JP2005320262A - Agent for suppressing elevation of blood sugar level containing acerola seed extract, age formation inhibitor and food containing them - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an agent effective for suppressing the elevation of blood sugar level and derived from a natural material, an AGE formation inhibitor and a food containing the agent. <P>SOLUTION: The agent for suppressing the elevation of blood sugar level, the AGE (advanced glycation endproducts) formation inhibitor and the food containing the agent, or the like, contain an acerola seed extract and/or its treated material as an active component. This invention further provides a method for the production of the acerola seed extract. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a blood sugar level increase inhibitor and an AGE production inhibitor.

  With recent changes in diet and lifestyle, the number of diabetic patients is increasing. The current number of diabetic patients in Japan is 7.4 million, and it is said that it will reach 16.2 million including the diabetes reserve.

  Diabetes is a group of metabolic diseases in which a hyperglycemic state continues for a long time due to a lack of action of a hormone called insulin. If the hyperglycemic state continues, various complications such as neuropathy, cataract, renal disorder, retinopathy, arteriosclerosis, atherosclerosis, diabetic gangrene and the like may occur. Complications are mainly due to vascular disorders caused by non-enzymatic glycation, where proteins in the patient's blood bind to sugar, and cell destruction due to the accumulation of sorbitol when the sugar is metabolized It is divided into neuropathy. A glycated protein formed by binding a protein to a sugar forms a compound called an advanced glycation end product (AGE) as the reaction proceeds further. AGEs are thought to contribute to the development of diabetic vascular disorders by binding to specific receptors (RAGE) in vascular endothelial cells.

  For this reason, suppressing the increase in blood glucose level and inhibiting the generation of AGE are considered to be one of the methods for treating and preventing diabetes and diabetic complications, respectively.

  In this regard, many drugs have been developed for the treatment and prevention of diabetes and diabetic complications.

  For example, an inhibitor of blood sugar level increase for administration to diabetic patients whose blood sugar level increases after meals and an α-glucosidase inhibitor that inhibits digestion and absorption of carbohydrates to prevent blood sugar level from rising Drugs are being developed. As typical α-glucosidase inhibitors, voglibose and acarbose are known. As an AGE production inhibitor, for example, aminoguanidine, which is a carbonyl reagent, is known, and various clinical experiments have been conducted with attention as antidiabetic drugs and antidiabetic complication drugs.

  However, while these drugs are powerful, various side effects on patients such as abdominal bloating when taken, inducing hypoglycemic conditions when combined with other hypoglycemic drugs, nausea and headaches are problems. Become.

  On the other hand, drugs derived from natural ingredients, which are moderately effective but have no side effects, have been developed. Natural component-derived α-glucosidase inhibitors include perilla extract (patent document 1), mate leaf extract (patent document 2), rabu hemp leaf extract (patent document 3), and bamboo leaf extract (patent document 4). Etc. are known. Further, as an AGE production inhibitor derived from natural ingredients, for example, an anti-diabetic active substance and an anti-diabetic complication active substance using an extract from rice such as wild rice and a production method thereof (Patent Document 5) are known. However, the number is still small.

  Acerola, on the other hand, is a tropical fruit belonging to the genus Hollytranoaceae, which is native to the Caribbean Islands. Acerola pulp is now used in beverages and health foods around the world. When beverages and health foods are produced from acerola pulp, a large amount of residues including pulp, pericarp or seeds are generated. So far, it is known that acerola seed extract contains quercetin 3-O-α-L-rhamnoside and quercetin 3-O-arabinosyl galactoside, which are polyphenols. It is known to have an oxidizing action and a collagenase activity inhibiting action and a hyaluronidase activity inhibiting action (Non-patent Document 1). However, other actions are not known for the acerola seed extract, and most of the acerola seed is disposed of as industrial waste. It is desired to effectively utilize these unused resources to find industrial value.

JP 2000-102383 A JP 2003-146900 A JP 2002-053486 A JP 2003-128571 A Japanese Patent No. 3333416 Abstracts of the 123rd Annual Meeting of the Pharmaceutical Society of Japan (Nagasaki), General Academic Presentation 27 (P1) I-255

  An object of the present invention is to provide a natural product-derived blood glucose level increase inhibitor, an AGE production inhibitor, and foods containing them.

