JP2010202580A - alpha-GLUCOSIDASE INHIBITOR DERIVED FROM NATURAL MATERIAL - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an α-glucosidase inhibitor derived from natural materials, having high safety, producible from an easily available raw material and having high α-glucosidase inhibition activity. <P>SOLUTION: The α-glucosidase inhibitor derived from natural materials contains, as an active component, an extract obtained by extracting a pericarp of Trapa japonica with a solvent such as water, methanol, ethanol and a mixture of arbitrary two or more kinds of these solvents. There are provided the α-glucosidase inhibitor having high safety, producible from easily available natural raw materials and having high α-glucosidase inhibiting activity, and pharmaceutical compositions, foods, health foods and feeds produced by using the inhibitor. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to an α-glucosidase inhibitor, and more particularly to a natural product-derived α-glucosidase inhibitor that is highly safe and can be produced from readily available raw materials and has high α-glucosidase inhibitory activity.

Diabetes is a group of diseases characterized by chronic hyperglycemia due to decreased insulin supply or lack of action, and various metabolic abnormalities. In severe cases, coma can lead to death in the worst case. When metabolic abnormalities persist for a long period of time, protein glycation triggers the production of intracellular oxidative stress substances, which are specific to the retina, kidneys, and nerves. Cause complications and atherosclerosis. The number of diabetic patients, one of lifestyle-related diseases, has been increasing in recent years, and including the reserve army is estimated to be about 13.7 million, or about 1 in 10 people (Ministry of Health, Labor and Welfare 1997) Annual diabetes survey).

Diabetes is treated by controlling blood glucose levels through diet therapy, exercise therapy, and drug therapy. Examples of oral agents for diabetes include sulfonylurea (SU) agents, biguanide (BG) agents, insulin resistance agents, α-glucosidase inhibitors, and the like. Of these, α-glucosidase inhibitors are drugs that competitively inhibit carbohydrate hydrolases such as maltase and sucrase present in the small intestinal mucosa microvilli and delay the absorption of carbohydrates in the intestinal tract. Alfa-glucosidase inhibitors such as acarbose and voglibose have already been used clinically. The α-glucosidase inhibitor has the effect of suppressing a significant increase in blood glucose after meals and reducing the daily fluctuation range of blood glucose, and is also considered to be applicable to patients with insulin-dependent diabetes mellitus. Patent Document 1). Acarbose was found in the culture solution of actinomycetes Actinoplanes strain SE 50, and voglibose was found in the culture solution of the actinomycetes Streptomyces hygroscopicus sub sp. Limoneus (see Patent Document 1).
As an alternative to these pharmaceuticals, there are reports of substances having an α-glucosidase inhibitory action present in plants. For example, Patent Document 2 discloses a preventive and therapeutic agent for diabetes comprising a powder and / or extract of a plant belonging to the genus Lilyaceae. Patent Document 3 discloses an α-glucosidase inhibitor characterized by containing a chestnut skin extract and a food containing the same. Patent Document 4 discloses a carbohydrase inhibitor obtained by extracting chestnut astringent skin with ethanol or an ethanol solution.

JP-A-9-67271 JP 2003-81858 A JP 2006-1872 A Special table 2008-512345 gazette

Ota Ikuo, “Recent Advances in the Treatment of Diabetes Mellitus”, Journal of Nippon Medical University, Nippon Medical School Medical Association, June 1999, Vol. 66, No. 3, p. 195-198

However, as a specific example of a plant belonging to the genus Lilyaceae used as a preventive and therapeutic agent for diabetes comprising a powder and / or extract of the lilyaceae genus plant described in Patent Document 2, Paris polyphylla is exemplified. ), Heavy towers (Palis petiolata), and grandchildren (Paris tetraphylla) are not readily available in Japan. In addition, the chestnut skin (demon skin and astringent skin), which is a raw material for the α-glucosidase inhibitor described in Patent Document 3 and the saccharide-degrading enzyme inhibitor described in Patent Document 4, is poor in eating experience, and the contained components are sufficiently safe. Sex has not been proven. Chestnuts take time to grow and it is difficult to secure a large amount of astringent skin as a raw material.

