JP2007131599A - Plant extract containing glycation-inhibiting ability and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a glycation inhibitor which is a substance originated from a material having daily been eaten, can safely be taken for a long period, exhibits an aldose reductase inhibition action and a dicarbonyl compound production-inhibiting action in living bodies, can inhibit the production of AGE, and is excellent in absorbability into bodies, to provide a method for producing the same, and to provide a food, a drink, a raw material therefor, a medicine or a raw material thereof, which contains such the glycation inhibitor and can prevent or improve diabetes and complications thereof. <P>SOLUTION: This method for producing a plant extract having a glycation-inhibiting ability is characterized by comprising extracting one or more materials selected from (A) the pericarp, seed, and fruit strained lees of a Vitis coignetiae plant, (B) the pericarp, seed, and fruit strained lees of a Pseudocydonia sinensis plant, and (C) a processed product of one of the above-mentioned materials with water or an aqueous liquid at a temperature of 70°C. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention leaves the fruit of Vitis coignetiae squeezed, which has the ability to inhibit the production of late glycation end product (also called advanced glycation end products; hereinafter sometimes referred to as “AGE”). The present invention relates to an extract of plant material such as pomace and a production method thereof, and a composition containing or using this extract.

  Diabetes mellitus is a lifestyle-related disease in which complications such as kidney damage and cataract become a major problem in social life. Due to the increase in the number of patients with diabetes, the cost of treatment is said to exceed 1 trillion yen. If diabetic complications can be suppressed, not only maintenance of a healthy social life for patients but also reduction of medical expenses, etc. The social and economic effects are also great.

  It has been shown that all diabetic complications are caused by non-enzymatic glycation reactions (glycation) of the kidney, lens, and neuroprotein associated with hyperglycemia (Non-patent Document 1).

  Glycation is a browning reaction that has been known for a long time as an aminocarbonyl reaction, and consists of two stages: the above-mentioned stage and a late stage. In the above step, the amino group of the protein and the aldehyde group or ketone group of the reducing sugar react non-enzymatically, and an Amadori transfer product is generated via a Schiff base. This reaction is reversible. In the late stage, AGE is generated through a complicated reaction including generation of active oxygen. This reaction is irreversible.

  These reactions also occur in vivo, and it is suggested that AGE accumulates in the living body with aging and causes various tissue damages. Furthermore, in vivo, it is also known that a dicarbonyl compound that is a precursor of AGE is produced through a polyol pathway activated in a hyperglycemic state (Non-patent Document 2).

  Aminoguanidine is known as a drug that inhibits glycation. Aminoguanidine has been proven by experimental animals to be effective for diabetic complications (Non-Patent Document 1), but at the same time, it has been found to have strong side effects. In addition, a drug that inhibits aldose reductase, which is a rate-limiting enzyme of the polyol pathway, has been developed (Non-Patent Document 2), but the effectiveness of such drugs for inhibiting AGE production or accumulation is not well understood.

  Substances that have fewer side effects, inhibit glycation, and can prevent diabetes or its complications are widely found in plant materials existing in nature (Patent Documents 1 to 4). For example, rutin, which is one of the flavonoids that are included in buckwheat and is a habitual eating habit, is orally administered to diabetic model rats, and the early stage and complicated late stage of AGE production may be inhibited. It has been suggested (Non-Patent Document 3).

Recently, it has been found that yama grape extract also has anti-glycation activity and suppresses AGE production by ingestion (Non-Patent Documents 4 and 5). Yama grapes are generally known to contain 4 to 5 times more abundant polyphenols than grapes, and are particularly rich in low molecular weight polyphenol components.
Moreover, there is no report that kalin has anti-glycation activity.

JP-A-11-130671 JP 2004-250445 A JP 2004-217544 A JP 2004-217545 A Singh, R., Barden, A., Mori, T., and Beilin, L. (2001) Diabetologia 44: 129-146 Dunlop, M. (2000) Kidney Int. 58 suppl77: S3-S12 Nagasawa, T., Tabata, N., Ito, Y., Aiba, Y., Nishizawa, N. and Kitts, D. D. (2003) Mol. Cell. Biochem. 252: 141-147 Abstracts of the 57th Annual Meeting of the Japan Society of Nutrition and Food (May 17-19, 2003), p. 236, 3D-08a Japanese Society for Food Science and Technology 51st Conference Lecture (September 2-4, 2004), p. 51, 2Ca12

  The inventors of the present invention are substances derived from materials with daily eating habits, which can be safely ingested for a long period of time, exhibit aldose reductase inhibition and dicarbonyl compound production inhibition in vivo, and inhibit AGE production. An object of the present invention is to provide a glycation inhibitor excellent in absorbability into the body and a method for producing the same. Another object of the present invention is to provide a food / beverage product or a material thereof, a pharmaceutical product or a material thereof, or the like that can prevent or ameliorate diabetes and its complications, including such a glycation inhibitor.

