JP5897796B2 - Hypoglycemic agent and food and drink for preventing diabetes or improving symptoms comprising the same - Google Patents

Description

  The present invention relates to a hypoglycemic agent and a food or drink for preventing diabetes or improving symptoms to which it is added. The present invention also relates to a method for treating diabetes and a method for screening a therapeutic agent for diabetes.

  Ziou has long been an important Chinese medicine known for its nutrition and tonic due to its blood-enhancing effect, and has been widely used as a medicine for numbness due to poor circulation, dry skin and itching, and prostate enlargement in combination with other Chinese medicines. It is also known to have a blood glucose lowering effect and has been studied as a component of diabetes therapeutic agents (for example, Patent Document 1). Moreover, it has been reported that the ethanol precipitation fraction of the hot water extract of Geow has a blood glucose lowering effect (Non-patent Document 1). However, until now, the main body of the blood glucose lowering effect exhibited by Ziou has not been clarified, and its proper usage has not been established.

  Diabetes mellitus is a disease that shows chronic hyperglycemia and abnormal glucose tolerance. Diabetes has few subjective symptoms at an early stage and leads to various organ disorders (nephropathy, peripheral neuropathy, retinopathy, etc.), which are serious complications. It is a disease that causes many problems, such as requiring many social resources. And since the number of the sick is increasing rapidly globally, it has become a big social problem to be resolved by the United Nations.

  Diabetes can be roughly divided into type 1 and type 2, but type 2 is regarded as a social problem. Type 2 diabetes is mainly caused by genetic background and obesity, and is based on a decrease in insulin secretion and a decrease in sensitivity to insulin (insulin resistance). 95% of diabetic patients are considered to be of this type 2. Type 2 diabetes has a strong aspect of lifestyle-related diseases, and is on an increasing trend with the recent obesity and aging.

  As a treatment for type 2 diabetes, dietary therapy and exercise therapy are generally performed, but when good blood glucose control cannot be achieved, oral hypoglycemic drugs and insulin injection are performed. Depending on the mechanism of action, oral hypoglycemic drugs include insulin secretagogues, insulin resistance improvers, α-glucosidase inhibitors and the like.

  However, any of these may cause obesity, worsening of insulin resistance, blood glucose level control failure such as severe hypoglycemia, etc. due to excessive administration or long-term use. Therefore, new therapeutic / preventive drugs, functional foods, etc. are strongly demanded and are being developed. Some of them contain sugar as a structural unit. For example, Patent Document 2 discloses galactomannan degradation products such as guar gum, dietary fibers such as indigestible dextrin, and diabetes prevention or polyphenol-containing diabetes. A therapeutic composition is shown, and Patent Document 3 discloses a hypoglycemic agent containing heparin or heparan sulfate as an active ingredient.

  However, the effects of these therapeutic / prophylactic drugs, functional foods, etc. have not been sufficiently confirmed, and development of a drug that can achieve better blood glucose control has been desired.

  On the other hand, the present inventors have focused on silkworms as experimental animals that can replace mammals, and have reported that they can be used in evaluation systems for the development of therapeutic agents against bacteria and viruses that infect humans and the like (patents). Document 4 and Patent document 5). This evaluation system uses invertebrates for the first time, has fewer problems in terms of cost and ethics than conventional experimental animals, and can be used for quality control animals. This is a method that has many advantages such as being able to evaluate a wider range of objects, including objects that have not been included in studies using animals, and making an accurate evaluation.

  Furthermore, the present inventor has already filed a new application that the above evaluation system using silkworms can be used as an evaluation system for a therapeutic agent for type 2 diabetes in mammals. However, a novel type 2 diabetes therapeutic / prophylactic agent, functional food, etc., which can control blood glucose satisfactorily by using the evaluation system, has not yet been found.

Japanese Patent Laid-Open No. 08-198768 JP 2006-052191 A JP 2004-168771 A JP 2007-327964 A WO2005 / 116269

J. Pharm. Soc. Jpn., T. Kihoet al., 112 (6), 393-400 (1992)

  The present invention has been made in view of the above-mentioned background art, and its problem is that there are few side effects such as causing serious hypoglycemia of conventional therapeutic agents for type 2 diabetes, It is an object of the present invention to provide a hypoglycemic agent that has almost no problems in use and is suitable for use in foods and drinks.

  In order to solve the above-mentioned problems, the present inventor has treated silkworm larvae (hereinafter referred to as “Silkworm”) type 2 diabetes mellitus for the long-established nourishing tonic effect, hypoglycemic effect, safety, etc. As a result of intensive investigations using a drug evaluation system, it was confirmed that the hot water extract fraction of Jiou reduced the blood glucose level of hyperglycemic silkworms.

  Moreover, when this hot water extraction fraction was hydrolyzed to monosaccharide, it discovered that the main component sugar contained therein was galactose. Then, it was confirmed that the component in the “hot water extract fraction of diau” that lowers the blood sugar level of hyperglycemic silkworms is a galactose homopolymer, and that the isolated galactose homopolymer lowers the blood sugar level. To the present invention.

  Surprisingly, the present inventors have found that galactose, which is a monosaccharide that is not contained in Zeo itself, reduces the blood glucose level of hyperglycemic silkworms and hyperglycemic mice.

  That is, the present invention provides a hypoglycemic agent characterized by containing galactose or a galactose derivative as an active ingredient.

  The present invention also provides a hypoglycemic agent characterized by containing an isolated galactose homopolymer as an active ingredient.

  The present invention also provides a food or drink for preventing diabetes or improving symptoms characterized by adding the above-mentioned hypoglycemic agent, and galactose or a galactose derivative is effective in reducing blood glucose. The present invention provides a food or drink characterized by being added as a component. Moreover, the isolated galactose homopolymer is added as an active ingredient of a blood glucose lowering, and the food-drinks characterized by the above-mentioned are provided.

  The present invention also provides a method for treating diabetes characterized by administering a drug containing galactose or a galactose derivative as an active ingredient, and a drug containing an isolated galactose homopolymer as an active ingredient It is intended to provide a method for treating diabetes, which comprises administering

The present invention also provides a method for screening a therapeutic agent for diabetes, comprising at least the following steps (a) to (d), wherein the test substance is a galactose derivative.
(A) a step of increasing the concentration of sugar (B) in the fat body or blood of the invertebrate by ingesting sugar (A) to the invertebrate (b) obtained in step (a) above In addition, a step of administering a test substance to an invertebrate in which the concentration of sugar (B) in the fat body or blood is increased (c) in the fat body or blood of the invertebrate to which the test substance is administered (D) a step of selecting a substance that lowers the concentration of sugar (B) in the fat body or blood of the invertebrate from among the test substances

  The present invention has solved the above-mentioned problems and problems, and the hypoglycemic agent of the present invention has an effect of lowering the blood glucose level of an animal in a hyperglycemic state, and makes the hyperglycemic state due to diabetes extremely remarkable. Has the effect of improving. That is, the hypoglycemic agent of the present invention is extremely effective particularly for therapeutic / preventive agents for type 2 diabetes and functional foods.

  Further, since the galactose or galactose derivative, which is an essential active ingredient of the hypoglycemic agent of the present invention, is a very safe substance used as an essential component of life activity, the hypoglycemic agent of the present invention is also extremely safe and has side effects. There is nothing. Moreover, since the isolated galactose homopolymer is also hydrolyzed to galactose, it is as safe as galactose. In addition, since it can be easily made into various dosage forms and can be easily added to foods and drinks as much as it can be used as a sweetener, it is extremely effective as a safe hypoglycemic agent and functional food. Prevention and improvement of symptoms.