The present invention includes the following inventions.
(1) A blood glucose level increase inhibitor comprising an acerola seed extract and / or a processed product thereof as an active ingredient.
(2) The blood sugar level increase inhibitor as described in (1) above, which contains a component derived from acerola pulp as an active ingredient.
(3) The blood glucose level elevation inhibitor according to (1) or (2) above, wherein the acerola seed extract is extracted with water at 80 to 100 ° C.
(4) An AGE production inhibitor containing an acerola seed extract and / or a processed product thereof as an active ingredient.
(5) The AGE production inhibitor according to the above (4), which contains a component derived from acerola pulp as an active ingredient.
(6) The AGE production inhibitor according to (4) or (5) above, wherein the acerola seed extract is extracted with water at 80 to 100 ° C.
(7) The blood glucose level increase inhibitor or AGE production inhibitor according to any one of (1) to (6) above, for use in the treatment or prevention of diabetes and / or diabetic complications.
(8) A method for preparing an acerola seed extract, which comprises contacting water at 80 to 100 ° C. with acerola seeds.
(9) A food for suppressing blood glucose level increase or inhibiting AGE production, comprising the blood sugar level increase inhibitor or AGE production inhibitor according to any one of (1) to (7) above.

  ADVANTAGE OF THE INVENTION By this invention, the blood glucose level increase inhibitor and AGE production | generation inhibitor which contain a natural product as an active ingredient, and the foodstuff containing them are provided.

  Hereinafter, the present invention will be described in more detail.

  The present invention relates to a blood glucose level increase inhibitor and an AGE production inhibitor containing an acerola seed extract and / or a processed product thereof as an active ingredient. Since the blood glucose level elevation inhibitor and the AGE production inhibitor of the present invention are derived from natural products, there is a low possibility of adversely affecting the human body. In addition, when used as a medicine, it is less likely to cause side effects. The present invention is also preferable in that it contributes to effective use of acerola seeds.

  The production area and variety of the acerola from which the acerola seed used in the present invention is collected are not particularly limited, and examples of the production area include Okinawa and Brazil.

  In the present invention, the acerola seed refers to a non-edible portion contained in the acerola fruit. Acerola seeds may be dried, semi-dried, or undried. The acerola seed may be in the form of the seed itself or may be appropriately pulverized. For example, a frozen product of acerola seeds may be crushed. The pulverized product is preferable because of its high extraction efficiency.

  The acerola seed used in the present invention is typically provided as a residue (byproduct) of the acerola juice squeezing process. For example, the residue generated when processing the acerola fruit with a pulper finisher includes a large amount of uncrushed acerola seeds. In addition, the residue includes a part of the pulp. Therefore, the present invention is also preferable from the viewpoint of effective utilization of industrial waste.

  As a method for obtaining an acerola seed extract, for example, a method of extracting with water, a method of extracting with water containing alcohol, a crude extract extracted using an organic solvent is allowed to act on a synthetic adsorbent and adsorbed, There are methods for obtaining an extract having flavonoids. The type of water that can be used as the extraction solvent is not particularly limited, and may be pure water, purified water, or the like. When an organic solvent is used as the extraction solvent, either a hydrophilic organic solvent or a hydrophobic organic solvent may be used. Examples of the hydrophilic organic solvent include alcohols such as methyl alcohol, ethyl alcohol, glycerin, propylene glycol, 1,3-butylene glycol, acetone, tetrahydrofuran, acetonitrile, 1,4-dioxane, pyridine, dimethyl sulfoxide, N, N -Well-known organic solvents, such as a dimethylformamide and acetic acid, are mentioned. The hydrophilic organic solvent may be used as a mixture with water. Examples of the hydrophobic organic solvent include known organic solvents such as hexane, cyclohexane, carbon tetrachloride, chloroform, dichloromethane, 1,2-dichloroethane, diethyl ether, ethyl acetate, benzene, and toluene. Said hydrophilic organic solvent and hydrophobic organic solvent can also be used individually, respectively, and can also be used in combination of 2 or more type. The amount of the extraction solvent used is usually about 100 to 1000 parts by weight and more preferably about 200 to 500 parts by weight with respect to 100 parts by weight of the acerola seed (undried product). Further, the extraction temperature is preferably about 0 to 130 ° C, more preferably 80 to 121 ° C from the viewpoint of efficient extraction. In addition, when extraction is performed with water having a temperature exceeding 100 ° C., the extraction needs to be performed under pressure. Extraction with water at 80 to 100 ° C. is preferable because it is not necessary to pressurize and the amount of polyphenol recovered in the extract becomes sufficiently high. The amount of polyphenol recovered in an acerola seed extract extracted with 80 to 100 ° C. water, which is 5 times (by weight) the acerola seed, varies depending on the extraction time, but is typically 100 to 400 mg. / 100g. Refer to Example 1 for the definition of polyphenol recovery and the measurement method. The fact that the amount of polyphenol recovered in the extract increases when extraction is performed using water at 80 ° C. or higher is a surprising finding that has not been known so far. The extraction time is preferably about 1 to 24 hours, more preferably about 1 to 2 hours.