The present invention has been made in view of such circumstances, and an object thereof is to provide a natural product-derived α-glucosidase inhibitor that can be produced from a highly safe and easily available raw material and has high α-glucosidase inhibitory activity. And

As a result of extensive research, the present inventor has found that an extract obtained by solvent extraction of persimmon pericarp (solvent extract of persimmon pericarp) exhibits high natural product-derived α-glucosidase inhibitory activity. The invention has been completed.

Thus, the first aspect of the present invention that meets the above-described object is to provide a natural product-derived α-glucosidase inhibitor characterized by containing, as an active ingredient, an extract obtained by solvent extraction of persimmon peel (Trapa Japonica). By providing, the above-mentioned problems are solved.
Hoshi (Trapa Japonica) is an annual grass that grows naturally in ponds and swamps throughout Japan and throughout Asia, so it is easy to obtain and can be harvested in one year, so it can be stably supplied in large quantities. In addition, the fruit contains a lot of starch, has been used for food since ancient times, and is said to have nourishing tonic and healthy stomach action, making it a medicinal herb, herbal medicine and healthy tea. As described above, Hishi has abundant dietary experience and has demonstrated safety for the human body. Therefore, the α-glucosidase inhibitor provided by the present invention can be produced in a large amount and at a low cost, is highly safe for the human body, and has a high α-glucosidase inhibitory activity.

In the natural product-derived α-glucosidase inhibitor according to the first aspect of the present invention, the extraction solvent is preferably water, methanol, ethanol, and any two or more mixed solvents thereof.
These solvents have high extraction efficiency of the α-glucosidase inhibitor, are inexpensive, and have a relatively low environmental load and toxicity to the human body, and therefore do not require much labor in handling and treatment of waste liquid.

The natural product-derived α-glucosidase inhibitor according to the first aspect of the present invention may have one or more hydrolyzed tannins as a main component.
A solvent extract of persimmon pericarp contains a large amount of hydrolyzed tannin, and particularly a product obtained by subjecting persimmon pericarp to solvent extraction exhibits high α-glucosidase activity. Further, it is different from a saccharide-degrading enzyme inhibitor derived from chestnut astringent peel mainly composed of condensed tannin, and has higher α-glucosidase inhibitory activity.

The second aspect of the present invention solves the above-mentioned problem by providing a pharmaceutical composition comprising one or more of the natural product-derived α-glucosidase inhibitors according to the first aspect of the present invention. Is.

A third aspect of the present invention solves the above problem by providing a food comprising one or more of the natural product-derived α-glucosidase inhibitors according to the first aspect of the present invention. is there.

4th aspect of this invention solves the said subject by providing the feed characterized by including 1 or more of the natural product origin alpha-glucosidase inhibitor which concerns on 1st aspect of this invention. is there.

According to the present invention, there is provided a natural product-derived α-glucosidase inhibitor that can be produced in a large amount and at low cost, has high safety to the human body, and has high α-glucosidase inhibitory activity. In addition, according to the present invention, food, health food, supplements and pharmaceutical compositions that are highly safe and have high diabetes preventive effect and hypoglycemic action, and are preferably used for prevention of diabetes and alleviation of hyperglycemic state, etc. Is provided.