  As a result of diligent studies focusing on fruits and vegetables rich in flavonoids and having daily eating habits, the present inventors have found that fruits of karin plants that contain a large amount of flavonoids, and especially fruit juices in vivo. Extracts that are rich in glycation-inhibiting substances and that have high absorbability in the body, such as squeezed fruit of a vine grape plant that is known to have a strong glycation-inhibiting action, and efficiently produce them A method was developed to complete the present invention.

That is, according to the present invention,
[1] A) Vitis coignetiae (Vitis coignetiae) pericarp, seeds, and fruit pomace plants, B) Karin (Pseudocydonia sinensis plant fruits of), pericarp, seeds, and pomace fruit, and C) said one A process for producing a plant extract having a glycation-inhibiting ability, characterized by extracting one or more materials selected from the above-mentioned processed product with water or an aqueous liquid at a temperature of 70 ° C. or higher;
[2] The method according to [1] above, wherein the temperature is in the range of 70 ° C. or higher and 120 ° C. or lower;
[3] A plant extract produced by the method described in [1] or [2] above and having glycation-inhibiting ability;
[4] A glycation inhibitor comprising the plant extract according to [3] above;
[5] Food / beverage products, pharmaceuticals or feed material composition comprising the plant extract according to [3];
[6] A food and drink composition comprising as a raw material the plant extract according to [3] or the material composition according to [5];
[7] A pharmaceutical composition comprising, as a raw material, the plant extract according to [3] or the material composition according to [5];
[8] A feed composition comprising the plant extract according to [3] or the material composition according to [5] as a raw material,
Is provided.

  According to the present invention, an extract containing abundant flavonoids such as polyphenols, particularly catechins and anthocyanidins, which exhibit aldose reductase inhibitory action and dicarbonyl production inhibitory action in vivo and have strong glycation inhibitory ability. It can be efficiently extracted from a pomace of a Yama grape fruit or a fruit of Karin or the like by a simple process. The extract of the present invention has excellent absorption efficiency of active ingredients in living bodies, is effective for diabetes and diabetic complications, and can be used as a safe raw material for foods and beverages.

As a raw material for producing the extract of the present invention, A) Vitis coignetiae (Vitis coignetiae) pericarp of the plant, seed, and pomace fruit; B) Karin (Pseudocydonia sinensis) fruit of the plant, pericarp, seeds, and fruit And C) one or more selected from any one of the above-mentioned processed products can be used. Fruit squeezed is a residue after squeezing fruit juice by pressing, etc., and generally contains seeds, fruit peels, etc., but has little fruit juice content.

  The raw material is preferably fruit pomace. By using pomace as a raw material, it is possible to obtain an extract having a further superior physiological action as compared with a fruit juice-derived extract, and this extract is also excellent in absorbability into the body. Furthermore, using pomace as a raw material is superior to the case of using fruit juice from the viewpoint of productivity and effective use of plants (use of waste).

  Examples of the processed product include, but are not limited to, dried plant materials and freeze-dried powder.

  An extraction process is performed at 70 degreeC or more using water or an aqueous liquid for a plant raw material. The extraction temperature is preferably 70 ° C. or higher and 120 ° C. or lower. When the temperature is lower than 70 ° C., the extraction efficiency of the active ingredient is inferior, and when the temperature exceeds 120 ° C., it is difficult to expect higher extraction efficiency. From the balance of extraction efficiency, operability, economy, etc., the temperature is most preferably 80 ° C. or higher and 100 ° C. or lower.

  As the liquid used for extraction, water is preferable, but lower alcohols (methanol, ethanol, propanol, isopropanol), ketones (acetone, etc.), esters (diethyl ether, ethyl acetate), etc. are appropriately added to water or You may mix and use as an aqueous liquid. When there is a concern about the residual of these solvents, such as when used for foods and drinks or pharmaceuticals, it is preferable to use water, ethanol, or a mixed solvent thereof.

The extraction is usually performed under normal pressure, but may be performed under pressure or reduced pressure. The extraction time is usually about 10 minutes to overnight (18 hours), preferably 10 minutes to 3 hours, and most preferably about 30 minutes to 1 hour.
Note that water or an aqueous liquid may be in the form of vapor or gas instead of liquid depending on the extraction temperature or pressure condition.