  The extraction operation is not particularly limited and may be performed according to a conventional method. In order to improve extraction efficiency, it is also possible to perform extraction using an extractor equipped with a shake extraction or a stirrer. For example, acerola seeds are immersed in an extraction solvent, or extracted with stirring and shaking with the extraction solvent without being immersed, and the processing liquid is separated into an extraction liquid and an extraction residue by filtration, centrifugation, decantation, etc. Thus, the extraction process can be performed, and the extraction residue may be further subjected to the same extraction process. The obtained extract may be used as it is, but may be further subjected to treatments such as concentration, solid-liquid separation, and purification as necessary. The “processed product of acerola seed extract” in the present invention typically includes those subjected to these processes. Concentration treatment means include solvent removal, soluble content recovery processing utilizing solubility in water and / or organic solvents, insoluble content recovery processing, liquid-liquid partition processing with water-hydrophobic organic solvent, recrystallization processing, Examples include, but are not limited to, precipitation treatment and treatment for recovering precipitates generated by cooling. Examples of the solid-liquid separation processing means include, but are not limited to, filtration and centrifugation. Examples of the purification treatment means include normal phase or reverse phase chromatography, ion exchange chromatography, gel filtration and the like, but are not limited thereto. These concentration, solid-liquid separation, and purification means may be used alone or in appropriate combination.

  The acerola seed extract used in the present invention typically contains polyphenols. Examples of polyphenols that can be included in the acerola seed extract of the present invention include, but are not limited to, quercetin 3-O-α-L-rhamnoside and quercetin 3-O-arabinosyl galactoside. The polyphenol content in the acerola seed extracted solid is preferably 0.1 to 100% by weight, particularly preferably 10 to 100% by weight.

  The content of the acerola seed extract in the blood sugar level increase inhibitor or AGE production inhibitor of the present invention is not particularly limited as long as it is a content at which a desired action is exerted. Moreover, the blood sugar level increase inhibitor or AGE production inhibitor of the present invention may further contain any component other than the acerola seed extract, preferably a component derived from acerola pulp. The acerola pulp contains about 1500 mg / 100 g or more of vitamin C, and further, polyphenols are also contained in large amounts as measured by the inventors.

  The blood glucose level elevation inhibitor or AGE production inhibitor of the present invention can be prepared in a food form or a pharmaceutical form by a conventional method using a suitable carrier or the like as necessary.

  Examples of food forms include beverages, solid foods, semi-solid foods, and the like, and can also be food for specified health use (for example, food for preventing diabetes / diabetic complications). Specific examples of the beverage include fruit juice beverages, soft drinks, and alcoholic beverages. Further, it may be in a form of being diluted with water or the like when ingested. Examples of solid food include tablets (tablets) and sugar-coated tablets including candy, troches, etc., granules, powdered beverages, powdered soups, powdered blocks, confectionery such as biscuits, capsules, jelly, etc. Various forms of food such as Examples of the semi-solid food include a paste form such as jam and a gum form such as chewing gum. The content of the blood sugar level elevation inhibitor or AGE production inhibitor of the present invention in food can be appropriately selected according to the type of food and the type, amount, form, etc. of other components contained in the food, Usually, it is 0.001 to 20% by weight, preferably 0.01 to 10% by weight in terms of dry matter of the acerola seed extract with respect to the total amount of food. In the food of the present invention, various other components that are usually used as food ingredients can be blended within a range where the desired effects of the present invention are not impaired. Examples of other components include water, alcohols, sweeteners, acidulants, colorants, preservatives, fragrances, and excipients. These components can be used alone or in combination.