It is a graph which shows the relationship between the density | concentration of the hot water extract of a persimmon peel and a chestnut peel, and alpha-glucosidase inhibition efficiency. It is a graph which shows the result of having compared the inhibition efficiency of (alpha)-glucosidase of the distilled water of the hot water extract of the persimmon skin peeled using the crosslinked polystyrene column, 50% methanol aqueous solution, and a methanol fraction. It is a graph which shows the result of having compared the inhibitory efficiency of (alpha)-glucosidase in the solvent extract of the persimmon and chestnut peel obtained using various extraction solvents. It is the chromatogram obtained by the high performance liquid chromatography (HPLC) analysis of the 50% methanol aqueous solution fraction of the hot water extract of a persimmon peel. It is the chromatogram (UV) obtained by the HPLC analysis of the product which carried out the acid hydrolysis of the 50% methanol aqueous solution fraction of the hot water extract of a persimmon peel. It is the chromatogram (RI) obtained by the HPLC analysis of the product which carried out the acid hydrolysis of the 50% methanol aqueous solution fraction of the hot water extract of a persimmon peel. It is the chromatogram (PDA, Maxplot method) obtained by the HPLC analysis of the acid hydrolyzate of the hot water extract of a persimmon peel. It is the chromatogram (PDA, Maxplot method) obtained by the HPLC analysis of the acid hydrolyzate of the hot water extract of chestnut peel.

Subsequently, an embodiment of the present invention will be described to provide an understanding of the present invention.
The natural product-derived α-glucosidase inhibitor according to one embodiment of the present invention contains, as an active ingredient, an extract (solvent extract of persimmon pericarp) obtained by solvent extraction of persimmon pericarp (Trapa Japonica) pericarp. Yes. Hoshi peels can be collected from raw fruits, dried after collection, or collected from dried fruits, and any method prior to solvent extraction can be used to improve extraction efficiency. It may be used for pretreatment such as crushing or crushing.

As the solvent used for the solvent extraction, water, an arbitrary solution solvent that is liquid at normal temperature, and a mixed solvent in which any two or more of these are mixed in an arbitrary ratio can be used. Preferred extraction solvents are water, methanol, and the like. , Ethanol and a mixed solvent in which any two or more of these are mixed in an arbitrary ratio, and particularly preferable extraction solvent is water, ethanol which is an organic solvent recognized as a food additive, and these mixed in an arbitrary ratio Mixed solvent. Alternatively, a supercritical fluid (SCF) such as water or carbon dioxide can be used.

When water or a mixed solvent of water and another solvent is used as the extraction solvent, an acid, a base, a salt, or the like may be appropriately included as necessary in order to improve extraction efficiency. Although there is no restriction | limiting in particular about the temperature and pH of the water used for extraction, About pH, it is preferable that it is near neutrality in consideration of the use to a biological body, More specifically, it is pH 4-9, 6-8 More preferably. If necessary, a heated extraction solvent may be used in order to improve the extraction efficiency.

Solvent extraction can be carried out by any known method. For example, after mixing a persimmon peel in a solvent for a predetermined time, separation from solids by filtration, centrifugation, decantation, etc., continuous Soxhlet extraction method, etc. A method such as an extraction method can be used.

The solvent extract of the persimmon peel may be used as it is in the extract, or may be powdered by freeze drying or spray drying. In any case, pretreatment by dialysis, ultrafiltration, filtration, column chromatography or the like may be performed as necessary in order to remove high molecular weight components, insoluble components, and the like. Alternatively, a fraction having high α-glucosidase inhibitory activity may be fractionated by reverse phase chromatography, ion chromatography or the like.

The natural product-derived α-glucosidase inhibitor thus obtained has an action of inhibiting the activity of α-glucosidase. α-Glucosidase is an enzyme that catalyzes a reaction of hydrolyzing an α-glycoside bond of a sugar, and maltase (EC 32.0.) that hydrolyzes an α-glycoside bond of maltose to produce two molecules of glucose. 20) Glucamylase (EC 3.2.1.3) which produces glucose by degrading the 1,4-α glycoside bond at the non-reducing end of the sugar chain, and glucose by hydrolyzing the α-glycoside bond of sucrose. And fructose-producing sucrase (EC 32.0.48), and isomaltase (EC 3.2.1.10) that hydrolyzes the α-glycoside bond of isomaltose to produce three molecules of glucose. Broadly divided. The α-glucosidase inhibitor has high inhibitory activity against any of these.