  The liquid extract thus obtained can be used as it is as a glycation inhibitor or a raw material for foods, drinks, pharmaceuticals, feeds and the like. The extract may be concentrated and / or purified using techniques such as adsorption chromatography, gel filtration chromatography, ion exchange chromatography and the like. Moreover, you may utilize as forms, such as powder form and paste form, by spray drying, freeze drying, drying by membrane filtration, etc., concentration.

When the extract of the present invention is used as a food or drink, it is used alone or mixed with other fruit juices to form a liquid composition, mixed into bread or cookies to form a solid composition, or yogurt or jam. Or a creamy composition.
Moreover, it can be fermented and used as liquor, vinegar, etc. by using an extract as a raw material.

The extract of the present invention can also be used for drugs and quasi drugs. For example, in the case of a pharmaceutical composition, the extract is used as it is, or a commonly used inorganic or organic carrier or pharmaceutical excipient is added, and it is a solid, semi-solid, or liquid orally administered agent, enteral agent, or external use. It can be a parenteral administration agent such as an agent.
The effective dosage of the pharmaceutical composition of the present invention having such a glycation-inhibiting action can be appropriately changed according to the age, weight, general physical condition, etc. of the administration subject. In terms of the total polyphenol content, 0.01 to 10 g, generally 0.05 to 5 g, more generally 0.1 to 0.5 g can be orally administered.

  EXAMPLES The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

Example 1: Manufacture of an extract of squeezed vine grape fruit and test of its glycation-inhibiting ability Yama vine fruit was heated at 70 ° C for 10 minutes and pressed to prepare fruit juice. The same amount of water (10 ml) was added to the pomace (10 g) obtained after this pressing, and the mixture was extracted at 100 ° C. for 1 hour to obtain the extract of the present invention. This extract and the previously prepared fruit juice were lyophilized and powdered.

  In order to examine the glycation-inhibiting ability of these powders, each of the above powders (final concentration 0.1 mg) was added to 200 mM potassium phosphate buffer (pH 7.4) containing BSA (20 mg / ml) and D-Fructose (500 mM). / Ml, 0.5 mg / ml, or 1 mg / ml) or aminoguanidine (final concentrations 10 μM, 50 μM, 100 μM) were prepared and incubated at 37 ° C. for 5 days. Thereafter, Nε- (carboxymethyl) lysine (abbreviated as CML), which is an AGE structure contained in each sample, was detected using Western blotting. The amount of CML detected was expressed as 100% (ie, inhibition rate 0%) of the AGE produced by reacting without adding powder such as pomace (relative value ± standard error (n = 4)).

  The results are shown in FIG. The extract of vine grapes according to the present invention strongly inhibits AGE production in a concentration-dependent manner under the condition that even 100 μM aminoguanidine does not show any inhibition, and the inhibitory activity is also significant to the inhibitory activity of yama grape juice extract. It was more than.

Example 2: Production of extract using karin and test of its ability to suppress glycation 10 ml of water was added to each 1 g of powder obtained by freeze-drying the raw fruit of karin as it was, and extracted at 100 ° C for 1 hour. And the extract of the present invention was obtained. Moreover, the extract was similarly prepared using the raw fruits of Aronia and Cassis which are known to have anti-glycation activity.
Each of the resulting extracts was added in an amount of 1% (V / V) to 200 mM potassium phosphate buffer (pH 7.4) containing BSA (20 mg / ml) and D-Fructose (500 mM). Incubated for 3 days at ° C. For comparison, a sample to which rutin was added instead of the extract was treated in the same manner. Thereafter, the glycation suppression ability was measured by ELISA using an anti-AGE monoclonal antibody having CML as an epitope.

  As a result, the relative glycation inhibition rate when the value when rutin 10 mM was added was taken as 100 was 90 for Aronia and 241 for Cassis, whereas 284 for Karin. Therefore, the quinine extract of the present invention has a higher glycation-inhibiting ability than the aronia and cassis extract, and has a glycation inhibitory action equivalent to about three times that of 10 mM rutin at a concentration of 1% (V / V). Had.

Example 3: Examination of Extraction Temperature of Yama Grape Pomace 10 times (500 ml) of water is added to 50 g of Yama Grape pomace obtained in the same manner as in Example 1, and room temperature, 45 ° C., 70 ° C., 100 ° C., or 120 Extraction was carried out for 1 hour at each degree. In addition, about 120 degreeC, it processed using the autoclave.
The obtained extract was freeze-dried, and the dry weight, the total polyphenol amount by the Folin-Denis method, and the total flavanol amount by the Vanillin-HCl method were measured.