  As a formulation form, the formulation for oral administration or the formulation for parenteral administration is mentioned, for example.

  Examples of preparations for oral administration include powders, tablets, granules, fine granules, solutions, capsules, pills, troches, liquids for internal use, suspensions, emulsions, syrups, and elixirs. it can. Examples of preparations for parenteral administration include preparations for nasal administration, enteral administration, and transdermal administration. Injections, drops, suppositories, inhalants, transdermal absorption agents, transmucosal absorption agents, patches, ointments Or the like. These preparation forms may be used alone or in combination depending on the symptoms.

  Preparation into various forms is carried out by conventional methods. The carriers, excipients, binders, preservatives, oxidation stabilizers, disintegrants, lubricants, flavoring agents, and diluents used at that time are also appropriately selected from those conventionally used.

  As described above, the blood sugar level increase inhibitor or AGE production inhibitor of the present invention can be prepared in various forms. Furthermore, various conventional additives can be added and blended as necessary when preparing these food forms or preparation forms. Examples of additives include stabilizers, pH adjusters, sugars, sweeteners, fragrances, various vitamins, minerals, antioxidants, excipients, solubilizers, binders, lubricants, and suspensions. Agents, wetting agents, film-forming substances, flavoring agents, flavoring agents, coloring agents, preservatives, surfactants, fluidity promoters and the like can be exemplified.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to these examples.

Study on extraction method of polyphenols from seeds The recovery method of polyphenols from seeds, which are residues generated during squeezing of acerola fruit, was investigated. First, seeds were collected from acerola fruit and used in this experiment without drying. Five times the amount (by weight) of distilled water was added to the acerola seeds, homogenized with polytron for 10 minutes, and the following extraction treatment method was examined using this suspension.

1.1 was extracted treatment at study 100 ° C. Extraction time by the hot water extraction method 15,40,60 and 120 min.
1.2 Examination of extraction time by heat and pressure extraction method Using an autoclave, extraction treatment was performed at 121 ° C for 30, 60, 90, and 120 minutes.
1.3 Examination of extraction temperature Extraction treatment was performed at 25, 50, 75, 80, 85, 90, 95 and 100 ° C. for 60 minutes, respectively.

  The extraction results were evaluated by measuring the amount of polyphenol recovered (mg / 100 g). The amount of polyphenol recovered is the amount of polyphenol obtained by extraction from 100 g of acerola seed, which is the starting material. Here, the amount of polyphenol was measured by the Folin-Denis method.

  In the examination of the extraction time by the hot water extraction method (1.1 above), the polyphenol recovery amount reached the maximum when the extraction time was 60 minutes, and thereafter the recovery amount tended to decrease (A in FIG. 1). . In the examination of the reaction time by the heat-pressure extraction (121 ° C. extraction) method (1.2 above), there was a tendency that the amount of polyphenol recovered increased as the extraction time increased (FIG. 1B). In addition, the amount recovered was higher than that in the case of hot water extraction. On the other hand, in the examination of the extraction temperature (1.3 above), the amount of polyphenol recovered increased as the extraction temperature increased, and a favorable result was obtained particularly when the extraction temperature was 80 ° C. or higher (C in FIG. 1). From these results, it was found that it is efficient to extract polyphenols from acerola seeds under heating and pressurization at 121 ° C. or with hot water at 100 ° C. for about 1 hour. Therefore, in the following examination, the extraction was performed at 100 ° C. for 60 minutes.