When removing an insoluble matter etc. by filtration, you may add adsorbents and filter aids, such as activated carbon, bentonite, and celite, in order to remove an impurity as needed. In particular, when used in the state of an extract, it is preferable to perform sterilization filtration using a membrane filter or the like.

By mixing a natural product-derived α-glucosidase inhibitor with a carrier or the like, it can be used as a pharmaceutical composition having one or both of a therapeutic effect and a preventive effect for diabetes and related diseases and symptoms. Examples of the dosage form of the pharmaceutical composition for humans or animals include oral, rectal, parenteral (for example, intravenous administration, intramuscular administration, subcutaneous administration, etc.), and the dosage is the formulation of the pharmaceutical composition. However, in the case of humans, the active ingredient generally contained in the formulation is difficult to determine and uniquely determined depending on the administration method, purpose of use, and age, weight, and symptoms of the subject to be applied. The amount is preferably 0.1 to 2000 mg / day per day for adults. Of course, since the dosage varies depending on various conditions, an amount smaller than the above dosage may be sufficient or may be necessary beyond the above range.

When it is prepared as an oral preparation, it can be prepared in the form of tablets, granules, powders, capsules, coatings, liquids, suspensions, etc. When it is made into a parenteral preparation, injections, drops, It can be prepared in the form of a suppository or the like. Any known method can be used for formulation. For example, an α-glucosidase inhibitor and a pharmaceutically acceptable carrier or diluent, a stabilizer, and other desired additives can be blended to obtain the above desired dosage form.

The food containing a natural product-derived α-glucosidase inhibitor is a food containing a natural product-derived α-glucosidase inhibitor, or any form normally used for food or health food such as capsules and tablets. Can do. There are no particular restrictions on the type of food to be blended, for example, liquid (fluid) foods such as coffee, fruit juice, soft drinks, beer, milk, miso soup, soup, tea, tea, nutrients, syrup, margarine, jam, etc. , Cooked rice,
It can also be added to staple foods, side dishes, confectionery, and seasonings such as solid foods such as bread, potato products, rice cakes, strawberries, chocolate, sprinkles, ham, sausage, and candy. Depending on the application, it may be formed into a powder, granule, tablet or the like. Moreover, you may mix suitably with an excipient | filler, a filler, a binder, a thickener, an emulsifier, a coloring agent, a fragrance | flavor, a food additive, a seasoning etc. as needed.

In addition to foods for human consumption, when a natural product-derived α-glucosidase inhibitor is mixed in feed and administered to animals such as livestock and pets, it is mixed with feed materials in advance. It can be prepared as a feed with added functionality. In addition, a natural product-derived α-glucosidase inhibitor may be added to the feed for administration. That is, a food containing a natural product-derived α-glucosidase inhibitor as an active ingredient is added to livestock such as pigs, chickens, cows, horses, sheep, and feed such as fish, pets (dogs, cats, birds). It can be used as a functional feed that is safe and has one or both of therapeutic and / or prophylactic effects of diabetes and related diseases and symptoms.

Next, examples carried out for confirming the effects of the present invention will be described.
The present invention is not limited to these examples.
(1) Preparation of hot water extract of persimmon skin 20 parts by weight of pure water was added to 1 part by weight of persimmon pericarp collected from freeze-dried persimmon fruit and sonicated for 5 minutes. Thereafter, the mixture was stirred at 95 ° C. for 1 hour for extraction. After recovering the supernatant by centrifugation, 10 parts by weight of pure water was further added to the insoluble matter, and the supernatant was recovered again by centrifugation. The combined supernatant was freeze-dried to obtain a freeze-dried powder of hot water extract.

(2) Preparation of cold water extract of persimmon peel 20 parts by weight of pure water was added to 1 part by weight of persimmon pericarp collected from freeze-dried persimmon fruit and sonicated for 5 minutes. Thereafter, the mixture was stirred overnight at 4 ° C. for extraction. After recovering the supernatant by centrifugation, 10 parts by weight of pure water was further added to the insoluble matter, and the supernatant was recovered again by centrifugation. The combined supernatant was freeze-dried to obtain a freeze-dried powder of cold water extract.