  Further, the obtained powder was added to a 200 mM potassium phosphate buffer (pH 7.4) containing BSA (20 mg / ml) and D-Fructose (500 mM) to a final concentration of 0.5 mg / ml. After 3 days of incubation at 0 ° C., glycation suppression ability was measured by ELISA using an anti-AGE monoclonal antibody with CML as an epitope, and the amount of AGE produced when no extract was added was 100 (ie, inhibition rate 0%). ) And the inhibition rate.

  As a result, as shown in Table 1, glycation having a polyphenol content of about 6% or more and an AGE production inhibition rate of about 20% or more by performing extraction at a temperature of 70 ° C. or more using water. It was revealed that an extract having an inhibitory ability was obtained. Moreover, the powder which has the high glycation suppression ability of about 40% or more was able to be obtained by extracting at 100 degreeC. On the other hand, for the extraction at 120 ° C., the total polyphenol content was higher than 100 ° C., but the total flavanol content and the AGE production inhibition rate were not significantly higher than 100 ° C.

Example 4 Concentration of Yama Grape Pomace Extract 250 ml of the extract obtained by extraction at 100 ° C. in Example 3 was adsorbed on a Sephadex LH20 column (2.0 × 20 cm) swollen with water and washed with water. Wash and elute with 150 ml of 70% (V / V) ethanol followed by 150 ml of 60% (V / V) acetone. Each of the obtained fractions was dried, and the amount of dry powder obtained from each fraction and the content of polyphenol were measured.

  As a result, the dry weight of 250 ml of 100 ° C. extract was 5 g (polyphenol 260 mg), whereas the dry weight of 150 ml of 70% (V / V) ethanol elution fraction was 240 mg (polyphenol 177 mg), 60% (V / V V) The dry weight of 150 ml of acetone-eluted fraction was 40 mg (polyphenol 20 mg). Therefore, using a column and elution with ethanol and acetone, a powder enriched with polyphenols could be obtained.

  These powders were added to a 200 mM potassium phosphate buffer (pH 7.4) containing BSA (20 mg / ml) and D-Fructose (500 mM) so that the polyphenol concentration was 0.1 mg / ml. Similarly, glycation suppression was measured by ELISA.

  As a result, the AGE production inhibition rate was 72% for the 100 ° C. extract, 85% for the 70% ethanol eluate and 92% for the 60% acetone eluate, and the glycation suppression ability by Sephadex LH-20 column fractionation. It became clear that it was possible to concentrate the fraction with

Example 5: Inhibition of glycation of diabetic rats ingesting yama grape squeezed extract About lyophilized powder of yama grape squeezed extract prepared at 100 ° C. in the same manner as in Example 3, using diabetic rats in vivo We investigated glycation suppression.

  Wistar male rats (130-150 g) were divided into normal casein diet group (AIN93G composition 20% (W / W) casein diet, NC group), diabetic casein diet group (AIN93G composition 20% (W / W) casein diet, DC Group), Diabetes 2.5% (W / W) Yama grape squeeze extract added food group (AIN93G composition 20% (W / W) Casein diet group 2.5% (W / W) After dividing into 3 groups, diet with added waste extract powder (DV group), streptozotocin (50 mg / kg body weight) was administered to the diabetic group to induce type I diabetes and reared for 30 days. Food and water were taken freely. Blood was collected at the time of dissection, and the glucose concentration in plasma was measured using a commercially available measurement kit (“Glucose B-Test Wako”, Wako Pure Chemical Industries, Ltd.).

  The results are shown in FIG. The plasma glucose concentration (mean ± standard error (n = 5-6)) was significantly increased in the DC and DV groups compared to the NC group (P <0.1; FIG. 2, panel A). . However, when AGE of liver protein was detected by Western blotting, it was found that AGE accumulation was suppressed in the DV group to the same extent as in the NC group compared to the DC group (FIG. 2, panel B).

Further, 3-deoxyglucosone (3-DG), which is a dicarbonyl compound, was derivatized to 2,3-diaminonaphthalene and then analyzed by HPLC. Aldose reductase activity was measured from the amount of NADPH oxidized using glyceraldehyde as a substrate.
As a result, both 3-DG and aldose reductase activity were significantly lower in the DV group than in the DC group (FIG. 3, panels A and B).

Example 6: Polyphenol component of Yama grape pomace extract and its absorbency To identify the components contained in the polyphenols of the 70% (V / V) ethanol elution fraction (VL) obtained in Example 4, Component analysis was performed by LC / MS / MS, NMR, and TOF-MS.