It is a graph showing the blood glucose level or sugar concentration in the silkworm which gave the food containing 15% (w / w) glucose to the silkworm of the 1st day of 5th age for 1 day and 3 days (evaluation experiment example 1). (A): Blood sugar level in blood (hemolymph) (B): Sugar concentration in fat body It is a graph showing the time-dependent transition (time course) of a blood glucose level when the food containing glucose 33% (w / w) is ingested (evaluation experiment example 2). From the top, they are “33% (w / w) glucose-added feed feeding group”, “normal feed feeding group”, and “fasting group”. It is a graph which shows the blood glucose level 60 minutes after a feed start when changing the amount of glucose contained in a bait (evaluation experiment example 3). It is a graph which shows the fall effect of the blood glucose level of the silkworm of a hyperglycemic state by insulin (evaluation experiment example 4). From the left, “administer physiological saline to the normal feed group”, “given 16% (w / w) glucose-added feed and administer physiological saline 60 minutes later”, “16% (w / w) glucose-added feed "Insulin is administered 60 minutes after giving". It is a graph which shows the fall action of the blood glucose level of the hyperglycemic state silkworm by metformin (evaluation experiment example 5). From the left, “administer physiological saline to the normal feed group”, “given 16% (w / w) glucose-added feed and administer physiological saline 60 minutes later”, “16% (w / w) glucose-added feed "Metformin is administered 60 minutes after giving". It is the graph used for calculation of ED50 value in the blood glucose level reduction effect | action of the silkworm of an insulin (evaluation experiment example 6). It is a graph showing a time-dependent transition of the blood glucose level when the food containing glucose 15% (w / w) is ingested, and the glucose concentration in the blood (evaluation experiment example 7). (A): Blood glucose level (b): Glucose concentration in blood It is a graph showing the time-dependent transition of the blood glucose level of a silkworm when a silkworm is made to fast after ingesting the food containing glucose 12% (w / w) (evaluation experiment example 7). It is a graph which shows the blood glucose level 180 minutes after a feed start when changing the amount of glucose contained in a bait (evaluation experiment example 3). It represents the body length, body weight, and blood glucose level of silkworms after feeding them with or without giving normal feed or 5%, 10%, 15%, 30% (w / w) glucose-added feed to silkworms, respectively. (Evaluation Experiment Example 8). (A): Photograph showing silkworm state (b): Body weight (c): Body length (d): Blood glucose level (A) It is a graph showing the time-dependent transition of the blood glucose level after administering a glucose-added feed and “physiological saline” or “recombinant human insulin” to silkworms (Evaluation Experimental Example 9). (B) It is a graph showing a blood glucose level when each amount of recombinant human insulin is administered to a silkworm after feeding a glucose-added feed (Evaluation Experiment Example 9). (C) shows the results of Western blotting using anti-Akt antibody and anti-phosphorylated Akt antibody. (D) It is a graph showing the quantity of phosphorylated Akt of the fat body extracted from the silkworm (evaluation experiment example 9). (E) It is a graph showing the sugar uptake | capture amount of the fat body extracted from the silkworm (evaluation experiment example 9). It is a graph which shows the change of the blood glucose level and AMPK activity of a silkworm by AICAR administration (evaluation experiment example 10). (A): Blood glucose level (b): AMPK activity It represents changes in body weight and body length of silkworms by recombinant human insulin injection (Evaluation Experimental Example 11). (A): Outline of evaluation experiment example (b): Photograph showing the state of silkworm (c): Body weight (d): Body length It is a figure which shows the analysis result of the saccharide | sugar which comprises the ethanol precipitation fraction of a geothermal hot water extract, and is a figure which shows the result which compared the mobility on TLC with standard saccharides, such as D-galactose (Example 1). (Example 2) which is a figure which shows the effect of the blood glucose level fall in a silkworm body fluid when the ethanol precipitation fraction of a hot water extract is administered to a hyperglycemia silkworm model. It is a figure which shows the effect of the glucose level fall in a silkworm body fluid when D-galactose is administered to a hyperglycemic silkworm model (Example 2). It is a figure which shows the effect of the glucose level fall in a mouse | mouth blood when D-galactose is administered to a hyperglycemia mouse model (Example 3). It is the figure which showed the method of obtaining the ethanol precipitation fraction of a geothermal hot water extract from a geological (Example 1).

  Hereinafter, the present invention will be described. However, the present invention is not limited to the following embodiments, and can be arbitrarily modified and implemented.

[Galactose or galactose derivative]
The “galactose or galactose derivative” in the present invention refers to D-galactose and its derivatives. In addition, α type and β type are included. That is, in the case of D-galactose, [α] _D ²⁰ = + 144 ° α-type and [α] _D ²⁰ = + 52 ° β-type are included. The “galactose or galactose derivative” in the present invention is a monosaccharide, and the “galactose derivative” in the present invention does not include a polysaccharide such as a disaccharide or a trisaccharide having galactose as a constituent component.

  Examples of the galactose derivative include those in which a substituent is bonded to the galactose ring. Examples of the substituent include alkyl groups such as methyl group, ethyl group, and propyl group; aryl groups such as phenyl group; arylalkyl groups such as benzyl group; alkanoyl groups such as acetyl group (ethanoyl group) and the like. The “galactose or galactose derivative” in the present invention (hereinafter sometimes abbreviated as “galactose (derivative)”) includes both a pyranose type and a furanose type.

  In addition, if the galactose derivative is used in the same metabolic pathway as galactose, it is considered that the galactose derivative has the same blood glucose lowering effect as galactose. Examples thereof include galactose hydrate, aldehyde, O-methylated, O-sulfated, pharmacologically acceptable salts thereof, UDP (uridine, which is a metabolic intermediate of galactose, and the like. And diphosphoric acid) galactose. In other words, one of the fundamentals of the present invention is that a monosaccharide called galactose, which could not be predicted to have a blood glucose lowering effect by itself, has been found to have a blood glucose lowering effect. Any monosaccharide galactose derivative utilized in the pathway is within the scope of the present invention.

  The reason why galactose exhibits a blood glucose lowering effect is that galactose acts on unidentified cells. It is considered that the cells have an effect of lowering blood glucose level by some mechanism. A general understanding in modern biology is that when a biological substance such as galactose acts on a cell, it binds to a receptor protein on the cell surface and the signal is transmitted into the cell. is there. Therefore, as long as it is a substance having an affinity for the receptor on which galactose acts, it is considered that there is a substance that exhibits a blood glucose lowering action as galactose, even if it is not galactose. Some derivatives of galactose should have an affinity for such receptors. Accordingly, such galactose derivatives are also within the scope of the present invention. If there is such a galactose derivative that is more advantageous than galactose (for example, good stability in blood), it can be said that it is superior to galactose as a hypoglycemic agent. That is, it is logically obvious that there can be a galactose derivative equivalent to or more convenient than galactose having the same performance as galactose.

  Galactose exists in nature as α-D-galactose as a constituent sugar of lactose, which is a disaccharide mainly bonded to D-glucose. Agarose forming agar is a polysaccharide in which 1 → 3 linked β-D-galactose and 1 → 4 linked 3,6-anhydro-α-L-galactose are alternately arranged in a straight chain. . In this way, polysaccharides containing galactose as a constituent component are extremely abundant in nature. However, galactose, which is a monosaccharide, is extremely rare in nature, and is therefore present in ordinary foods and beverages. Not done.

  Since galactose is a basic sugar necessary for animal life activities and is generally supplied via dairy products, the effect of galactose on blood sugar reduction in hyperglycemic animals has been studied so far. There was no suggestion of such an effect. It was discovered that sugar, which has a fundamental role in the life activity of such animals, has a hypoglycemic effect on hyperglycemic animals. become. In addition, galactose has not been reported to have a particularly problematic toxicity, except for those who have impaired glucose metabolism such as galactosemia.

  In addition, galactose has a sweetness of 0.3 when the sweetness of sucrose is 1, and is not as sweet as glucose of 0.5, but has a moderate sweetness and energy, so it is nutritious. It can also be used as a sweetener. Galactose (derivative) is also effective in preventing diabetes or improving symptoms by being able to be used as an alternative to sucrose for sweetness. In addition, the fact that the sweetness is not so strong is excellent in that even if the amount added as a hypoglycemic agent is increased, there is little influence on the taste if considered as a substitute for the commonly used sweetener.

[Isolated galactose homopolymer]
The “galactose homopolymer” in the present invention is a polysaccharide in which two or more galactoses are bound. The molecular weight (number of bonds) is not limited, but the number of bonds is preferably 10 or more, more preferably 100 or more, and particularly preferably 1000 or more. We found an effect of lowering blood glucose level using “isolated galactose homopolymer” extracted from Diou, but if it was “isolated galactose homopolymer”, it was not extracted from Diou However, since the effects are considered to be the same, the “isolated galactose homopolymer” in the present invention is not limited to those extracted from diau. In addition, “isolated galactose homopolymer” includes those artificially synthesized from galactose.

[Evaluation system for blood glucose lowering effect and method for determining galactose (derivative) having blood glucose lowering effect]
The blood glucose lowering agent of the present invention is preferably one in which the galactose (derivative) or the isolated galactose homopolymer is determined by a process including at least the following steps (a ′) to (d ′).
(A ′) a step of increasing the concentration of sugar (B) in the fat body or blood of the invertebrate by ingesting the sugar (A) to the invertebrate (b ′) in the above step (a) A step of administering a test substance to the invertebrate in which the concentration of sugar (B) in the fat body or blood is increased (c ′) the fat body of the invertebrate to which the test substance is administered A step of measuring the concentration of sugar (B) in blood or blood (d ′) a substance that lowers the concentration of sugar (B) in the fat body or blood of the invertebrate from among the test substances Process to select

The galactose (derivative) is particularly preferably determined at least by the steps including the following steps (a) to (d).
(A) a step of increasing glucose concentration in the fat body or blood of the invertebrate by ingesting sugar to the invertebrate (b) in the fat body or blood obtained in the step (a) (C) a step (d) of measuring a glucose concentration in a fat body or blood of an invertebrate to which the test substance is administered (c). A step of selecting a substance that lowers the glucose concentration in the fat body or blood of the invertebrate from the test substance.

