JP2008266215A - Post-ischemic angiogenesis promoter and blood flow improving agent - Google Patents

Abstract

Provided is a novel therapeutic agent useful for the treatment and prevention of various ischemic diseases.
SOLUTION: General formula (1)

(In the formula, Glc represents a glucose residue, and n represents an integer of 0 or 1 or more.) A post-ischemic angiogenesis promoter comprising a quercetin glycoside represented by the formula as an active ingredient. In particular, the total amount of quercetin glycosides containing n = 3 quercetin glycosides and n = 1 to 3 quercetin glycosides is 50 mol% or more, and n is 4 or more. It is.
[Selection] Figure 1

Description

  The present invention relates to a post-ischemic angiogenesis promoting agent and a blood flow improving agent useful for the prevention or treatment of various ischemic diseases.

  In recent years, the number of obese people has been steadily increasing due to westernization of eating habits, lack of exercise due to convenience of living, disorder of lifestyle habits, etc.Furthermore, high blood sugar, high fat plasma, high blood pressure, etc. When concomitant, these are defined as lifestyle-related diseases, or metabolic syndrome, and are listed as one of the most modern diseases to be treated as greatly increasing the risk of serious cardiovascular diseases such as arteriosclerosis. Furthermore, arteriosclerosis is a major cause of chronic vascular occlusion along with thrombus formation, arteritis, trauma, etc. If this vascular occlusion persists, an ischemic state based on poor blood circulation has occurred in the surrounding tissues around the occlusion site and downstream. Eventually, tissue necrosis may result.

  In particular, limbs in diabetic patients frequently develop chronic obstructive arteriosclerosis, and this pathological condition also involves diabetic neuropathy, and presents a so-called diabetic foot pathology. If this condition progresses, it may develop into a diabetic skin ulcer, and it is not uncommon for infection to lead to amputation. As described above, skin ulcer is one of the important diabetic complications to be noted because it has a great impact on the prognosis of diabetic patients and causes a decrease in quality of life (QOL).

  In recent years, the treatment of chronic obstructive arteriosclerosis caused by vascular occlusion has led to the development of surgical techniques in the field of vascular surgery and the development of various intervention techniques to prevent severe ischemia. A good prognosis can be expected. However, there are still some cases in which revascularization is impossible, such as a wide range of obstruction cases and peripheral obstruction cases that are likely to be associated with diabetes, and this treatment method is naturally a large-scale operation. The burden on patients is still a problem.

  In recent years, “therapeutic angiogenesis”, which is an attempt to improve an ischemic state by inducing angiogenesis at an ischemic site and developing a collateral blood circulation, has attracted attention as a new treatment method. This method is largely divided into a method of administering vascular growth factor and a method of administering vascular endothelial progenitor cells. In the former case, treatment by administration of a protein of blood vessel growth factor such as VEGF (Vascular endothelial growth factor), HGF (Hepatic growth factor), bFGF (Basic fibroblast growth factor), or introduction of these genes is applicable. Although these treatments have been improved, there are some problems such as safety of protein recombinants, concerns about side effects caused by systemic circulation of highly physiologically active substances, and vector safety during gene transfer. In the latter case, autologous bone marrow mononuclear cell transplantation, including the transfer of peripheral blood-derived vascular endothelial progenitor cells (EPC), and this method has also been reported to be effective in increasing the number of cases gradually. However, the patient burden at the time of bone marrow collection and the complexity of cell fractionation remain as problems.

  In relation to the above, administration of human G-CSF (Granulocyte-colony stimulating factor), a differentiation and growth factor of hematopoietic progenitor, is expected to increase the level of hematopoietic stem cells in bone marrow and peripheral blood. Attempts have also been made to improve blood.

  Other treatment methods include exercise therapy and drug therapy. In the former case, it is limited to the case of so-called mild ischemic symptoms that can be walked or jogged, etc., and there is a current situation where a large improvement effect has not been obtained, and patients who are not satisfied with the therapeutic effect want revascularization Not a few. Regarding drug therapy, vasodilators and antiplatelet drugs are also applied, but there are not many examples in which a large therapeutic effect has been obtained by drug therapy alone, and it cannot be fundamental treatment.

Quercetin (3,3 ′, 4 ′, 5,7-pentahydroxyflavone) is a kind of polyphenol compound and is known to have a strong antioxidant activity (see Non-Patent Document 1) and has attracted attention. Various glycosides including rutin and quercitrin are known. These quercetins and quercetin glycosides are known to have different oral absorbability and different levels of antioxidant power due to differences in structure. For example, quercetin glycoside (Quercetin-4'-β-D-glucoside, Quercetin-3,4'-β-D-glucoside), which is abundant in onions, has been reported to be more absorbable than quercetin. (See Non-Patent Document 2). Similarly, it has been reported that isoquercitrin in which glucose is β-bonded at the 3-position of quercetin (Quercetin-3-β-D-glucoside) has higher absorbability than quercetin and rutin (Non-patent Document 3). reference).
Middlton EJ. Et al., Pharmacol Rev., 52, 673-751, 2000 Hollman PC et al., Arch Toxicol Suppl., 20, 237-248, 1998 Morand C et al., Free Rad Res., 33, 667-676, 2000

  An object of this invention is to provide the novel therapeutic agent useful for the prevention or treatment of various ischemic diseases.

  The present inventors have conducted extensive research on the action of a specific quercetin glycoside, and found that it promotes angiogenesis after ischemia and improves blood flow, thereby completing the present invention.

