtained in Example 1 is not contained or the component powder of less than 3 kDa is contained in the extract so that the final concentration becomes 40 mg/mL, the results of adding to the reaction solution The α-amylase activity was analyzed when the relative concentration of the substrate test solution was changed, and the horizontal axis was the relative concentration of the substrate test solution, and the vertical axis was a graph of the Michaelis curve when the reaction rate was indicated. Fig. 6 is a graph showing the conditions for adding to the reaction solution under the condition that the extract obtained in Example 1 does not contain the component powder less than 3 kDa or contains the component powder less than 3 kDa in the extract so that the final concentration becomes 40 mg/mL The results of the measurement of α-amylase activity observed when the relative concentration of the matrix test solution changed were analyzed. - A graph of the Lineweaver-Burk curve and a graph showing the rate theory parameters of the enzyme reaction obtained from the curve. Fig. 7 is a graph showing the conditions for adding to the reaction solution under the condition that the extract obtained in Example 1 does not contain the component powder less than 3 kDa or contains the component powder less than 3 kDa in the extract so that the final concentration becomes 40 mg/mL The α-amylase activity observed when the relative concentration of the matrix test solution changes in the matrix is analyzed, and the horizontal axis is the relative concentration of the matrix test solution, and the vertical axis is the relative concentration of the matrix test solution divided by the reaction rate. A graph of the Hanes-Woolf curve and a table showing the rate theory parameters of an enzyme reaction obtained from the curve. Fig. 8 is a graph showing the observed α-amylase activity when the relative concentration of the matrix test solution was changed, and the component powders of which the extract solution obtained in Example 1 was less than 3 kDa at various concentrations were added to the reaction solution. For analysis, according to the relative concentration of the matrix test solution, the horizontal axis is set as the final concentration of the component powder that is less than 3 kDa in the extract added to the reaction solution, and the vertical axis is set as the reciprocal of the reaction rate Dixon (Dixon) curve chart. Fig. 9 is a graph showing the α-glucosidase activity and the time-dependent change in the amount of the product (absorbance at 400 nm) observed when the fraction powder of the extract obtained in Example 1 less than 3 kDa was added to the reaction solution at various concentrations chart. Fig. 10 is a graph showing the inhibition rate with respect to α-glucosidase activity when the fraction powder of which the extract solution obtained in Example 1 is less than 3 kDa is added to the α-glucosidase reaction solution at various concentrations .

Claims (3)

A method for producing a saccharolytic enzyme inhibitor, which has an activity-inhibiting effect on a specific saccharolytic enzyme, wherein the specific saccharolytic enzyme is α-glucosidase, and the following process is performed to obtain the above-mentioned saccharolytic enzyme inhibitor, that is, an extraction process , take the earthworm or its pulverized material or the dry powder of the earthworm or its pulverized material as raw material and add water to obtain the earthworm extract; and the separation process, obtain the molecular mass derived from the earthworm extract less than 3kDa, that is, the extract is less than 3kDa components. The method for producing a saccharolytic enzyme inhibitor according to claim 1, wherein the specific saccharolytic enzymes are α-glucosidase and α-amylase. The method for producing a saccharolytic enzyme inhibitor according to the claim 1 or 2, wherein the components of the extract recovered by the aforementioned separation process less than 3 kDa are selected from the group consisting of freeze-thaw treatment, acid treatment, heat treatment, At least one of organic solvent treatment is used to obtain the aforementioned saccharolytic enzyme inhibitor.

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