(3) Preparation of ethanol extract of persimmon peel 20 parts by weight of ethanol was added to 1 part by weight of persimmon pericarp collected from freeze-dried persimmon fruits and sonicated for 5 minutes. Thereafter, the mixture was stirred at room temperature for 1 hour for extraction. After recovering the supernatant by centrifugation, 10 parts by weight of ethanol was further added to the insoluble matter, and the supernatant was recovered again by centrifugation. The collected supernatant was used as an ethanol extract (hereinafter, referred to as “ethanol extract of persimmon peel”).

(4) Measurement of α-glucosidase inhibition efficiency Ten times as much 0.1 M maleic acid buffer (pH 6.0) was added to rat small intestine acetone powder (manufactured by Sigma), and homogenized in ice. The supernatant obtained by centrifuging this at 4 ° C. (3000 rpm for 10 minutes) was diluted 5-fold with 0.1 M maleate buffer (pH 6.0) to prepare an enzyme solution. 0.25, 0.5 for maltose 10 mg / ml and hot water extract of persimmon peel prepared in (1) above or cold water extract of persimmon pericarp prepared in (2) above for 100 μl of enzyme solution The total volume was 200 μl. As a control sample, distilled water was used instead of the extract. Moreover, about the ethanol extract of the persimmon peel prepared by said (3), it added so that it might become 10% capacity | capacitance, and ethanol was used as a control sample. These mixtures were kept at 37 ° C. for 60 minutes and then treated at 100 ° C. for 10 minutes to stop the reaction. In addition, it changed into the thing deactivated by previously processing the enzyme solution at 100 degreeC for 10 minute (s), it reacted similarly, and it was set as the blank of the test sample and the control sample.

The produced glucose was measured, and the inhibition efficiency (%) for α-glucosidase was calculated by the following formula. The amount of glucose produced was measured by quantitative HPLC (column: YMC-pac PA3, eluent: 70% acetonitrile-water).

Inhibition efficiency (%) = 100 − {(A−Ab) / (B−Bb)} × 100
However, A, Ab, B, and Bb in the formula are respectively:
A: Glucose production amount in reaction system containing extract of persimmon pericarp Ab: Glucose production amount in reaction system (blank) containing persimmon pericarp extract B: Glucose production amount in reaction system of control sample Bb: Control sample This represents the amount of glucose in the reaction system (blank).

As a comparative example, α-glucosidase inhibitory activity was similarly measured for a hot water extract, a cold water extract and an ethanol extract obtained by solvent extraction of chestnut peel as in the methods described in (1) to (3).

(5) A hot water extract of persimmon peel prepared in (1) above is adsorbed onto a fractionated crosslinked polystyrene column (Mitsubishi Chemical Diaion HP-20) using a crosslinked polystyrene column, and distilled as an eluent. Water, 50% aqueous methanol, and methanol were used to fractionate each elution. About each fraction, the inhibition efficiency of (alpha)-glucosidase was measured using the method as described in said (2).

The results of (4) and (5) were as follows.
The relationship between the concentration of the hot water extract of the persimmon peel and the chestnut peel and the inhibition efficiency of α-glucosidase (hereinafter sometimes abbreviated as “inhibition efficiency”) is shown in FIG. As a general tendency, the hot water extract of persimmon peel showed a higher inhibition efficiency, and the difference was particularly remarkable when the concentration was low. This result indicates that the hot water extract of persimmon pericarp has higher α-glucosidase inhibitory activity than the conventionally known extract of persimmon pericarp.

With respect to distilled water, 50% aqueous methanol solution, and methanol fraction of hot water extract of persimmon peels fractionated using a cross-linked polystyrene column, the inhibition efficiency of α-glucosidase was determined using the method described in (4) above. . The result of normalization so that the sum of the inhibition efficiencies of the fractions is 100 is shown in FIG. It was confirmed that the 50% aqueous methanol fraction showed the highest inhibition efficiency.