  That is, the first aspect of the present invention is the general formula (1):

(Wherein Glc represents a glucose residue, and n represents an integer of 0 or 1 or more) is a post-ischemic angiogenesis promoting agent comprising a quercetin glycoside represented by the formula as an active ingredient.

  The post-ischemic angiogenesis promoter preferably has the following aspects. The quercetin glycoside is a composition that includes at least n = 3 quercetin glycoside and satisfies the following requirement (a). (A) The total amount of quercetin glycosides with n = 1 to 3 contained in the composition is 50 mol% or more, and the total amount of quercetin glycosides with n of 4 or more is 15 mol% or less. . In the present specification, “a quercetin glycoside with n = 1 to 3” means “a quercetin glycoside with n = 1, 2, or 3.”

  The second aspect of the present invention is a compound represented by the general formula (1):

(Wherein Glc represents a glucose residue, and n represents an integer of 0 or 1 or more) is a blood flow improving agent comprising a quercetin glycoside as an active ingredient.

  The blood flow improving agent preferably has the following aspect. The quercetin glycoside is a composition that includes at least n = 3 quercetin glycoside and satisfies the following requirement (a). (A) The total amount of quercetin glycosides with n = 1 to 3 contained in the composition is 50 mol% or more, and the total amount of quercetin glycosides with n of 4 or more is 15 mol% or less. .

  According to the present invention, a novel post-ischemic pro-angiogenic agent useful for the treatment, amelioration or prevention of various ischemic diseases is provided, and novel blood useful for the improvement or prevention of blood flow reduction or failure. A flow improver is provided.

  The present invention is a post-ischemic angiogenesis promoting agent and a blood flow improving agent comprising quercetin glycoside as an active ingredient.

  The quercetin glycoside used in the present invention is represented by the following formula (1).

In formula (1), Glc represents a glucose residue, and n represents 0 or an integer of 1 or more.

  That is, the quercetin glycoside used in the present invention is isoquercitrin (Quercetin-3-β-D-glucoside) in which glucose is β-bonded at the 3-position of quercetin represented by the following formula (2) (hereinafter simply referred to as “IQC”). (The compound in which n is 0 in the formula (1)), and each α-glycosylisoquercitrin in which glucose is further added to the glucose residue of the IQC by, for example, about 1 to 15 by α-1,4 bonds. (A compound in which n is about 1 to 15 in Formula (1)) may be any, or a mixture thereof. In the present specification, when referring to a mixture of quercetin glycosides composed of a plurality of types of compounds, it may be referred to as a “quercetin glycoside composition” or “quercetin glycoside mixture”.

Since the IQC and α-glycosylisoquercitrin are both quercetin glycosides, in the present specification, they may be collectively referred to as “quercetin glycoside” without distinguishing both. In addition, α-glycosyl isoquercitrin corresponds to a glycoside of IQC. Therefore, in the present specification, it may be referred to as “IQC glycoside” in order to distinguish it from IQC.

  In the present specification, for convenience of explanation, in the above formula (1), IQC represented by n = 0 is simply “G0”, and IQC glycoside represented by n = 1 (glucose is α-1,4 bonded to IQC. GQ), an IQC glycoside represented by n = 2 (a glycoside in which two glucoses are bonded to IQC through α-1,4 bonds) and “G2”, n = G3 glycoprotein (Glucose in which 3 glucoses are linked to αC by α-1,4 bonds), and IQC glycoside (n = 4 is glucose -Glycoside with 4 linkages with -1,4 bonds) is "G4", and an IQC glycoside with n = 5 (glycoside with 5 glucoses with α-1,4 linkages attached to IQC) “G5”, an IQC glycoside represented by n = 6 (a glycoside in which 6 glucoses are bonded to IQC through α-1,4 bonds) is referred to as “G6”,... And IQC glycoside is used in which n is m to as "Gm" (the glucose to IQC are m bound glycoside by alpha-l, 4 bonds) (m is an integer of 7 or higher).

  In the present invention, the quercetin glycoside includes at least n = 3 quercetin glycoside and satisfies the following requirement (a) (hereinafter also referred to as “super quercetin glycoside composition”): Is preferably used. (A) The total amount of quercetin glycosides with n = 1 to 3 contained in the composition is 50 mol% or more, and the total amount of quercetin glycosides with n of 4 or more is 15 mol% or less. . This is because it is well absorbed by the body by oral administration.

  In the present specification, “total amount of isoquercitrin G (1-3)” or “total amount of IQC-G (1-3)” refers to n contained in the quercetin glycoside composition of interest. Means the total amount of 1 quercetin glycoside (G1), n is 2 quercetin glycoside (G2), and n is 3 quercetin glycoside (G3). In the present specification, “total amount of isoquercitrin G (4 ≦)” or “total amount of IQC-G (4 ≦)” means that n contained in the quercetin glycoside composition of interest is 4 or more. Means the total amount of quercetin glycoside.

  The post-ischemic angiogenesis promoting agent of the present invention promotes angiogenesis after ischemia and improves blood flow, thereby improving myocardial infarction, heart failure, angina pectoris, cerebral infarction, vascular dementia, obstructive arteriosclerosis. It can be used as a therapeutic agent for various ischemic diseases such as symptom and Buerger disease. Moreover, by administering when an ischemic state is observed, it can be used as a prophylactic agent for preventing the progression of these ischemic diseases and the damage caused by ischemia.

  The forms of the post-ischemic angiogenesis promoting agent and the blood flow improving agent according to the present invention are not particularly limited, and can be provided in the form of pharmaceuticals, quasi drugs, cosmetics, and foods, for example.