  Of the 741 mg of polyphenol in 1 g of powder, the confirmed components were procyanidin B1 (25 mg), (+)-catechin (77 mg), procyanidin B2 (38 mg), (−)-epicatechin (64 mg) for catechins. It was. As anthocyanins, cyanidin-3,5-diglucoside, malvidin-3,5-diglucoside, cyanidin-3-glucoside, malvidin-3-glucoside were confirmed, and the amount of cyanidin-3-glucoside equivalent was the largest amount. As 11 mg per 1 g of powder.

  Using this powder (VL) and a grape seed extract powder (245 mg of polyphenol per gram) commercially available as a control, the absorbability in diabetic rats was examined as follows. Six-week-old SD male rats were orally administered intragastrically with an aqueous solution of VL dissolved at 200 mg / ml and commercially available grape seed extract powder dissolved at a concentration of 750 mg / ml per 200 g body weight. Blood was collected 2 hours after the administration, and the concentration of catechins in plasma was examined. As a result, it was 68 μg / ml for VL and 17 μg / ml for commercially available grape seed extract powder. Thus, it was clear that VL was absorbed more efficiently compared to commercial grape seed extract powder.

  In addition, 6-week-old male SD rats were divided into two groups, and streptozotocin was administered to one body at a dose of 45 mg / kg body weight to induce type I diabetes, and a 0.2% (W / W) VL was obtained. The AIN93G composition contained was bred for 20 days on a 20% (W / W) casein diet. As a result of measuring the catechin content in the collected blood plasma, there was a significant difference of 0.394 ± 0.074 (μg / ml) in the diabetic group and 0.609 ± 0.384 (μg / ml) in the healthy group. I couldn't. Therefore, VL is absorbed even in the diabetic state, suggesting that the diabetic state is improved.

Example 7: Brewing of vinegar from yama grape wine and pomace extract After heating yama grape fruit at 70 ° C, brewing of vinegar using the squeezed fruit juice and pomace was carried out as follows. 10 L of the obtained fruit juice was inoculated with wine yeast Saccharomyces cerevisiae L-2226 and fermented at a product temperature of 15 ° C. During the fermentation, glucose was supplemented with Brix 11 ° to Brix 23 °, further fermented, fermentation stopped at Brix 9 °, and wine with 12% alcohol was obtained. On the other hand, twice the amount (V / V) of water was added to the pomace and heated at 70 ° C. for 30 minutes to obtain an extract. Since both the pH of the fruit juice and the pH of the wine were about 2.9, which is low for the growth of acetic acid bacteria, ammonia was added to adjust the pH to 3.5.
Add 10 ml of Acetobacter aceti NBRC3281 culture solution to 990 ml of 935 ml of pomace extract and 990 ml of wine diluted to an alcohol concentration of 5.5%. Then, culturing at 30 ° C. for 3 weeks gave vinegar having an acetic acid concentration of about 5%.

  As shown in Table 2, it was found that the vinegar prepared from the pomace extract had a sufficiently good color tone, the amount of polyphenol was larger than that of vinegar brewed from wine, and the DPPH radical scavenging activity was also very high. Therefore, the pomace extract is very useful as a food material.

FIG. 1 is a diagram showing the glycation-inhibiting ability of an extract of yama grape grape pomace according to the present invention. FIG. 2 is a diagram showing suppression of glycation in a diabetic rat ingesting an extract of a vine grape pomace of the present invention. Panel (A) shows plasma glucose concentration, and panel (B) shows AGE accumulation in the liver. FIG. 3 is a diagram showing suppression of glycation in a diabetic rat ingesting an extract of a vine grape pomace of the present invention. Panel (A) shows changes in the amount of 3-DG produced, and panel (B) shows aldose reductase activity.

Claims (8)

A) Peach of grape ( Vitis coignetiae ) plant, seed and fruit,
B) Karin ( Pseudocydonia sinensis ) plant fruit, pericarp, seeds and fruit pomace, and C) one or more materials selected from any of the above processed products at a temperature of 70 ° C. or higher, water or aqueous A method for producing a plant extract having glycation-inhibiting ability, characterized by being extracted with a liquid.   The method according to claim 1, wherein the temperature is in a range of 70 ° C. or higher and 120 ° C. or lower.   The plant extract which has the glycation suppression ability manufactured by the method of Claim 1 or 2.   A glycation inhibitor comprising the plant extract according to claim 3.   A food, beverage, medicine or feed material composition comprising the plant extract according to claim 3.   The food-drinks composition which contains the plant extract of Claim 3, or the raw material composition of Claim 5 as a raw material.   A pharmaceutical composition comprising the plant extract according to claim 3 or the raw material composition according to claim 5 as a raw material.   A feed composition comprising the plant extract according to claim 3 or the raw material composition according to claim 5 as a raw material.

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