  As shown in the following explanation and evaluation experiment examples in the examples, the above-mentioned “steps including steps (a ′) to (d ′)” and the “steps including steps (a) to (d)” are used. For example, it is possible to accurately screen for a hypoglycemic agent for humans and the like. Since galactose and galactose homopolymer were found by this process, they have an effect as a hypoglycemic agent for humans and the like. Furthermore, it is clear that the use of the above-mentioned “steps including steps (a ′) to (d ′)” enables screening of a hypoglycemic agent having an effect on humans and the like from various galactose derivatives.

  According to the above-mentioned “steps including steps (a ′) to (d ′)”, since it is possible to screen for a drug having an effect of lowering the blood glucose level of a hyperglycemic animal, screening for a drug that substitutes for insulin Insulin may already be in invertebrates, and if insulin is separately administered to invertebrates, it is possible to screen for insulin resistance improvers (substances that remove insulin resistance). is there.

  One embodiment of the present invention includes at least the “steps (a ′) to (d ′)”, preferably the “steps (a) to (d)”, and the test substance is a galactose derivative. This is a screening method for a therapeutic agent for diabetes.

<Process (a)>
Invertebrates are preferably those belonging to the arthropod phylum, such as insect class, crustacean class, centipede class, spider class, etc., Lepidoptera, Coleoptera, Diptera, Hymenoptera, Diptera, Reticulates More preferred are those belonging to the class of insects. There is no restriction | limiting in particular as what belongs to an insect class, According to the objective, it can select suitably. From the convenience of handling, larvae belonging to the class of insects are particularly preferred. Such larvae are not particularly limited. Specifically, lepidoptera (including moths, butterflies, etc.), Coleoptera (including beetles, etc.), Diptera (including flies), Hymenoptera (roaches) Larvae of those belonging to the order of (including), straight (including grasshopper), reticulated (including bee), and the like. Of these, fully transformed insect larvae are more preferred, and fully transformed insect substantially hairless larvae are particularly preferred. Completely metamorphic insects are eggs, larvae, moths, and adults that have undergone the growth process.Many of them have a virtually hairless and simple form specialized for nutritional supplementation called `` riceworms '' during larvae. In addition, the larvae of completely transformed insects are particularly suitable because they can be easily injected with drugs and the like.

Further, as such larvae, silkworms are particularly preferable from the following points.
(1) It is easy to obtain.
(2) A breeding method has already been established, and there is convenience in breeding.
(3) The properties similar to the internal organs and organs of mammals such as humans have been known to some extent in previous studies.
(4) Genetic lines have been established and genetic homogeneity has been maintained.
(5) Since it is relatively large, slowly moving, and substantially hairless, it is easy to administer drugs, such as being able to inject quantitatively.
(6) Having fat bodies, the fat bodies can be taken out and the contained substances can be quantified.
(7) It is cheaper than mice, rats, etc., and can be bred with a large number of individuals in a small space, and has few ethical problems, so it is easy to perform screening evaluation.
(8) Evaluation can be performed even when there is only a small amount of the test substance.
(9) It is easy to align individuals in the same state, such as aligning ages.
(10) It is possible to collect body fluids and analyze components such as sugars, lipids and enzymes.

  Among the above, (5) and later are the characteristics that can be said to be the larvae of the fully transformed insects, and the substantially hairless larvae of the completely transformed insects have excellent characteristics as experimental animals. The larvae are preferably large larvae from the viewpoint of easy intake of sugar (A), ease of administration of the test substance, ease of collection of blood and fat bodies, and the like. Here, the “large larva” is a larva having a body length of 1 cm or more, preferably 1.5 cm or more and 15 cm or less, particularly preferably 2 cm or more and 5 cm or less. In the case of silkworms, 4th to 5th instar larvae are preferable, and 5th instar larvae are particularly preferable.

  There is no restriction | limiting in particular as a method of ingesting sugar (A), According to the objective, it can select suitably. Specifically, for example, injection into blood, oral ingestion by addition to feed (food), injection into the intestine, etc. are mentioned, and in terms of simplicity and correspondence with human clinical practice. Oral ingestion by injection into the intestinal tract, addition to feed (food) or the like is preferable.

  The sugar (A) is not particularly limited as long as it increases the concentration of sugar (B) in the fat body or blood of the invertebrate. For example, glucose, heptose, hexose, pentose, tetrose Monosaccharides such as triose, disaccharides such as trehalose, maltose, lactose, sucrose, cellobiose, nigerose and sophorose; oligosaccharides such as fructooligosaccharide, galactooligosaccharide and dairy oligosaccharide; starch, glycogen, cellulose, pectin and glucomannan Polysaccharides such as deoxy sugars, oxidation products of sugars such as uronic acid, sugar alcohols such as sorbit and arabit, etc., may be mentioned as those that may increase the concentration of sugar (B). Among these, glucose, sucrose, oligosaccharide, glycogen or starch is particularly preferable from the viewpoint of increasing the glucose concentration and / or trehalose concentration in the silkworm blood, the point corresponding to human clinical practice, and the like. These may be used alone or in combination of two or more.

  In step (a), it is essential to increase the sugar (B) concentration in the fat body or blood of the invertebrate. The sugar (B) is not particularly limited as long as the concentration in the fat body or blood is increased by ingestion of the sugar (A) and the decrease in the concentration can be measured when the test substance is administered. For example, glucose, trehalose and the like can be mentioned. Moreover, although the total amount of several types of saccharide | sugar may be quantified depending on the determination method of saccharide | sugar (B), in that case, saccharide | sugar (B) is not limited to 1 type.

  Sugar (A) and sugar (B) may be the same or different. For example, in the case of silkworms, when sucrose is administered as sugar (A), the glucose concentration and trehalose concentration in the silkworm blood immediately increase. The sugar (B) in this case is glucose and trehalose.

  The sugar (B) quantification method includes phenol sulfate method, anthrone sulfate method, carbazole sulfate method and the like for quantification of all saccharides; glucose oxidase method and the like for quantification of glucose.

  The “degree of increase in the concentration of sugar (B)” in the “invertebrate in which the concentration of sugar (B) in fat body or blood is increased” obtained in step (a) is the effect of galactose (derivative). Is not particularly limited as long as it can be confirmed, but is preferably 1.5 times to 100 times, more preferably 1.5 times to 8 times, compared to a normal one that does not take sugar (A). .5 times to 3 times is particularly preferable. If the “degree of increase in the concentration of sugar (B)” is too low, the effect of galactose (derivative) may not be confirmed. On the other hand, the upper limit may not be higher than the above range, or may not be necessary. In addition, if an attempt is made to make it higher than necessary, an individual's disorder other than an increase in blood glucose level may occur, and the individual may die in a significant case.

  Specifically, in the case of the concentration of sugar (B) in blood, 7 mg / mL (μg / μL) to 200 mg / mL (μg / μL) is preferable, 8 mg / mL to 150 mg / mL is more preferable, 10 mg / mL to 100 mg / mL is particularly preferable. In the case of the glucose concentration in the fat body, 10 ng / μg to 1000000 ng / μg is preferable, 100 ng / μg to 10000 ng / μg is more preferable, and 1000 ng / μg to 20000 ng / μg is particularly preferable. The measurement method and expression method of the concentration are the same as those in the examples.

<Step (b)>
In the step (b), galactose (derivative) is administered to the “invertebrate in which the concentration of sugar (B) in the fat body or blood is increased” obtained in the step (a).

  There is no restriction | limiting in particular as a method of administering galactose (derivative), According to the objective, it can select suitably. Specifically, for example, injection into blood, oral intake by addition to feed (food), injection into the intestine, and the like can be mentioned. In terms of simplicity and compatibility with humans, injection into the intestinal tract and oral ingestion are preferred. There is no restriction | limiting in particular as dosage of galactose (derivative), According to the kind of invertebrate, administration method, etc., it can select suitably. It is also preferable to dilute and administer with physiological saline, water or the like.