  When provided as a pharmaceutical, its dosage form is not particularly limited, and is appropriately selected and used as necessary. Tablets, capsules, granules, fine granules, powders, sustained-release preparations, suspensions Liquids, emulsions, syrups, elixirs, and other oral preparations, injections, suppositories, coatings, patches, and other parenteral preparations. For example, when using a super quercetin glycoside composition as an active ingredient In particular, the form of an oral preparation is more preferable than having excellent oral absorbability.

  In the case of an oral preparation, it can be prepared by blending quercetin glycoside with a component such as a diluent, a carrier or an additive and carrying out any formulation treatment. The diluent or carrier used herein is not particularly limited as long as it does not interfere with the effects of the present invention. For example, sucrose, glucose, fructose, maltose, trehalose, lactose, oligosaccharide, dextrin, dextran, cyclodextrin Sugars such as ethanol, propylene glycol, glycerin; sugar alcohols such as sorbitol, mannitol, erythritol, lactitol, xylitol, maltitol, reduced palatinose, reduced starch degradation products; Examples include solvents such as triacetin; polysaccharides such as gum arabic, carrageenan, xanthan gum, guar gum, gellan gum, and pectin; or water. Examples of additives include auxiliaries such as chelating agents, fragrances, spice extracts, preservatives, and the like.

  The ratio of the quercetin glycoside compounded in the post-ischemic angiogenesis promoting agent and the blood flow improving agent is not particularly limited and can be appropriately selected from the range of 0.01 to 100% by weight. When the preparation is prepared using the diluent, carrier or additive for convenience in use, the quercetin glycoside is 0.01 to 100% by weight, preferably 0.00%, in 100% by weight of the preparation. It is desirable to prepare such that it is contained at a ratio of 1 to 50% by weight.

  The amount of post-ischemic angiogenesis promoting agent or blood flow improving agent is not particularly limited as long as it exhibits post-ischemic angiogenesis promoting action or blood flow improving action in vivo, but in the case of an adult weighing 60 kg, It can be appropriately selected from a range in which quercetin glycoside is included in the preparation at a ratio of 1 mg to 30 g per administration.

  The food containing the blood flow improving agent according to the present invention is prepared by adding the above diluent, carrier, additive or the like to the quercetin glycoside itself or the quercetin glycoside composition according to the present invention. The quercetin glycoside of the present invention is blended as a component in, for example, supplements or general foods such as pharmaceutical preparations (eg powders, granules, tablets, capsules, liquids, drinks, etc.), and blood for the living body is contained in the foods. Examples include functional foods (including specific health foods and conditionally specified health foods) that have a flow improving effect. These foods contain the quercetin glycoside according to the present invention and have a blood flow improving action, and are used for the blood flow improving action in a living body. Includes food with a label.

  In the case of a food having an effect of improving blood flow, the ratio of quercetin glycoside mixed in the food is not particularly limited as long as it has the function, but it is usually selected appropriately from the range of 0.001 to 100% by weight. it can.

  The target food is not limited, but ice cream, ice milk, lacto ice, sherbet, frozen desserts such as ice confectionery; milk beverage, lactic acid bacteria beverage, soft drink (including fruit juice), carbonated beverage, fruit juice beverage, vegetable Beverages such as beverages, vegetable and fruit beverages, sports beverages, powdered beverages; alcoholic beverages such as liqueurs; tea beverages such as coffee beverages and tea beverages; soups such as consomme soup and potage soup; custard pudding, milk pudding, Puddings such as pudding with fruit juice, desserts such as jelly, bavaroa and yogurt; gums such as chewing gum and bubble gum (board gum, sugar-coated granule gum); coated chocolate such as marble chocolate, strawberry chocolate, blueberry chocolate And chocolate with flavor such as melon chocolate Chocolate such as rate; hard candy (including bonbon, butterball, marble, etc.), soft candy (including caramel, nougat, gummy candy, marshmallow, etc.), drop, toffee and other caramels; Baked confectionery such as rice crackers; pickled in soy sauce, pickled in soy sauce, pickled in salt, miso pickled, pickled in salmon, pickled in bonito, pickled in cucumber, pickled in cucumber, pickled in moromi, pickled in plum, pickled in Fukujin, pickled in ginger, pickled in Korean, pickled in plum Pickles such as separate dressings, non-oil dressings, ketchup, sauce, sauces, etc .; jams such as strawberry jam, blueberry jam, marmalade, apple jam, apricot jam, prazabu; fruit wine such as red wine; Cherry, apricot, apple, strawberry, Processed fruits such as ham, sausages, grilled pork, etc .; fish meat ham, fish sausage, fish meat surimi, salmon, bamboo rings, hampen, fried Satsuma, Date roll, whale bacon, etc .; dairy products such as cheese Kinds of noodles such as udon, cold wheat, somen, buckwheat, Chinese noodles, spaghetti, macaroni, rice noodles, harsame and wonton; various other prepared foods and various processed foods such as rice cakes and rice fields.

  The manufacturing method of the quercetin glycoside used for this invention is not specifically limited. Hereinafter, an example of a quercetin glycoside composition that can be used as an active ingredient of the present invention and an example of a method for preparing the same will be described.

(I) Enzyme-treated isoquercitrin and its preparation method In the present invention, the term “enzyme-treated isoquercitrin” means that a glucose residue transferase is acted on IQC in the presence of a sugar donor (glucose source) according to a conventional method. Means a mixture of IQC and α-glycosyl isoquercitrin that is glucosylated to various extents, as shown in the following formula (for example, FFI Journal Vol. 209, No. 7, 2004, p. .622-628, Food Hygiene Journal, Vol.41, No.1, pp.54-60).