Examination of purification method of acerola seed extract Next, a method of purifying the acerola seed extract to increase the purity of polyphenol was examined. 3 types of anion exchange resin (DOWEX 1 × 2, DOWEX 2 × 8, DOWEX MARATHON WBA), 2 types of cation exchange resin (DOWEX 50W × 2, DOWEX MAC-3), 3 types of synthetic adsorption resin (Amberlite XAD16HP, Amberlite) XAD7HP and Amberlite XAD1180) were used in total. A column with a volume of 5 ml was prepared for each resin, and 10 ml of the residue extract (extracted at 100 ° C. for 60 minutes. Other condition extraction was as in Example 1) was applied to each column (flow rate 2 ml / min).

  Among the resins examined, polyphenols were most efficiently recovered when subjected to the synthetic adsorption resin Amberlite XAD7HP, and the polyphenol purity was about 40%. Here, the polyphenol purity means the ratio (% by weight) of polyphenol in the solid content in the column adsorption fraction. The amount of polyphenol was measured by the Folin-Denis method as in Example 1.

  From the above, it was found that the synthetic adsorption resin Amberlite XAD7HP is most suitable for the purification of polyphenols from acerola seeds among the resins studied.

Purification of acerola seed extract In this example, in response to the results of Examples 1 and 2, an extract was prepared from the acerola seed residue produced during squeezing, and the extract was further purified with a 500 ml column of Amberlite XAD7HP. did.

  First, 5 times the amount (by weight) of distilled water is added to the acerola seed residue, and a suspension is prepared by crushing with a blender mixer. The suspension is heated to 100 ° C. and maintained for 1 hour. And extracted. Subsequently, the suspension was centrifuged and filtered to obtain an acerola seed extract.

  This extract was applied to an Amberlite XAD7HP column (500 ml capacity) at a flow rate of 10 to 15 ml / min, washed with 5 times (2500 ml) distilled water of the column, and then eluted with 3 times (1500 ml) ethanol. An adsorption fraction was obtained. The adsorbed fraction was concentrated under reduced pressure, redissolved in distilled water, and lyophilized. Thus, 3.0 g of a roughly purified acerola seed extract (hereinafter referred to as “acerola seed extract crude purified product”) containing about 40% by weight of polyphenols from 1 kg (undried weight) of acerola seeds was obtained.

Antioxidant Activity of Acerola Seed Extract Crude Purified Product In this example, the antioxidant activity of the acerola seed extract crude purified product prepared in Example 3 was evaluated.
[Evaluation of antioxidant activity]
Antioxidant activity was evaluated by the superoxide anion radical scavenging activity by the xanthine-xanthine oxidase reaction system. Radical detection was performed using ESR (electron spin resonance).

  DMPO (5,5-dimethyl-1-pyrroline-N-oxide) 15 μl, 50 mM phosphate buffer (pH 7.4) 50 μl, sample solution 50 μl, 2 mM hypoxanthine / phosphate buffer solution 50 μl, 0.4 unit / ml Xanthine oxidase (XOD) / phosphate buffer (50 μl) was mixed, and ESR spectrum measurement was performed 45 seconds after the addition of XOD. Thus, the amount of superoxide anion radical was measured. Here, the sample solution was prepared by diluting the purified product with water so that the concentration of the crude purified product of the acerola seed extract prepared in Example 3 was 0.5 or 1.0 mg / ml. is there. Further, as a comparative control, (+)-catechin as a standard was similarly evaluated.

  As the ESR apparatus, an ESR apparatus (JES-FR30) manufactured by JEOL Ltd. was used, and the ESR spectrum analysis was performed under the following conditions.

  Magnetic field sweep width: 335.9 ± 5 mT, magnetic field modulation: 0.1 mT, amplification factor: 100, sweep time 2 minutes, response time: 0.1 seconds, measurement temperature: room temperature.

  In addition, the amount of superoxide anion radical was measured in the same manner using the same amount of distilled water instead of the sample solution as a control. The result of the control was set to 0% radical scavenging activity.

  The results are shown in FIG. It was confirmed that the acerola seed extract crude purified product prepared in Example 3 showed strong antioxidant activity like catechin.

Sugar hydrolase inhibitory action of crude acerola seed extract The α-glucosidase inhibitory action (maltase inhibition and sucrase inhibition) of the crude acerola seed extract prepared in Example 3 was measured by the following method. As a comparative control, a guava extract that was already known to have an α-glucosidase inhibitory action was also evaluated in the same manner.