  Galactose (derivative) is a water-soluble low-molecular-weight organic compound that is thought to be taken up into the blood in large quantities by the transporter-mediated pathway called paracellular pathway, that is, paracellular transport, from the intestinal tract . For example, the following documents (1) and (2) show that galactose is absorbed by a pathway called paracellular transport. “Parallel cell transport” refers to transport through which nutrients pass between cells in the intestinal tract and is transported efficiently even at high concentrations. Accordingly, since galactose (derivative) is absorbed very efficiently from the intestinal tract, the effect of galactose (derivative) can be obtained by any one of administration into the blood, injection into the intestinal tract, and oral ingestion. If so, it is reasonably considered that the effect can be obtained by other administration methods as well.

Reference (1):
In vivo
increase of passive intestinal absorption by cotransporter activation in rat.
P &eacute; rez M,
Barber A, Ponz F., Rev Esp Fisiol. 1993 Dec; 49 (4): 259-64.
Reference (2):
Galactose
transport inhibition by cytochalasin E in rat intestine in vitro.
D &iacute; ez-Sampedro
A, Urdaneta E, Lostao MP, Barber A., Can J Physiol Pharmacol. 1999
Feb; 77 (2): 96-101.

  The timing of administration of galactose (derivative) is not particularly limited. Immediately after the intake of sugar (A) in step (a), the concentration of sugar (B) in the fat body or blood of the invertebrate is almost maximum. And the time when the concentration of sugar (B) in the blood recovers to the normal value, the concentration of sugar (B) in the fat body of the invertebrate or blood The point at which the maximum value is reached is preferable in terms of obtaining reproducibility of the experimental results. Specifically, “immediately after the sugar (A) has been ingested” is particularly preferable.

<Step (c)>
In the step (c), the concentration of sugar (B) in the fat body or blood of the invertebrate animal to which galactose (derivative) is administered in the step (b) is measured. As for the method of collecting the measurement sample, it is preferable to dissect and collect the fat body, and for blood, the method of collecting from a cut is preferable.

  Examples of the method for measuring the type of sugar (B), the concentration of sugar (B), and the concentration of glucose include the same methods as those described in the step (a). That is, as sugar (B), the concentration in fat body or blood is increased by ingestion of sugar (A), and the decrease in the concentration can be measured when a test substance is administered. Although there is no limitation, glucose, trehalose, etc. are mentioned, for example. Moreover, sugar (B) is not limited to 1 type, You may measure the total amount of all the sugars (B). However, sugar (B) does not include galactose (derivative) or galactose homopolymer.

  For example, in the case of silkworms, the glucose concentration and trehalose concentration in the silkworm blood are often increased by ingestion of sugar (A). Therefore, the sugar (B) is preferably glucose or trehalose. Further, when the galactose (derivative) administered in step (b) is detected in the blood, the saccharide (B) alone is excluded from the amount of galactose (derivative), or the sugar (B) It is preferable to quantify only the glucose in the water.

  As described above, the sugar (B) quantification method includes phenol sulfate method, anthrone sulfate method, carbazole sulfate method and the like for quantification of all saccharides; It is done.

  There is no particular limitation on the time for measuring the concentration of sugar (B) in the fat body or blood. Immediately after galactose (derivative) is administered in step (b), sugar (B) by galactose (derivative) is used. It is sufficient to select from the period until the effect of decreasing the concentration of is not seen. Specifically, for example, 1 minute to 1 day is preferable from the time when the test substance is administered, and 30 minutes to 10 hours is particularly preferable.

  When measuring the concentration of sugar (B), “an invertebrate with an increased concentration of sugar (B) in the fat body or blood” to which galactose (derivative) is not administered is used as a negative control. It is preferable. For the negative control, for example, it is preferable to administer only the same amount of physiological saline. The galactose (derivative) is evaluated according to how much the concentration of the sugar (B) is decreased in the galactose (derivative) administered compared to the negative control.

<Step (d)>
In step (d), the concentration of sugar (B) in the fat body or blood of the invertebrate is selected from the galactose (derivative) used in steps (a), (b) and (c). Select the substance to be reduced.

  As described in detail in Examples, when hyperglycemic silkworms are used, the concentration of sugar (B) is reduced to 60% with insulin, 70% with metformin, 5-Aminoimidazole-4-Carboxamide Ribonucleoside , Abbreviated as “AICAR”) to 80%. This is because the substance screened in the steps including the steps (a) to (d), that is, the test substance selected in the step (d) has an effect as a hypoglycemic agent. Has been shown to be extremely effective in screening for drugs for the treatment and prevention of type 2 diabetes. Therefore, the blood glucose lowering effect of galactose (derivative) can also be evaluated by this method, and it is clear that one having a blood glucose lowering effect can be determined.

  Compared to the negative control, galactose (derivative) was administered, and how much the glucose concentration decreased was judged to be a significant difference and the test substance was selected, shown in the evaluation experiment example, Derived from insulin, metformin and AICAR results when the negative control glucose concentration is reduced to 95% to 80% or less.

  As will be described in detail in the examples, when D-galactose was ingested by a hyperglycemic silkworm, the glucose concentration became 60% or less to 70% or less. This indicates that D-galactose is effective as a hypoglycemic agent and is effective for preventing type 2 diabetes or improving symptoms. Moreover, this fact has shown that what is effective as a hypoglycemic agent in galactose (derivative) can be screened at the process including process (a)-(d).

[[Evaluation system action and principle]]
Although the action / principle for screening a substance that lowers blood glucose level such as humans in the steps including steps (a) to (d) is not clear, the following may be considered. That is, silkworms and the like have a mechanism to maintain homeostasis of blood sugar level, can store sugar in fat bodies corresponding to muscles and human liver, and have bombyxin which is an insulin-like peptide hormone. Downstream of bombyxin, there is an insulin signaling pathway including the MAPK signaling pathway similar to that in humans. In addition, recombinant human insulin has an action of enhancing the uptake of sugar in the fat body of silkworm through the activation of PI3 kinase. Furthermore, activation of AMP kinase, which is a pathway other than the insulin signal transduction pathway, reduces the blood glucose level of silkworms. That is, since silkworms have a mechanism similar to that of human blood glucose regulation, it is possible to screen for substances that lower blood glucose levels in humans, etc., and it is thought that galactose and galactose homopolymers have been discovered as blood glucose lowering agents. Therefore, it is considered that a blood glucose lowering agent can be screened from galactose (derivative).

[Hypoglycemic agent]
The present invention is a hypoglycemic agent characterized by containing galactose (derivative) or an isolated galactose homopolymer as an active ingredient. It is essential that the galactose homopolymer is isolated, and a hypoglycemic agent containing the galactose homopolymer without being isolated, such as an extract of a plant or the like, is not included in the present invention.

  Since galactose (derivative) and galactose homopolymer are stable substances and do not have high hygroscopicity, no special attention is required for handling. Therefore, regardless of whether it is used as the bulk powder; when it is formulated into various dosage forms such as liquids, granules, fine granules, powders, tablets, capsules, pills; Can be used in any form.

  When formulating galactose (derivative) or isolated galactose homopolymer, the excipient is not particularly limited, but inorganic salts, organic acid salts such as magnesium stearate, fillers such as talc, citric acid, etc. Organic acids and the like. Galactose (Derivatives and isolated galactose homopolymers can be formulated by themselves without using substances such as excipients that are usually used for formulation. The content of galactose (derivative) or isolated galactose homopolymer in each preparation is determined by the application target of the preparation; application purpose of treatment, improvement, prevention, etc .; What is necessary is just to adjust suitably by application methods;

[Food and Drink]
One aspect of the present invention is a food or drink for preventing diabetes or improving symptoms, characterized by adding a hypoglycemic agent containing galactose (derivative) or isolated galactose homopolymer as an active ingredient. is there. That is, galactose or a galactose derivative is a food or drink characterized by being added as an active ingredient for lowering blood glucose, and an isolated galactose homopolymer is added as an active ingredient for lowering blood sugar It is a food and drink.

  Here, “food and drink” includes all drinks and foods taken by humans. The food and drink of the present invention also includes feed for consumption by livestock, poultry, pets and the like. “Addition” means that a substance is added from the outside when producing a food or drink, and the substance is generated by a chemical reaction or the like over time in the already produced food or drink, resulting in the food or drink. It is not included when it comes to be contained. That is, for example, when lactose or the like is decomposed in a food and drink and, as a result, D-galactose is contained in the food or drink, it is not included in the present invention. In addition, the present invention is a food or drink to which a galactose homopolymer is added in an isolated form, and the galactose homopolymer is one of various multi-components from the beginning, such as “the extract itself of a plant body”. Foods and drinks contained as ingredients are not included in the present invention, and food and drink in which “extracts from plants etc.” containing galactose homopolymer as one of various multi-components from the beginning are added as they are. Products are not included in the present invention.