(In the formula, Glc represents a glucose residue, and n represents 0 or an integer of 1 or more)
Specifically, “enzyme-treated isoquercitrin” is an IQC in which α-1,4-bonded glucose number (n) is 0 in the above formula, and α-1,4-bonded glucose number (n) is 1 or more, Usually a mixture with α-glycosyl isoquercitrin in the range of 1-15, preferably 1-10.

  Examples of the glucose residue transferase used for the glycosylation treatment of IQC include glucosidases such as α-amylase (EC 3.2.1.1) and α-glucosidase (EC 3.2.1.20); or cyclodextrin glucanotransferase ( EC2.4.1.19) (hereinafter abbreviated as CGTase) and the like.

  All of these glycosyltransferases are commercially available enzymes. Examples of such commercially available enzyme agents include Contizyme (trade name) (manufactured by Amano Enzyme Co., Ltd.). The amount of glycosyltransferase used is, for example, CGTase [enzyme specific activity of about 100 units (the amount of enzyme that produces 1 mg of β-cyclodextrin from soluble starch per minute is defined as 1 unit)] isoquercitrin. The range of 0.001-20 weight part of glycosyltransferases can be mentioned with respect to 1 weight part. Preferably, it is about 0.005 to 10 parts by weight, more preferably about 0.01 to 5 parts by weight.

  Any sugar donor (glucose source) used for glycosylation may be used as long as at least one molecule of the glucose residue can be transferred to one molecule of IQC. Examples thereof include glucose, maltose, amylose, amylopectin, starch, starch liquefaction product, starch saccharification product, and cyclodextrin. The amount of the glucose source used is usually 0.1 to 20 parts by weight, preferably 0.5 to 15 parts by weight, more preferably 1 to 10 parts by weight based on 1 part by weight of isoquercitrin present in the reaction system. The ratio of a weight part can be mentioned.

  “Enzyme-treated isoquercitrin” is usually 80 ° C. or lower, preferably about 20 to 80 ° C., more preferably about 40 to 75 ° C., and usually about pH 3 to 11, preferably pH 4 to 8, and the above sugar donor. It can be prepared by allowing glucose residue transferase to act on IQC in the presence of (glucose source), and usually has the following composition.

However, the total amount of IQC of G1 to G3 is 65% by weight or less, and the total amount of IQC of G4 or more is 15% by weight or more.

  The above reaction can be performed while standing or stirring or shaking. In order to prevent oxidation during the reaction, the head space of the reaction system may be replaced with an inert gas such as nitrogen, and an antioxidant such as ascorbic acid may be added to the reaction system.

  The enzyme-treated isoquercitrin can be prepared starting from rutin as well as using isoquercitrin as a raw material as described above. In this case, α-1,6-rhamnosidase (E.C.3.2.1.40) is allowed to act on rutin to change to isoquercitrin, and then enzyme-treated isoquercitrin can be prepared according to the above method. The α-1,6-rhamnosidase may have any activity, and commercially available products include hesperidinase and naringinase (both manufactured by Tanabe Seiyaku Co., Ltd.), and cellulase A “Amano” 3 (Amano Enzyme Co., Ltd.). ))).

  In the post-ischemic angiogenesis promoting agent or blood flow improving agent according to the present invention, as an active ingredient, for example, the enzyme-treated isoquercitrin itself, the enzyme-treated isoquercitrin is further purified, What extracted the component of length, or what combined them arbitrarily can be used.

(II). Super Quercetin Glycoside Composition A quercetin glycoside composition (super quercetin glycoside composition) preferably used in the present invention is a mixture of quercetin glycosides represented by the following formula (1): Including n = 3 α-glycosylisoquercitrin.

(In the formula, Glc represents a glucose residue, and n represents 0 or an integer of 1 or more)
More specifically, the super quercetin glycoside composition includes α = glycosylisoquercitrin (G3) of formula (1), and satisfies the requirement (a) below:
(a) A quercetin glycoside in which the total amount of n = 1 to 3 quercetin glycosides (IQC-G (1-3)) contained in the composition is 50 mol% or more and n is 4 or more The total amount of the body (IQC-G (4 ≦)) is 15 mol% or less.

  A composition containing a large amount of α-glycosylisoquercitrin (G1, G2, G3) having an α-1,4-bonded glucose number (n) of 1 to 3 to IQC is a known enzyme-treated isoquercitrin or glucose number. A composition containing a large amount of (n) 4-6 α-glycosylisoquercitrin (G4, G5, G6) or an α-glycosylisoquercitrin (G3, G4, G5, Compared with a composition containing a large amount of G6), the absorbability into the body (transferability into blood) by oral administration is high. Furthermore, among α-glycosylisoquercitrins having a glucose number (n) of 1 to 3, particularly α-glycosylisoquercitrin (G3) having a glucose number (n) of 3 and then a glucose number (n) of 2. α-Glycosyl isoquercitrin (G2) has high in vivo absorbability (transferability to blood). On the other hand, when α-glycosylisoquercitrin (G4) having a glucose number (n) of 4 is absorbed into the body (transferability into blood) compared to α-glycosylisoquercitrin (G3) having a glucose number (n) of 3. ) Tend to decrease.

  As described above, the super quercetin glycoside composition contains G3, and the total amount of IQC-G (4 ≦) is 15 mol% or less, and IQC-G (1-3) The total amount is 50 mol% or more, preferably 55 mol% or more, more preferably 60 mol% or more, still more preferably 65 mol% or more, still more preferably 70 mol% or more, still more preferably 75 mol% or more. Particularly preferably, it is contained in a proportion of 80 mol% or more, more preferably 85 mol% or more.