  A 9-fold amount of 56 mM maleate buffer (pH 6.0) was added to commercially available rat intestinal acetone powder, homogenized with a glass homogenizer, centrifuged, and the supernatant was collected to obtain a crude enzyme solution. . In the maltase reaction, the crude enzyme solution was diluted 20 times and used in the sucrase reaction after being diluted 2 times.

First, 0.6 ml of a sample solution having a concentration of 2 mg / ml is added to 0.6 ml of 2% maltose solution or 2% sucrose solution, and the mixture is incubated at 37 ° C. for 5 minutes, and then 0.6 ml of the crude enzyme solution is added. The reaction was carried out at 120 ° C. for 120 minutes. After heating in boiling water for 10 minutes to inactivate the enzyme, centrifugation was performed and the amount of glucose in the supernatant was measured by HPLC.
The HPLC conditions are as follows.
Column: Shim-pack CLC-NH2 column (6.0 × 150 mm)
Column temperature: Room temperature Flow rate: 2 ml / min Mobile phase: 75% acetonitrile Detection: RI

  In addition, the amount of glucose was measured in the same manner using the same amount of distilled water instead of the sample solution as a control. The control result was defined as a maltase or sucrase inhibition rate of 0%.

  The results are shown in FIG. The acerola seed extract crude purified product prepared in Example 3 was shown to have a strong inhibitory action on maltase.

AGE production inhibitory action of acerola seed extract crude purified product The AGE production inhibitory action of the acerola seed extract crude purified product prepared in Example 3 was measured by the following method.

  1 ml of 16 mg / ml bovine serum albumin, 1 ml of 4 M glucose, 1 ml of 1/15 M phosphate buffer (pH 7.2) and 1 ml of 0.3 mg / ml sample solution were mixed and stored at 60 ° C. After 7 days, the AGE produced by protein and glucose was analyzed by fluorescence spectrometer. The fluorescence conditions were an excitation wavelength of 325 nm and a fluorescence wavelength of 405 nm in the analysis of the AGE initial product, and an excitation wavelength of 370 nm and a fluorescence wavelength of 440 nm in the analysis of the late AGE product. For comparison, an experiment using aminoguanidine was performed in the same manner. Further, a fluorescence analysis was performed in the same manner using a control using the same amount of distilled water instead of the sample solution. The result of the control was defined as an AGE production inhibition rate of 0%.

  The results are shown in FIG. It was shown that the crudely purified acerola seed extract prepared in Example 3 has an AGE production inhibitory effect in the same manner as aminoguanidine.

It is a figure which shows the collection | recovery amount of the polyphenol component in each extraction process condition (A: 100 degreeC extraction, B: 121 degreeC pressurization extraction, C: Examination of extraction temperature). It is a figure which shows the superoxide anion radical scavenging activity of the acerola seed extract refined | purified substance. It is a figure which shows the maltase inhibitory activity and sucrase inhibitory activity of the acerola seed extract crude refined | purified substance. It is a figure which shows the AGE production | generation inhibitory effect of the acerola seed extract crude refined material.

Claims (9)

  A blood glucose level increase inhibitor comprising an acerola seed extract and / or a processed product thereof as an active ingredient.   The blood sugar level rise inhibitor of Claim 1 which contains the component derived from acerola pulp as an active ingredient.   The blood glucose level increase inhibitor according to claim 1 or 2, wherein the acerola seed extract is extracted with water at 80 to 100 ° C.   An AGE production inhibitor containing an acerola seed extract and / or a processed product thereof as an active ingredient.   The AGE production | generation inhibitor of Claim 4 which contains the component derived from an acerola pulp as an active ingredient.   The AGE production inhibitor according to claim 4 or 5, wherein the acerola seed extract is extracted with water at 80 to 100 ° C.   The blood glucose level increase inhibitor or AGE production inhibitor according to any one of claims 1 to 6, for use in the treatment or prevention of diabetes and / or diabetic complications.   A method for preparing an acerola seed extract comprising contacting water at 80 to 100 ° C. with acerola seeds.   A food for suppressing an increase in blood glucose level or inhibiting an AGE production, comprising the blood glucose level increase inhibitor or AGE production inhibitor according to any one of claims 1 to 7.

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