  One embodiment of the present invention is a food or drink product in which galactose (derivative) or an isolated galactose homopolymer is added as an active ingredient for lowering blood glucose. The “active ingredient for lowering blood sugar” is an ingredient that lowers blood sugar level and is effective in preventing diabetes or improving symptoms. The definition of “addition” is the same as described above. Moreover, even if it does not claim that the galactose (derivative) or the isolated galactose homopolymer is contained as an active ingredient for lowering blood glucose in food and drink, it is included in the present invention. Further, unless it is added only for a completely different purpose, it is included in the present invention even if it is not recognized that the addition of the galactose (derivative) or the isolated galactose homopolymer causes a decrease in blood glucose. .

  When adding galactose (derivative) or isolated galactose homopolymer to foods and drinks containing feed, the addition amount may be determined in consideration of taste, calorie intake, cost, etc., and galactose (derivative) ) Etc., there is no particular limitation on the amount added. As described above, if it is a form that substitutes for other sweeteners, a relatively large addition amount can be selected even when added as a hypoglycemic agent.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to these Examples, unless the summary is exceeded.

  For the completion of the present invention, the existence of an evaluation system for a hypoglycemic agent using an invertebrate such as a silkworm is important. A book that galactose, a monosaccharide that has a fundamental role in life activity, which has not been studied until the stage of using an animal model, can be used alone as an active ingredient of a hypoglycemic agent having a hypoglycemic effect. The basis of the invention has been conceived. Therefore, the evaluation system of the hypoglycemic agent using silkworms is first described in detail in Evaluation Examples and Evaluation Experiment Examples 1-11.

  The following evaluation examples and evaluation experimental examples 1 to 11 show that this evaluation system is correct in screening an active ingredient for lowering blood glucose, and at the same time, lower blood sugar from galactose (derivative). It also shows that things can be accurately screened. Moreover, it shows that the galactose used in the Example has an effect of lowering blood glucose level reliably in humans, livestock, poultry, pets and the like.

[Evaluation example]
<Type of silkworm, breeding conditions>
Silkworm fertilized eggs (a hybrid Hu / Yo x Tukuba / Ne) were purchased from Ehime Seed Co., Ltd. The hatched larvae were given artificial feed silk mate 2S (manufactured by Nippon Nosan Kogyo Co., Ltd.) at room temperature to grow to 5th instar larvae. As the breeding container, a square type No. 2 petri dish (manufactured by Eiken Equipment Co., Ltd.) was used from the egg to the second instar larva, and the disposable plastic food pack (Food Pack FD Daifuka, Chuo Chemical Co., Ltd.) was used thereafter. The breeding temperature was 27 ° C.

  The feed used for normal feed feeding used in the following evaluation experiment examples is artificial feed silk mate 2S (manufactured by Nippon Agricultural Industrial Co., Ltd.), and the feed used for feeding glucose-added feed is artificial feed silk mate 2S (Nippon Agricultural Industrial Co., Ltd.). It is prepared by mixing D-glucose with a predetermined mass ratio. In this evaluation experiment example, the “fasting group (hereinafter sometimes referred to as“ St ”” as appropriate) is a silkworm group that did not feed at all for a predetermined period of time. The term “ND” may be appropriately described) ”is a silkworm group fed with the artificial feed silk mate 2S for a predetermined period, and is referred to as a“ glucose-added feed fed group (hereinafter sometimes referred to as “GD” as appropriate). "Is a group of silkworms fed with a feed mixed with glucose at a predetermined mass ratio for a predetermined period.

<Reagent>
“Recombinant insulin” used in the following evaluation experiment examples was prepared by dissolving insulin (manufactured by Sigma) in physiological saline (0.9% NaCl) containing 0.1% acetic acid. Metformin (1,1-Dimethylbiguanide Hydrochloride, manufactured by Wako Pure Chemical Industries, Ltd.) was used after being dissolved in physiological saline. Wortmannin was purchased from CALBIOCHEM and used in DMSO.

<Measurement method of blood sugar concentration>
The method for measuring the sugar concentration in silkworm blood (hereinafter abbreviated as “blood glucose level”) is as follows. About 100 μL of silkworm blood was collected from where the first abdominal limb was cut and mixed with 9 volumes of 0.6 N (normal) perchloric acid to precipitate the protein. Centrifugation was performed at 3000 rpm for 10 minutes to obtain a body fluid extract (supernatant).

  The blood glucose level was quantified by the phenol sulfate method (Hodge et al). 100 μL of the body fluid extract diluted 10-fold with distilled water and 100 μL of 5% (w / v) phenol aqueous solution were mixed, and 500 μL of concentrated sulfuric acid was added and stirred vigorously. After standing at room temperature for 20 minutes, absorbance at 490 nm was measured. The blood glucose level was calculated using an aqueous glucose solution as a standard sugar solution. The denominator of the unit “mg / mL” is the volume of silkworm blood.

<Measurement method of blood glucose concentration>
The glucose concentration in the silkworm blood was quantified according to the usual glucose oxidase method. In the same manner as the measurement of the sugar concentration in the blood, a body fluid extract (supernatant) was obtained, and then diluted to an appropriate concentration with distilled water to 20 μL of the enzyme reaction solution (0.12 M 400 μL of Na-phosphate buffer (pH 7.4), 4 units / mL of glucose oxidase, 3 units / mL of peroxidase, 9 mM o-dianisidine) was mixed and allowed to stand at room temperature for 40 minutes. Thereafter, 100 μL of 70% concentrated sulfuric acid was added and stirred vigorously, and the absorbance at 530 nm was measured. The glucose concentration was calculated using an aqueous glucose solution as a standard sugar solution. The denominator of the unit “mg / mL” is the volume of the silkworm body fluid extract.

<Measurement method of sugar concentration in fat body>
The method for measuring the sugar concentration in the fat body of the silkworm is as follows. Silkworm fat body is opened with scissors and removed with tweezers, washed in physiological saline, and then added with potassium hydroxide (KOH) to a final concentration of 30% (w / v) at 100 ° C. Heat treated for 5 minutes. Further, ethyl alcohol was added to a final concentration of 60% (v / v), boiled to precipitate the sugar, allowed to stand at 4 ° C. overnight, collected by centrifugation (3000 rpm, 15 minutes), and quantified. The method for measuring the sugar concentration is the same as the method for measuring the sugar concentration in the blood. The denominator of the unit “μg / mg” (or “mg / g”) is the mass of the fat body of the silkworm.

<Measurement method of fat body sugar uptake>
The fasted 5th day old silkworm was dissected from the dorsal side and the fat pad was removed. The excised fat body was washed with Insect saline (130 mM NaCl / 5 mM KCl / 1 mM CaCl ₂ ), and then the mass was measured. In an experiment using wortmannin, wortmannin was added to the medium at the same time. After 30 minutes of pre-culture, 50 μL of insulin (3 mg / mL) was added to the medium, and the culture was continued at 27 ° C. After culturing, a fat pad extract was prepared by the above method, the sugar concentration in the fat pad was measured, and the sugar uptake per fat pad mass was calculated. The denominator of the unit “μg / mg” is the mass of the fat body of silkworm.

<Method of measuring the amount of phosphorylated Akt of fat body>
The fat pad excised from the silkworm was washed with Insect saline (130 mM NaCl / 5 mM KCl / 1 mM CaCl ₂ ) and then conditioned at 27 ° C. in 200 μL of Grace's insect medium supplemented with penicillin and streptomycin. In an experiment using wortmannin, wortmannin was added to the medium at the same time. After 30 minutes of pre-culture, 50 μL of insulin (3 mg / mL) was added to the medium and cultured at 27 ° C. for 1 hour. After the fat pad was washed with Insect saline (130 mM NaCl / 5 mM KCl / 1 mM CaCl ₂ ), NP-40 lysis buffer (10 mM Tris / HCl (pH = 7.5), 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT (dithiothreitol), 1% NP-40, 10 mM NaF, 1 mM Na ₃ VO ₄ ) was mixed with 250 μL of solution and sonicated for 20 seconds. Thereafter, TCA precipitation was performed, the protein was subjected to SDS electrophoresis, and transferred to a PVDF membrane. Western blotting was performed using an anti-Akt antibody and an anti-phosphorylated Akt antibody, and the amount of phosphorylated Akt (phosphorylated Akt amount / total Akt amount) in the fat pad was measured.

<Method of administration of test substance>
In Evaluation Experiment Example 4, Evaluation Experiment Example 5 and Evaluation Experiment Example 9, 0.05 mL of physiological saline containing a predetermined amount of each test substance was injected into the pattern part of the fifth somite of the 5th day-old silkworm. did. As a control, only 0.05 mL of physiological saline having the same amount was injected. The syringe (1 mL) and the injection needle (27Gx3 / 4) used the Terumo Corporation make.