  As described above, when the quercetin glycoside composition contains α-glycosylisoquercitrin [IQC-G (4 ≦)] having an n of 4 or more in a high ratio, the absorbability into the body (transferability into blood) is reduced. There is a tendency. For this reason, it is preferable that the content ratio (total amount) of IQC-G (4 ≦) in the super quercetin glycoside composition is lower than 15 mol%. For example, the content ratio of IQC-G (4 ≦) can be 10 mol% or less, preferably 6 mol% or less.

  The super quercetin glycoside composition may contain isoquercitrin (IQC) (G0) in which n is 0. However, the lower the content of IQC (G0), the higher the total amount of α-glycosyl isoquercitrin (IQC-G (1-3)) of n = 1 to 3 occupying the entire quercetin glycoside composition. It is preferable because it can be performed. The proportion of IQC (G0) contained in the quercetin glycoside composition of the present invention is, for example, 45 mol% or less, preferably 30 mol% or less, more preferably 20 mol% or less, and even more preferably 10 mol% or less. Can be illustrated.

The super quercetin glycoside composition preferably satisfies the following requirement (b) in addition to the requirement (a):
(b) The amount of quercetin glycoside with n = 0 contained in the composition is 20 mol% or less.

As another preferred embodiment of the super quercetin glycoside composition, in addition to the requirement (a) or in addition to the requirements (a) and (b), the following requirement (c) is satisfied. You can mention:
(C) α-glycosylisoquercitrins (G2 and G3) of n = 2 and 3 are included, and the total amount of these (IQC-G (2-3)) is 50 mol% or more of the whole.

  More preferably, the total amount of IQC-G (2-3) is 55 mol% or more, 60 mol% or more, 65 mol% or more, 70 mol% or more, or 75 mol% or more.

Further, as another preferred embodiment of the super quercetin glycoside composition, a composition containing G3 and having a total amount of IQC-G (4 ≦) of 15 mol% or less and satisfying the following requirement (d) is mentioned. Can:
(D) A quercetin glycoside in which the total amount of n = 1 to 3 quercetin glycosides (IQC-G (1-3)) contained in the composition is 60 mol% or more and n is 0 (IQC) (G0) is 20 mol% or less.

  As a super quercetin glycoside composition satisfying the above requirement (d), the total amount of quercetin glycosides (IQC-G (1-3)) of n = 1 to 3 is more preferably 70 mol% or more, preferably 80 The mol% or more, more preferably 85 mol% or more. Preferably, the total amount of quercetin glycosides with n of 4 or more is 10 mol% or less, preferably 6 mol% or less. More preferably, the quercetin glycoside (IQC) (G0) with n = 0 is 10 mol% or less.

As another embodiment of the super quercetin glycoside composition, in the formula (1), an α-glycosylisoquercitrin (G3) having n = 3 and satisfying the requirement of the following (e) can be exemplified. :
(e) A quercetin glycoside in which the total amount of n = 1 to 3 quercetin glycosides (IQC-G (1-3)) contained in the composition is 70 mol% or more and n is 4 or more The total amount of the body (IQC-G (4 ≦)) is 10 mol% or less, and the quercetin glycoside (IQC) (G0) in which n is 0 is 20 mol% or less.

  As a quercetin glycoside composition satisfying the above requirement (e), the total amount of IQC-G (1-3) is more preferably 75 mol% or more, preferably 80 mol% or more, more preferably 85 mol% or more. . Preferably, the total amount of IQC-G (4 ≦) is 6 mol% or less. More preferably, the quercetin glycoside (G0) where n is 0 is 10 mol% or less.

(III) Method for Preparing Super Quercetin Glycoside Composition A super quercetin glycoside composition having high oral absorbability is obtained by using general formula (3):

(In the formula, Glc represents a glucose residue, and n represents an integer of 4 or more.)
The quercetin glycoside (IQC-G (4 ≦)) represented by the formula (1) can be prepared by reducing the total amount to 20 mol% or less.

  Here, regardless of the method for reducing the amount of IQC-G (4 ≦), for example, a method for fractionating and removing IQC-G (4 ≦) from enzyme-treated isoquercitrin, which is included in enzyme-treated isoquercitrin Any method such as a method of decomposing IQC-G (4 ≦) may be used. Preferably, a method of treating enzyme-treated isoquercitrin with amylase can be mentioned.

  The amylase used here may be an enzyme having amylase activity, and its origin is not particularly limited. For example, α-amylase (EC3.2.1.1), β-amylase (EC3.2.1.2), α-glucosidase (EC3.2.1.20), glucoamylase (EC3.2.1.3), malto-oligosaccharide-generating enzymes such as maltotriohydrolase Can be mentioned.

Preferable examples include β-amylase. When β-amylase is used as the amylase, the content ratio of α-glycosylisoquercitrin (IQC-G (4 ≦)) in which the number of α-1,4-linked glucose residues (n) is 4 or more is selectively set. It can be reduced to increase the content of n = 1-3 α-glycosylisoquercitrin (IQC-G (1-3)) contained in the composition. Therefore, the quercetin glycoside composition of the invention which contains G3 and satisfies the requirements of the following (a) can be easily prepared.
(a) The total amount of IQC-G (1-3) contained in the composition is 50 mol% or more, and the total amount of IQC-G (4 ≦) is 15 mol% or less.