FIG. 3 shows the results of comparing the inhibition efficiency of α-glucosidase in solvent extracts of castor and chestnut peels obtained using various extraction solvents. For any of the extraction solvents, it was confirmed that the solvent extract of persimmon peel showed higher inhibition efficiency than the solvent extract of chestnut peel.

High performance liquid chromatography (HPLC) analysis (column: YMC J) of the 50% methanol aqueous solution fraction showing the highest inhibition efficiency among the fractions of hot water extract of persimmon peel obtained in (5) above The chromatogram obtained by using 'sphere ODS-H80, eluent: water containing 0.1% TFA-acetonitrile gradient (gradient conditions: see Table 1), detector: UV detector (280 nm) is shown in FIG. Show. From this result, it is presumed that the component having α-glucosidase inhibitory activity contained in the hot water extract of persimmon peel is a mixture composed of a large number of compounds having strong absorption around 280 nm. Further, as a result of multi-wavelength analysis using a photodiode array (PDA) detector, no strong absorption band was observed except in the vicinity of 280 nm. From these results, tannins are considered as candidates for components having α-glucosidase inhibitory activity.

Therefore, HPLC analysis was performed on a hydrolyzate obtained by hydrolyzing a 50% aqueous methanol fraction under acidic conditions (heating at 100 ° C. in 0.5 M HCl for 4 hours). FIG. 5 shows chromatograms obtained using a UV detector (280 nm) (column: column: YMC J'sphere ODS-H80, eluent: water containing 0.1% TFA-acetonitrile gradient (gradient conditions). 6), and a chromatogram (column: YMC-pac PA3, eluent: 70% acetonitrile-water) obtained using a differential refractive index (RI) detector is shown in FIG. Thus, in the sample after hydrolysis, a strong peak that was not observed in the sample before hydrolysis was observed at a retention time of about 6.5 minutes, and this peak was found by comparison with the chromatogram using the sample. In Fig. 6, a new peak was observed at a retention time of about 8 minutes in the sample after hydrolysis. However, it was confirmed that this was derived from glucose, and from these results, the active ingredient contained in the 50% aqueous methanol fraction of the hot water extract of persimmon peel was hydrolyzed tannin ( It is suggested that the mixture is rich in gallotannin).

HPLC analysis of acid hydrolyzate of hot water extract of Hoshi peel and chestnut peel (column: column: YMC J'sphere ODS-H80, eluent: water containing 0.1% TFA-acetonitrile gradient (gradient FIG. 7 and FIG. 8 show chromatograms obtained by conditions: see Table 2), detector: PDA detector (200 to 600 nm), Maxplot method).

From this result, it was confirmed that the hot water extract of persimmon peel is different from the hot water extract of chestnut peel known to be mainly composed of condensed tannin.

INDUSTRIAL APPLICABILITY The present invention can be used for the production of foods, health foods, supplements, and pharmaceutical compositions that have a diabetic preventive effect and a hypoglycemic action, and are suitably used for the prevention of diabetes and the mitigation of hyperglycemic conditions.

Claims (6)

A natural product-derived α-glucosidase inhibitor comprising, as an active ingredient, an extract obtained by solvent extraction of persimmon peel (Trapa Japonica). 2. The natural product-derived α-glucosidase inhibitor according to claim 1, wherein the extraction solvent is water, methanol, ethanol, or a mixed solvent of any two or more thereof. The natural product-derived α-glucosidase inhibitor according to any one of claims 1 and 2, comprising one or more hydrolyzable tannins as a main component. A pharmaceutical composition comprising one or more of the natural product-derived α-glucosidase inhibitors according to any one of claims 1 to 3. A food comprising one or more of the natural product-derived α-glucosidase inhibitors according to any one of claims 1 to 3. A feed comprising one or more of the natural product-derived α-glucosidase inhibitors according to any one of claims 1 to 3.

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