Evaluation Experiment Example 1
It was examined whether or not the blood sugar level of silkworms was increased by feeding them with sugar-containing food. Silkworms on the first day of age 5 are divided into “fasting group”, “normal feed feeding group”, and “15% (w / w) glucose-added feed feeding group”, and 15% at 27 ° C. for 10 animals each. A diet containing (w / w) glucose was fed for 1 and 3 days. Thereafter, blood sugar level (mg / mL) and sugar concentration (μg / mg) in the fat body were measured. The results are shown in FIG. 1 (A) and FIG. 1 (B), respectively.

  As can be seen from FIG. 1 (A), the glucose-added feed-feeding group showed a blood glucose level about 3 times higher than that of the normal feed-feeding group in both the first and third day silkworms after the start of feeding. . In addition, the blood glucose level of the fasting group was about ½ of the normal feed feeding group.

  Moreover, as can be seen from FIG. 1 (B), the glucose concentration in the fat body of silkworms on the first day after the start of feeding is about twice as high in the glucose-added feed group as compared to the normal feed group. It was. In addition, the sugar concentration of the fasting group was 1/10 or less of the normal feed feeding group.

  Therefore, it was found from the above that the blood sugar level of the silkworm and the sugar concentration of the fat body are increased by giving the glucose-added feed to the silkworm. From these results, it was suggested that, similarly to humans, silkworms are taken up and stored in fat bodies due to an increase in blood glucose level in these silkworms.

Evaluation Experiment Example 2
With respect to silkworms on the first day of the fifth age, the time course of blood glucose level was examined when 3 mice were fed with food containing 33% glucose (w / w) (33% Glucose Diet). That is, the blood sugar levels of silkworms were measured in the same manner before starting feeding, after 30 minutes, after 60 minutes, and after 180 minutes. The results are shown in FIG.

  As can be seen from FIG. 2, the blood glucose level increased approximately 30 times after the start of feeding and approximately 3 times before the start of feeding in both about 60 times and 180 minutes after the start of feeding. . The above results show that, similarly to mammals, the blood sugar level rises rapidly by oral intake of sugars in silkworms. In addition, in the time passage of this range, the change in blood glucose level was not seen in the normal feed feeding group (Normal Diet) and the fasting group (Stavation).

Evaluation Experiment Example 3
Changes in the blood sugar level of silkworms were examined by changing the sugar content in the diet. That is, silkworms on the first day of age 5 are designated as “normal feed feeding group”, “8% (w / w) glucose-added feed feeding group”, “16% (w / w) glucose-added feed feeding group”, “33 % (W / w) glucose-added feed-feeding group ”, and 3 each were reared at 27 ° C., and blood glucose level was measured 60 minutes after the start of feeding. The results are shown in FIG. Similarly, the blood glucose level 180 minutes after the start of feeding was measured. The results are shown in FIG.

  As can be seen from FIGS. 3 and 9, the average blood glucose level of each group increased depending on the glucose content ratio of the feed without being saturated even if the glucose content was 33%.

Evaluation Experiment Example 4
In Evaluation Experimental Examples 1 to 3 and Evaluation Experimental Examples 7 to 9, conditions for making silkworms hyperglycemic were found. Next, it was examined whether or not a hypoglycemic drug used clinically reduces the blood glucose level of a hyperglycemic silkworm. A 5% -day-old silkworm (body weight 1 ± 0.1 g) was fed with a diet supplemented with 16% (w / w) glucose and bred at 27 ° C. for 60 minutes. “0.05 mL was administered” and “0.05 mL physiological saline solution containing 0.36 mg of recombinant human insulin was administered”, blood was collected 6 hours after the administration, and the sugar concentration was quantified. The results are shown in FIG.

  As can be seen from FIG. 4, the blood glucose level of the insulin administration group was reduced to about 60% compared to the blood glucose level of the physiological saline administration group. From this, it was shown that recombinant human insulin has an action to lower the blood sugar level of silkworm.

Evaluation Experiment Example 5
We examined whether metformin, a human hypoglycemic drug used in clinical practice, reduces the blood glucose level of hyperglycemic silkworms. Metformin is known as a biguanide hypoglycemic agent whose action mechanism is the promotion of glycolysis. That is, in Evaluation Experiment Example 4, except that “Metformin 0.1 mg / Saline 0.05 mL” was administered instead of “Recombinant human insulin 0.36 mg / Saline 0.05 mL”, the evaluation experiment example The same operation as in No. 4 was performed, and then the sugar concentration was quantified. The results are shown in FIG.

  As can be seen from FIG. 5, the blood glucose level of the metformin administration group was reduced to about 70% compared to the blood glucose level of the physiological saline administration group. From this, it was shown that metformin has the effect | action which reduces the blood glucose level of a silkworm.

  The amount of metformin administered (100 mg / g) was about 10 times the daily maximum human dose (12.5 mg / g). Metformin is a drug that lowers blood glucose levels by a mechanism of action different from that of insulin. Therefore, it was found that the method using silkworms is effective not only for type 1 diabetes but also for evaluation, screening, and production of therapeutic agents effective for type 2 diabetes that do not depend on a decrease in insulin secretion.

Evaluation Experiment Example 6
Next, an attempt was made to calculate the ED50 value of the action of insulin to reduce the blood sugar level of silkworms. A silkworm supplemented with 9% (w / w) glucose was given to 5th day old silkworms, and after feeding at 27 ° C. for 60 minutes, physiological saline and recombinant human insulin (0.005 mg to 0.5 mg) were administered. After 6 hours, blood was collected and the sugar concentration in the blood was quantified. FIG. 6 shows a graph of the relative blood glucose level reduction rate with the time when the 3% (w / w) glucose-added feed group was reduced to the blood glucose level being 100%.

  As can be seen from FIG. 6, the ED50 value of the hypoglycemic effect of silkworm by insulin was 0.015 mg / g. In clinical practice, the amount of insulin administered to a human per day is 0.14 to 3.53 mg. The calculated ED50 value was about 1000 times the daily dose for a human weighing 60 kg.

Evaluation Experiment Example 7
Examining the time course of blood glucose level and glucose concentration in silkworm blood when 3 years old were fed with food containing 15% (w / w) glucose for each 5th day silkworm It was. That is, the blood sugar level of the silkworm and the glucose concentration in the blood of the silkworm were measured in the same manner before starting feeding, after 30 minutes, after 60 minutes, and after 180 minutes. The results are shown in FIG. Next, the time course of the blood sugar level of the silkworm was examined when the silkworm was fed with a diet containing glucose 12% (w / w) at 27 ° C. for 60 minutes and then fasted. That is, the blood sugar levels of silkworms were measured in the same manner before feeding, 1 hour after feeding (0 hours after fasting), 2 hours, 5 hours, and 8 hours after fasting. The results are shown in FIG.

  As can be seen from FIG. 7 (a), after 30 minutes from the start of feeding, it is about twice before feeding, and after 60 and 180 minutes after feeding, it is about 3 times or more before feeding, and about 6 times or more. Blood sugar level was rising. Similarly, in the 15% glucose-added feed-fed group, the glucose concentration in the blood rapidly increased (FIG. 7 (b)). The above results show that, in silkworms, as in mammals, blood sugar levels and blood glucose concentrations increase rapidly by ingestion of sugar. In addition, in the time passage of this range, the change of a blood glucose level and the glucose level in blood was not seen in the normal feed feeding group (Normal Diet) and the fasting group (Stavation). Further, as can be seen from FIG. 8, the blood sugar level of the silkworm began to decrease 2 hours after the start of fasting. These results suggest that silkworms, like mammals, increase blood sugar levels due to excessive sugar intake and decrease over time.

Evaluation Experiment Example 8
The 5th day, 1st day silkworms, 3 each, were given normal diet or 5%, 10%, 15%, 30% (w / w) glucose supplemented diet, and the silkworms that had been raised for 3 days at 27 ° C. Body length, body weight and blood glucose level were measured. In addition, the silkworms of the first day of age 5 were reared without feeding any feed under the same conditions, and the body length, body weight and blood glucose level were measured in the same manner. The results are shown in FIG.

  From FIG. 10, each of the glucose-added feed feeding groups (5%, 10%, 15%, 30% GD) was shorter in body length and lighter than the normal feed feeding group (Normal Diet; ND) (FIG. 10). (A) to (c)). The blood glucose level at this time increased with the amount of glucose added (FIG. 10 (d)). It was also found that the same growth inhibition occurred when a glucose solution was injected into silkworms (data not shown). From these results, silkworms suggest that growth is inhibited by an increase in blood glucose level.