  It is known that β-amylase suitably used is contained in soybean, barley, wheat, radish, sweet potato, Aspergillus oryzae, Bacillus cereus, Bacillus polymyxa, Bacillus megaterium, etc. Can be used freely. β-amylase is a commercially available enzyme. For example, as β-amylase derived from soybean, “β-amylase # 1500” (manufactured by Nagase ChemteX Corp.), “Biozyme M5” (Amano Enzyme Ltd.) Barley-derived β-amylase includes “β-amylase L” (manufactured by Nagase Chemtech Co., Ltd.), “Biozyme / ML” (manufactured by Amano Enzyme Co., Ltd.), and “Maltoteam 206” (Nagase). Chemtex Co., Ltd.): "Biozyme M" (manufactured by Amano Enzyme Co., Ltd.) as the β-amylase derived from the germ: "Uniase L" (Yakult Pharmaceutical Co., Ltd.) as the β-amylase derived from Aspergillus oryzae ))).

  The β-amylase is not necessarily purified, and may be a crude product as long as it is possible to prepare a superquercetin glycoside composition. For example, a fraction containing enzyme-treated isoquercitrin and β-amylase (for example, an extract such as soybean or barley) may be mixed and reacted. Alternatively, β-amylase may be immobilized and reacted with enzyme-treated isoquercitrin in a batch or continuous manner.

  The reaction conditions for β-amylase are not particularly limited as long as β-amylase acts on enzyme-treated isoquercitrin. Preferably, G3 is included, and the total amount of IQC-G (4 ≦) is 15 mol% or less, and IQC-G (1-3) as a total amount is 50 mol% or more, preferably 55 mol% or more. More preferably, it is 60 mol% or more, more preferably 65 mol% or more, still more preferably 70 mol% or more, still more preferably 75 mol% or more, particularly preferably 80 mol% or more, still more preferably 85 mol%. The conditions which produce | generate the quercetin glycoside composition contained in the above ratio can be mentioned. Further, the conditions for producing a quercetin glycoside composition containing IQC-G (4 ≦) in a content (total amount) lower than 15 mol%, for example, 10 mol% or less, preferably 6 mol% or less. Can be mentioned.

  As a reaction condition of β-amylase, for example, when 4000 U / g enzyme is used, the amount of β-amylase used is in the range of 0.0001 to 0.5 parts by weight with respect to 1 part by weight of enzyme-treated isoquercitrin. Can be appropriately selected and used. Preferably it is about 0.0005-0.4 weight part, More preferably, it is about 0.001-0.3 weight part. The amount of the enzyme-treated isoquercitrin in the reaction system is not particularly limited, but for the purpose of efficiently carrying out the reaction, it is usually 0.1 to 20% by weight, preferably 0. It is desirable to contain 5 to 10% by weight, more preferably 1 to 10% by weight.

  As the reaction temperature, a range of about 80 ° C. or less can be mentioned, and it can be appropriately selected and used within this range. Within this range, industrially advantageous is about 20-80 ° C, preferably about 40-75 ° C. The pH condition is usually about pH 3 to 11 or less, preferably pH 4 to 8.

  The reaction can be performed with standing or stirring or shaking. In order to prevent oxidation during the reaction, the head space of the reaction system may be replaced with an inert gas such as nitrogen, and an antioxidant such as ascorbic acid may be added to the reaction system.

  In addition, the process which further reduces isoquercitrin can also be performed with respect to the reaction product acquired by said method as needed. Such a method is not particularly limited as long as it is a method capable of removing and desorbing isoquercitrin (IQC) (G0) from the reaction product obtained by the above method, and a conventional purification method is arbitrarily combined. be able to.

  For example, a method of precipitating and removing IQC (G0) by adjusting the reaction product to be acidic and cooling, various resin treatment methods (adsorption method, ion exchange method, gel filtration method, etc.), membrane treatment method, etc. (Ultrafiltration membrane treatment method, reverse osmosis membrane treatment method, ion exchange membrane treatment method, zeta potential membrane treatment method, etc.), electrodialysis method, salting out, acid precipitation, recrystallization, solvent fractionation method, activated carbon treatment method, etc. Can be illustrated.

  The IQC removal step may be performed on the reaction product after the amylase treatment as described above, but may be performed on the enzyme-treated isoquercitrin before the amylase treatment, for example. In particular, when β-amylase is used as the amylase, there is almost no change in the IQC content before and after the amylase treatment. Therefore, even when β-amylase treatment is performed after removing IQC from enzyme-treated isoquercitrin in advance, and when IQC is removed and reduced after treatment with enzyme-treated isoquercitrin with β-amylase, the resulting reaction product is obtained. There is almost no difference in things.

  The quercetin glycoside composition thus obtained further increases the ratio of IQC-G (1-3) in the whole as a result of the decrease in the content ratio of IQC-G (4 ≦) and IQC (G0). Can do. As such a quercetin glycoside composition, the ratio of IQC-G (1-3) is preferably 60 mol% or more, preferably 65 mol% or more, more preferably 70 mol% or more of the whole (100 mol%), More preferred is a composition of 75 mol% or more, still more preferred is 80 mol% or more, and particularly preferred is 85 mol% or more. Among them, a composition in which the ratio of IQC-G (2-3) is 50 mol% or more of the whole (100 mol%), preferably 55 mol% or more, more preferably 60 mol% or more, and further preferably 65 mol% or more. Further, a quercetin glycoside composition that further accounts for 70 mol% or more, particularly preferably 75 mol% or more is a more preferable composition in terms of absorbability in the body. The ratio of IQC-G (4 ≦) and IQC (G0) in the quercetin glycoside composition can be determined according to the content of the IQC-G (1-3). Examples of the ratio of G (4 ≦) are preferably 10 mol% or less, preferably 2 mol% or less, and the ratio of IQC (G0) is 20 mol% or less, preferably 10 mol% or less.