Evaluation Experiment Example 9
5th day old silkworms (body weight 1 ± 0.1 g) were fed with a diet supplemented with 12% (w / w) glucose and raised at 27 ° C. for 60 minutes. "0.05 mL was administered" or "0.05 mL physiological saline solution containing 0.36 mg of recombinant human insulin was administered", and blood was collected 1 hour, 3 hours, and 6 hours after Was measured. Furthermore, 12% (w / w) glucose-added feed was given to silkworms on the first day of 5 years old, and after breeding at 27 ° C. for 60 minutes, 3 each of them were “recombinant human insulin (0.005 mg˜ Administration of 0.05 mL of physiological saline solution containing 0.5 mg) ”, blood was collected 6 hours later, and blood glucose level was measured. The denominator of the unit “μg / g” in FIG. 11B is the body weight of the silkworm. The results are shown in FIG.

  Next, fat bodies were excised from 3 each of 5th and 1st day silkworms, and physiological saline or recombinant human insulin 0.7 mg was administered to Grace's medium supplemented with 1 mass% glucose. After incubation at 3 ° C. for 3 hours, the amount of phosphorylated Akt and the amount of sugar uptake of the extracted fat pad were measured. In addition, a fat body extracted from silkworms on the first day of age 5 was treated with wortmannin in the same manner, glucose was added, and recombinant human insulin was administered and cultured, and the amount of phosphorylated phosphorylated Akt and The amount of sugar uptake per fat body mass was measured. Furthermore, in order to qualitatively confirm the amount of phosphorylated Akt in the fat body, each sample was measured using an anti-Akt antibody and an anti-phosphorylated Akt antibody. The results are shown in FIG.

  As can be seen from FIGS. 11 (a) and 11 (b), it was found that the blood glucose level of the insulin administration group was reduced to about 60% as compared to the physiological saline administration group (FIG. 11 (a)). Moreover, the blood sugar level of the silkworm decreased depending on the dose of human insulin (FIG. 11 (b)). These results suggest that recombinant human insulin has an action to lower the blood sugar level of silkworm.

  As can be seen from FIGS. 11 (c) and 11 (d), the amount of phosphorylated Akt increased by adding recombinant human insulin to the in vitro tissue culture solution using the extracted fat pad. In addition, the addition of recombinant human insulin also increased the sugar uptake per fat body mass (FIG. 11 (e)). Further, it was found that phosphorylation of Akt and increase in fat body sugar uptake by this recombinant human insulin were suppressed by treatment with wortmannin, an inhibitor of PI3 kinase (FIGS. 11 (c) to (e)). ). From these results, it is suggested that recombinant human insulin has an action of enhancing sugar uptake of silkworm fat body through activation of PI3 kinase. Therefore, the hypoglycemic action of silkworms by recombinant human insulin is considered to be the same mechanism as that in humans.

Evaluation Experiment Example 10
Three silkworms on the first day of age 5 were given a feed supplemented with 12% (w / w) glucose, respectively, and reared at 27 ° C. for 60 minutes, then “saline 1 mL only” or “AICAR 4 mg / saline 1 mL” After 6 hours, blood was collected and blood glucose level was measured. In addition, after removing fat bodies of silkworms on the first day of age 5 and adding 50 μL of 500 μM AICAR and treating for 6 hours, AMPK activity in the fat bodies was converted to SAMs peptide, an artificial substrate of AMPK, and γ32P-ATP phosphorous. Measured using a reaction to add acid. The results are shown in FIG.

  As can be seen from FIG. 12 (a), the blood sugar level of silkworms in the AICAR administration group was lower than that in the physiological saline alone administration group (FIG. 12 (a)). From this result, it was found that AICAR, which is an activator of AMP kinase, has a blood glucose lowering effect on silkworms. It was also found that the AMPK activity was enhanced by treating the extracted fat pad with AICAR (FIG. 12 (b)). From the above, it is suggested that AICAR administered acts on the fat body of silkworm by the same mechanism as human and promotes AMPK activity.

  On the other hand, the injection of glibenclamide did not reduce the blood glucose level of silkworms (data not shown). Glibenclamide, a type of SU agent, is a drug that promotes insulin release by binding to the pancreatic β-cell SU receptor. In the silkworm, an organ having the same function as that of the pancreas has not been confirmed. The reason that the blood glucose level of silkworms was not decreased by the administration of glibenclamide is considered to be one of the reasons that silkworms do not have mammalian pancreatic β-cell-like cells. Therefore, when silkworms are used, it is considered that a drug having an insulin release promoting action such as glibenclamide cannot be evaluated.

Evaluation Experiment Example 11
Three silkworms on the first day of 5 years old, each with 3 normal feeds (Normal Diet; ND), 12% (w / w) glucose-added feed (Glucose Diet; GD) and 12% (w / w) glucose-added feed (Glucose Diet; GD) and recombinant human insulin 3.5 mg / mL were injected 50 μL, 5 times every 12 hours, and raised at 27 ° C. for 4 days, and then the body length and body weight of silkworms were measured. The results are shown in FIG.

  Silkworms fed with 12% glucose-added feed (GD) for 4 days had a smaller body length, decreased body weight, and growth inhibition was observed compared to silkworms in the normal feed (ND) feeding group (FIG. 13 (b) ) To (d)). When 12% glucose-added feed (GD) and recombinant human insulin were injected 5 times every 12 hours, an increase in body length and an increase in body weight were observed compared to those in which recombinant human insulin was not injected (FIG. 13). (A) to (d)). This result suggests that the inhibition of silkworm growth caused by hyperglycemia is recovered by the reduction of blood glucose level caused by recombinant human insulin.

  According to Evaluation Experiment Example 1 to Evaluation Experiment Example 11, the blood sugar level of the silkworm rises depending on the concentration of sugar (A) added to the feed and the feeding time, and insulin is a hypoglycemic drug for the hyperglycemic silkworm. , Metformin and AICAR were found to decrease the blood sugar level in silkworms. The method of the present invention is capable of lowering at least the blood glucose level of human beings, as it has been clarified that the arbitrarily selected “the above three substances effective for humans” lower the blood glucose level of silkworms in a specific state. It is a well-established method for evaluating and screening all drugs that have sex.

  Inhibition of silkworm growth caused by an increase in blood glucose level was recovered by the blood glucose lowering effect of human insulin. In diabetic mouse models, long-term rearing is known to cause uremia and paralysis of the feet, but inhibition of silkworm growth due to hyperglycemia can be observed within a few days. This is a method for simply and accurately evaluating and screening a substance that lowers blood glucose level.

  In clinical practice, a post-meal blood glucose level is determined to be hyperglycemic if it is twice or more that of a healthy person. By adjusting the concentration of sugar contained in the bait and the feeding time, we found a condition for making silkworms into a hyperglycemic state (twice the blood sugar level when feeding normal bait) (Evaluation Experiment Example 2 (FIG. 2)). Evaluation Experiment Example 3 (FIG. 3)). In clinical practice, administration (injection) of recombinant human insulin or metformin, which is used as a hypoglycemic agent, reduces the blood glucose level of hyperglycemic silkworms, and the hypoglycemic effect of insulin is the same in silkworms and humans. Therefore, it is possible to evaluate and screen for human blood glucose lowering agents using silkworms. Using this silkworm-based evaluation method and screening method for a blood sugar-lowering agent, it is possible to search for and determine a blood glucose-lowering agent for humans using the blood sugar-lowering activity of the silkworm as an index.

  A method of screening a compound that is a candidate for a hypoglycemic agent using a cultured cell system is generally performed as a method for developing a therapeutic agent for diabetes. However, most compounds that are generally effective in cultured cell lines do not exhibit a hypoglycemic effect in animal individuals. The reason is that the cultured cell system cannot reflect the pharmacokinetics in the individual. Therefore, it is essential to use an evaluation system using individual animals.

  As for all the results using the above silkworms, invertebrates such as “completely transformed insect larvae”, “larvae having a fat body in the body”, “animals belonging to insect class”, etc. Since it is common sense to think that similar or similar results can be obtained, it is obvious that the present invention is not limited to “Silkworm”.

Hereinafter, in the Example using the said evaluation system, it shows that galactose and the isolated galactose homopolymer are effective as a hypoglycemic agent.
Example 1
[Examination of the main component of the blood glucose lowering effect of Ziou]
[[Preparation of ethanol-precipitated fraction of Diou hot water extract]]
238 g of Diou (manufactured by Uchida Wakayaku Co., Ltd.) was extracted with 500 mL of 100% ethanol, and this extraction residue was pulverized and extracted twice with 500 mL of 70% aqueous ethanol in a boiling water bath for 8 hours. This extraction residue was subjected to hot water extraction with 500 mL of water in a boiling water bath three times for 8 hours. A quarter of this extract was subjected to subsequent operations. The hot water extract was dialyzed in distilled water, concentrated, added with 3 volumes of 100% ethanol with stirring, and allowed to stand at 4 ° C. for 24 hours. The precipitate fraction was collected by centrifugation to obtain an ethanol precipitate fraction of 1 g of Geothermal hot water extract as a lyophilized product. The flow of the preparation operation is shown in FIG.