Hereinafter, the present invention will be described with reference to preparation examples, experimental examples, and formulation examples, but the present invention is not limited to these examples.
Reference Preparation Example 1: Preparation of enzyme-treated isoquercitrin (1) Preparation of isoquercitrin A 250 g Enju bud, a leguminous plant, was immersed in 2500 mL of hot water (95 ° C or higher) for 2 hours, and then filtered. Was obtained as the “first extract”. On the other hand, the residue separated by filtration was further immersed in hot water and extracted to obtain a “second extract”. These first and second extracts are combined, cooled to 30 ° C. or lower, the precipitated components are filtered, and the precipitate is washed with water, recrystallized, and dried to obtain 22.8 g of rutin having a purity of 95% or more. Obtained.

  20 g of this rutin was dispersed in 400 mL of water and adjusted to pH 4.9 using a pH adjuster. To this was added 0.12 g of Naringinase (Amano Enzyme Co., Ltd., trade name “Naringinase“ Amano ””, 3,000 U / g) to start the reaction, which was maintained at 72 ° C. for 24 hours. Thereafter, the reaction solution was cooled to 20 ° C., and the precipitate produced by cooling was separated by filtration. The obtained precipitate (solid content) was washed with water and dried to recover 13.4 g of isoquercitrin.

(2) Preparation of enzyme-treated isoquercitrin To 10 g of isoquercitrin obtained above, 500 mL of water was added and 40 g of corn starch was added and dispersed. To this was added 15 g of cyclodextrin glucanotransferase (CGTase: Amano Enzyme Co., Ltd., trade name “Contizyme”, 600 U / mL) to start the reaction, and this was continued for 24 hours under the conditions of pH 7.25 and 60 ° C. Retained. After cooling the obtained reaction solution, it was added to a column (Φ3.0 × 40 cm) of Diaion HP-20 (Mitsubishi Chemical Industry Co., Ltd.) and washed with 1000 mL of water. Next, 600 mL of 50% by volume ethanol aqueous solution was applied to the column, and the obtained eluate was concentrated under reduced pressure, and then freeze-dried, followed by enzyme-treated isoquercitrin [hereinafter referred to as “isoquercitrin G (mix)” or Obtained 12.8 g] (referred to as “IQC-G (mix)”). This was subjected to HPLC under the following conditions to separate each component, and each component was analyzed using a mass spectrometer (LC / MS / MS, Japan Waters, model Quattro Micro).

<HPLC conditions>
Column: Inertsil ODS-2 Φ4.6 × 250mm (GL Science)
Eluent: Water / acetonitrile / TFA = 850/15/2
Detection: Absorbance measurement flow rate at a wavelength of 351 nm: 0.8 mL / min.

  As a result, it was found that the enzyme-treated isoquercitrin [IQC-G (mix)] consists of a mixture of IQC and various IQC glycosides represented by the following formula.

(In the formula, Glc represents a glucose residue, and n represents 0 or an integer of 1 or more.)
From the results of HPLC analysis, the molar ratio (%) of IQC and each IQC glycoside contained in the above mixture was calculated based on the following formula, and the composition was as shown in Table 2.

Preparation Example 1: Preparation of isoquercitrin G (1-3) fraction and isoquercitrin G (3-6) fraction Enzyme-treated isoquercitrin (IQC-G (mix)) obtained in Reference Preparation Example 1 0.65 g was dissolved in water-containing methanol, and gel filtration chromatography was performed using a gel filtration resin (Sephadex LH-20: Amersham Bioscience KK.). After separating a certain amount of the flow-through solution, HPLC analysis was performed under the conditions described in Reference Preparation Example 1 above. G3 with 3 α1,4 glucose bonds, G4 with 4 bonds, and G5 with 5 bonds to IQC. , And a fraction rich in 6 linked G6 (hereinafter referred to as “isoquercitrin G (3-6) fraction” or “IQC-G (3-6)” fraction), and glucose in IQC Fraction rich in 1 α1,4 linked G1, 2 linked G2, and 3 linked G3 (hereinafter referred to as “isoquercitrin G (1-3) fraction” or “IQC-G (1 -3) Fractions ")). Subsequently, the solvent was removed from each of these two fractions by concentration under reduced pressure, and then lyophilized to obtain “isoquercitrin G (3-6) fraction” (“IQC-G (3-6) fraction”) 0 .15 g and “Isoquercitrin G (1-3) fraction” (“IQC-G (1-3) fraction”) 0.1 g were obtained. These fractions were subjected to HPLC analysis under the conditions described in Reference Preparation Example 1 described above, and the molar ratio (%) of IQC and each IQC glycoside contained in each fraction was calculated.

  The results are shown in Table 3. As can be seen from Table 3, the ratio of G1, G2 and G3 contained in the IQC-G (1-3) fraction was 94% in total, and G3 contained in the IQC-G (3-6) fraction, The ratio of G4, G5 and G6 was 86% in total.

Experimental example: Observation of angiogenesis promotion after ischemia (1) Preparation of unilateral lower limb ischemia model and administration of quercetin glycoside
One-sided femoral artery of 24-32 week old male mouse (C3H / HeJ) was ligated and dissected to create a lower limb ischemia model mouse. A total of 30 model mice were prepared and used as a quercetin glycoside administration group (N = 15) and a distilled water administration group (N = 15) according to the present invention. In the former, the IQC-G (1-3) fraction prepared in Preparation Example 1 was once a day, 100 mg / kg per day for a total of 6 weeks from 2 weeks before ischemic surgery to 4 weeks after surgery. It was given by oral gavage, and the latter was similarly given distilled water.