[[Analysis of Constituent Sugars in Ethanol Precipitation Fraction of Geothermal Hot Water Extract]]
The ethanol precipitate fraction of the hot water extract prepared above was hydrolyzed with trifluoroacetic acid, developed with TLC, and detected with sulfuric acid. As a result, an almost single spot showing the same mobility as D-galactose was detected (FIG. 14). From this result, it was considered that the constituent sugar contained in the hot water extract was mainly D-galactose.

  Moreover, as a result of measuring molecular weight etc., it turned out that the active ingredient in the ethanol precipitation fraction of a geothermal hot water extract is a D-galactose homopolymer which 1000 or more molecules of D-galactose couple | bonded.

Example 2
[Evaluation of blood glucose-lowering effect of ethanol hot water extract ethanol precipitate fraction and D-galactose]
[[Study using hyperglycemia silkworm model]]
In the hyperglycemic silkworm model, a 12 mass% glucose-added feed was given to a silkworm on the first day of 5 years of age, and was bred at 27 ° C. for 60 minutes, and used for the following examination.

  As a negative control, 0.9% physiological saline was used, and as a positive control, recombinant human insulin (Sigma) was dissolved in physiological saline containing 0.1% acetic acid and used as a 3.5 mg / mL solution. It was.

[Subject 1]
The lyophilized product of the above “ethanol precipitation fraction of Geothermal hot water extract” was dissolved in 0.9% physiological saline to give a 1 mg / mL solution.
[Subject 2]
D-galactose (WAKO (Wako Pure Chemical Industries)) was dissolved in 0.9% physiological saline to obtain a 1 mg / mL solution.

[Body fluid extract]
Silkworm body fluid 20 μL was collected from the wound on the first abdominal limb and mixed with 9 times 0.6N (normal) perchloric acid to precipitate the protein. Centrifugation was performed at 3000 rpm for 10 minutes to obtain a body fluid extract.

[Measurement of sugar concentration (blood glucose level)]
The phenol-sulfuric acid method (Hodge et al.) Was used as a method for evaluating the sugar concentration (blood glucose level) of all kinds of sugars (B) in the body fluid. 100 μL of the body fluid extract diluted with distilled water at an appropriate concentration was mixed with 100 μL of 5% (w / v) aqueous phenol, 500 μL of concentrated sulfuric acid was added, and the mixture was vigorously stirred. After standing at room temperature for 20 minutes, the absorbance at 490 nm was measured. The sugar concentration (blood glucose level) was calculated from the value of the glucose standard solution.

[Measurement of glucose concentration]
When administering D-galactose as a monosaccharide, there is a possibility that D-galactose may be affected by the phenol-sulfuric acid method, so the glucose oxidase method capable of evaluating the glucose concentration was used. 20 μL of a sample diluted to an appropriate concentration with distilled water and 400 μL of an enzyme reaction solution (0.12 mol Na-phosphate buffer pH 7.4, glucose oxidase 4 units / mL, peroxidase 3 units / mL, o-dianisidine 9 mM) Mixed and left at room temperature for 40 minutes. Thereafter, 100 μL of 70% sulfuric acid was added and stirred vigorously, and the absorbance at 530 nm was measured. The glucose concentration was calculated from the value of the glucose standard solution.

[Subject administration to hyperglycemic silkworm and measurement of sugar concentration (blood glucose level) and glucose concentration in body fluid]
Saline, human insulin and subject 1 (ethanol-precipitated ethanol extract) were each administered to 0.05 mL hyperglycemic silkworm, and after 6 hours, the body fluid was collected and the sugar concentration in the body fluid (blood glucose level) ) Was measured. Each measurement was performed using 6 to 10 silkworms per group. The obtained result is shown in FIG.

  Similarly, physiological saline, human insulin, and subject 2 (D-galactose) were each administered to 0.05 mL hyperglycemic silkworms, and the glucose concentration in the body fluid was measured. The obtained results are shown in FIG.

<Result>
The blood glucose concentration of hyperglycemic silkworms administered with the ethanol precipitation fraction of the hot water extract of Jiou was clearly reduced (FIG. 15). In addition, the glucose concentration of hyperglycemic silkworms administered with D-galactose, which is the main constituent sugar of the ethanol precipitation fraction of the Diou hot water extract, was also clearly reduced (FIG. 16). From this, it turned out that D-galactose homopolymer and D-galactose are effective as a hypoglycemic agent.

Example 3
[Study of blood glucose lowering effect of D-galactose]
[[Examination using hyperglycemic mouse model]]
Streptozotocin-treated hyperglycemic mice were used. The hyperglycemic mouse was an 8-week-old male C57BL / 6J Jcl mouse, purchased from CLEA Japan, and used for examination after confirming that the blood glucose level was 230 mg / mL or higher.

  As a negative control, 0.9% physiological saline was used, and the subject was D-galactose (WAKO (Wako Pure Chemical Industries)) dissolved in 0.9% physiological saline to obtain a 2.5 mg / mL solution. . The glucose concentration was measured in the same manner as described above.

  A physiological saline solution and a subject (D-galactose) were each administered in an amount of 0.5 mL, and after 6 hours, blood was collected from a cut cut with scissors on the tail of the mouse. The blood glucose concentration was measured using 20 μL of blood. Each measurement was performed using 3 mice per group. The obtained result is shown in FIG.

<Result>
When D-galactose was administered to mice that became hyperglycemic by treatment with streptozotocin, the blood glucose concentration was clearly reduced compared to the negative control. From this, it was confirmed that D-galactose has an effect of lowering the blood glucose level of a hyperglycemic mouse which is a mammal.

  In addition, as described above, galactose (derivative) is known to be transported paracellularly by a route that does not involve a transporter called a paracellular pathway and taken into blood in large quantities (the above-mentioned literature (1), ( 2)). Therefore, if the effect is obtained by any one of the administration methods of injection into blood, injection into the intestinal tract, and oral ingestion, the effect is considered to be obtained by other administration methods as well. Therefore, from the results of Example 3, D-galactose is considered to decrease the blood glucose level of hyperglycemic mice by oral ingestion.

  For the first time, the present invention has revealed that D-galactose and the isolated D-galactose homopolymer have an effect of lowering blood glucose level, and can provide a novel therapeutic agent for diabetes. D-galactose is clearly different from the conventionally reported hypoglycemic agents in that it contains a sugar essential for life activity as an active ingredient. Although the mechanism of the blood glucose lowering effect has not yet been clarified, by analyzing the mechanism of the blood glucose lowering effect of D-galactose in the future, diabetes treatment or prevention of D-galactose (derivative) or D-galactose homopolymer, etc. It is thought that further effective use will be planned.

  In addition, when a biological substance such as galactose acts on a cell, it binds to a receptor protein on the cell surface and the signal is transmitted into the cell. Therefore, it is self-evident that if the substance has an affinity for the receptor on which galactose acts, a galactose derivative other than galactose may exhibit a hypoglycemic action as in galactose.

  As described above, among galactose derivatives, a blood glucose-lowering agent can be screened by at least the steps including the steps (a ′) to (d ′). Since screening targets are narrowed down, blood sugar lowering agents can be screened extremely efficiently.

  The hypoglycemic agent of the present invention is safe, has properties suitable for formulation, addition to foods and beverages, and the like and can be expected to be used differently from conventional hypoglycemic agents. Can be widely used. Moreover, since it can be easily added to food and drink, it can be widely used as a functional food for preventing and improving diabetes.

  This application is based on Japanese Patent Application No. 2008-178450 filed on Jul. 8, 2008, the entire contents of which are incorporated herein by reference and disclosed in the specification of the present invention. It is taken in.

Claims (4)

Hypoglycemic agent characterized that you containing galactose as an active ingredient.   The hypoglycemic agent according to claim 1, which is used for the treatment of diabetes. By using a silkworm, a method for screening a candidate hypoglycemic agent from galactose derivatives, to not at least the following steps (a) (d),
(A) by ingesting sugar silkworm, obtained in step (b) the step (a) increasing the concentration of sugar in the fat or in the blood of the silkworm, sugar fat or in the blood of the silkworm concentration rises, the step (d) the test substance to measure the concentration of sugar step (c) above the test substance in the fat body of silkworm administered or blood of administering a test substance how from screening candidates for hypoglycemic agents, which comprises the step of selecting the agent that reduces the concentration of sugar in the fat or in the blood of the silkworm in.   A therapeutic agent for diabetes comprising the blood glucose lowering agent according to claim 1 or 2.