(2) Blood flow measurement with laser Doppler blood flow meter On the day of surgery, 1, 2, 3, and 4 weeks later, a total of 5 times, the mouse was anesthetized with pentobarbital and placed in a cage placed on a 40 ° C hot plate. After heating for about 10 minutes, the blood flow of both lower limbs was quantified using a laser Doppler blood flow meter (Moor) on a 40 ° C. hot plate, and an image reflecting the blood flow was obtained. FIG. 1 shows blood flow on the ischemic limb side / normal limb side, and FIG. 2 shows an image reflecting blood flow in the lower limb of one mouse belonging to each administration group ((a) quercetin glycoside administration group (B) Distilled water administration group).

(3) Quantification of capillary density 4 weeks after surgery (after measuring blood flow with a laser Doppler blood flow meter), the mice were sacrificed by deep anesthesia, and the cross-section of the muscle tissue of the ischemic limb was anti-CD31 antibody (BD Immunohistochemical staining was performed with Pharmingen. The number of capillaries per unit area was counted per 10 fields in an optical microscope 200-fold field and averaged. In FIG. 3, the capillary density of a quercetin glycoside administration group (a) and a distilled water administration group (b) is shown.

(4) Discussion From FIG. 1, the ratio of blood flow on the ischemic limb side / normal limb side in the distilled water administration group was about 0.6 in 2-4 weeks after the operation, whereas the quercetin glycoside according to the present invention. The administration group showed about 0.9, and it was found that the blood flow improvement (promoting angiogenesis) action was significant at 3 and 4 weeks after the operation.

  From FIG. 2, the blood flow on the ischemic limb side is not improved in the distilled water administration group, whereas the blood flow is improved in the quercetin glycoside administration group to about the same level as the healthy limb. It can be seen that the effect of (promoting angiogenesis) was exhibited.

FIG. 3 shows that the quercetin glycoside-administered group showed a significant increase in blood vessel density and promoted angiogenesis compared to the distilled water-administered group.
Formulation Example (1) Formulation Example 1: Tablet
(weight%)
IQC-G (1-3) fraction (Preparation Example 1) 18
Lactose 78
Sucrose fatty acid ester 4
The above ingredients were mixed uniformly to give a tablet of 250 mg per tablet.

(2) Formulation Example 2: Powder and granules
(weight%)
IQC-G (1-3) fraction (Preparation Example 1) 18
Lactose 60
Starch 22
The above components were mixed uniformly to obtain a powder or granule.

(3) Formulation Example 3: Capsule
(weight%)
Gelatin 70.0
Glycerin 22.9
Methyl paraoxybenzoate 0.15
Propyl paraoxybenzoate 0.35
Water remaining
The granules prepared in Formulation Example 2 were filled in the soft capsule skin made of the above components by a conventional method to obtain 250 mg soft capsules.

(4) Formulation Example 4: Drink
Taste: DL-sodium tartrate 0.10 g
0.009 g of succinic acid
Sweetness: liquid sugar 800.00 g
Sour: Citric acid 12.00 g
Vitamins: Vitamin C 10.00 g
IQC-G (1-3) fraction (Preparation Example 1) 1.80 g
Vitamin E 30.00 g
Cyclodextrin 5.00 g
Fragrance 15.00ml
Potassium chloride 1.00 g
Magnesium sulfate 0.50 g
The above ingredients were blended and water was added to make 10 liters. This drink is prepared so that the dose per administration is about 250 ml.

(5) Formulation Example 5: Sugar 98g
Minamata (Brix 75) 91g
75 g of concentrated solution of the IQC-G (1-3) fraction (Preparation Example 1) (Brix 40)
Each of the above components were mixed well and boiled until the water content reached 2% to produce 2 g of persimmon.

  The post-ischemic angiogenesis promoter of the present invention is useful as a pharmaceutical agent for preventing or treating various ischemic diseases. Moreover, the blood flow improving agent of the present invention is useful for preventing or improving blood flow reduction or failure.

It is a figure which shows the change of the blood-flow ratio of the ischemic limb side / normal limb side of a quercetin glycoside administration group and a distilled water administration group. It is a figure which shows the image by the laser Doppler blood flow meter of a quercetin glycoside administration group and a distilled water administration group. It is a figure which shows the capillary density of the 4th week after a surgery of a quercetin glycoside administration group and a distilled water administration group.

Claims (4)

General formula (1):
(In the formula, Glc represents a glucose residue, and n represents an integer of 0 or 1 or more.) A post-ischemic angiogenesis promoter comprising a quercetin glycoside represented by the formula as an active ingredient. The post-ischemic angiogenesis promoter according to claim 1, wherein the quercetin glycoside is a composition that includes at least n = 3 quercetin glycoside and satisfies the following requirement (a):
(A) The total amount of quercetin glycosides with n = 1 to 3 contained in the composition is 50 mol% or more, and the total amount of quercetin glycosides with n of 4 or more is 15 mol% or less. . General formula (1):
(Wherein Glc represents a glucose residue, and n represents an integer of 0 or 1 or more) A quercetin glycoside represented by the formula is used as an active ingredient. The blood flow improving agent according to claim 3, wherein the quercetin glycoside is a composition containing at least n = 3 quercetin glycoside and satisfying the following requirement (a):
(A) The total amount of quercetin glycosides with n = 1 to 3 contained in the composition is 50 mol% or more, and the total amount of quercetin glycosides with n of 4 or more is 15 mol% or less. .