JP2017100970A - Protein crosslinking decomposer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide protein crosslinking decomposers containing plant extract mixtures of the combination which is found by elucidating a plant extract combination having an α-dicarbonyl compound-decomposing action enhanced more than that of each plant extract, because the studies on how α-dicarbonyl compound-decomposing action of each raw material is reflected in blended tea was not sufficient, in the case of making a blended tea in combination with a plurality of plant extracts.SOLUTION: In order to solve the above mentioned subject, a protein crosslinking decomposer and the like which contain as active ingredients each extract of Rubus suavissimus, Ebenaceae, Sasa veitchii (Carr.)Rehd. and Lagerstroemia speciosa.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a protein cross-linking degrading agent that decomposes a cross-linking bond between proteins formed by saccharification of proteins, a food containing the protein cross-linking degrading agent, and the like.

  The protein saccharification reaction is described in L.L. C. It became known as Maillard reaction because Maillard discovered that heating amino acids and reducing sugars produced brown pigments. At present, the Maillard reaction is positioned as an embodiment of a protein saccharification reaction. In recent years, it has been revealed that this protein saccharification reaction is also involved in aging phenomenon, dementia, cancer, hypertension, skin diseases such as stains, arteriosclerosis and the like.

  Further, AGEs (advanced glycation endproducts), which are final products resulting from protein saccharification reactions, are accumulated in blood particularly in diabetic patients and cause complications such as neuropathy and retinopathy. Thus, the saccharification reaction of proteins leading to the generation of AGEs has an unfavorable effect on the human body, and various studies have been conducted on the inhibition of saccharification reactions.

  FIG. 1 is a diagram showing an outline of a process for producing AGEs by a saccharification reaction of a protein. Examples of “saccharification reaction intermediates” and “AGEs (final products of saccharification reaction)” generated from the reaction of protein and glucose are shown as the reaction proceeds.

  Here, glyoxal (GO), methylglyoxal (MG), and 3-deoxyglucosone (3DG) exemplified as saccharification reaction intermediates, which are intermediates leading to the generation of AGEs, all have two carbonyl groups ( an α-dicarbonyl compound having c = 0).

  This α-dicarbonyl compound is highly reactive and forms a cross-link between proteins, for example, a cross-link between collagen molecules in vivo. Normal skin and bone maintain proper flexibility and strength of the skin and bone by the cross-links (physiological cross-links) that are orderedly formed at sites genetically defined through the action of enzymes. However, disorderly and excessively formed cross-linking by the α-dicarbonyl compound leads to skin hardening and bone weakening.

  Degrading such an α-dicarbonyl compound suppresses the formation and accumulation of AGEs and also breaks up the disorderly and excessive cross-links formed by the saccharification reaction to restore skin hardening and bone weakness. It is considered effective for

  N-phenacyl thiazolium bromide (PTB) is known as a substance capable of decomposing α-dicarbonyl compounds, but it is said to have problems such as side effects. From the viewpoint of safe ingestion, a substance derived from a natural product and capable of decomposing an α-dicarbonyl compound is required, and citrus fruits such as yuzu have been reported as such a substance (Patent Document 1). ). As for extracts of plants that are drunk as health tea, it has been reported that, for example, evening primrose, guava, loquat, and kumazasa have a decomposing action of α-dicarbonyl compounds (Patent Document 2). Here, healthy tea refers to foods such as herbal tea, wild grass tea, etc. that are dried and then boiled with hot water.

Japanese Patent No. 4315650 JP 2007-119373 A

  Many plant extracts used as health tea ingredients have unique flavors, such as dokudami, rooibos, eucommia, and ginseng, and are not easy to drink. Therefore, health tea that is generally manufactured and sold has been devised to make it easy to drink by blending various plant extracts.

  By the way, when a plurality of plant extracts are combined into a blended tea, research on how the degradation action of α-dicarbonyl compounds of individual ingredients is reflected in the blended tea is sufficient. There wasn't. Therefore, an object of the present invention is to obtain a combination of raw materials that can exceed the above-described decomposition action of individual raw materials by blending.

  As means for solving the above problems, the following inventions and the like are provided. That is, the present invention provides a protein cross-linking and decomposing agent containing, as an active ingredient, a mixture containing each extract of Tenno-komushi, Oyster, Kumazasa and Banaba. The present invention also provides the above-mentioned protein cross-linking and degrading agent having the ability to inhibit pentosidine production, comprising as an active ingredient a mixture containing each extract of Tenno Kenko, Oyster, Kumazasa and Banaba. Also provided are foods, food additives, pharmaceuticals, quasi-drugs and cosmetics containing the protein crosslinking agent.

  According to the present invention, it is possible to provide a protein cross-linking and decomposing agent comprising a combination of raw materials that can exceed the protein cross-linking and decomposing action of individual raw materials by blending.

The figure which shows the outline of the production | generation process of AGEs by the saccharification reaction of protein Diagram showing test results The figure which shows the production | generation inhibitory action of the AGEs etc. of the blend tea which combined the persimmon tea, persimmon leaf tea, Kumazasa tea, and banaba tea The figure which shows the AGEs production inhibitory effect of the blend tea which combined the tea, dokudami tea, kashiwanoha tea, and the gaba tea

Examples of the present invention will be described below. In addition, this invention should not be limited at all to these Examples, and can be implemented with various aspects in the range which does not deviate from the summary.
<Example>
<Overview>

The protein cross-linking / degrading agent of the present example contains, as an active ingredient, a mixture containing each extract of Tenno-komushi, Oyster, Kumazasa and Banaba.
<Configuration>

  In the present embodiment, the “protein cross-linking agent” means an agent capable of decomposing a cross-link formed by 3DG or the like by decomposing an α-dicarbonyl compound such as 3DG produced by a saccharification reaction of the protein. To do.

  “Rubus suavissimus” is a plant belonging to the genus Rubiaceae in the family Rosaceae. It is boiled and dried as a tea.

  “Diospyros kaki” is a plant of the genus Oysteraceae. The dried leaves are boiled and drunk as tea leaves.

  “Sasa veitchii” is a plant of the genus Saceae. Boiled dried leaves are drunk as Kumazasa tea.

  “Banaba (Lagerstromia speciosa)” is a plant of the genus Crape myrtle. Boiled leaves are drunk as banaba tea.

  The solvent for obtaining the above-mentioned extract of each plant can be appropriately selected according to the standard method according to the type and aspect of the product containing the extract, for example, purified water, ethanol, methanol, propyl alcohol Various solvents such as isopropyl alcohol, butanol, and propylene glycol can be used. When each plant is decocted and drunk like tea, extraction with water is simple and preferable. Moreover, after obtaining the extract as a liquid, it may be further dried to obtain powder or granules.

  The protein cross-linking degradant of the present embodiment comprising the above plant extract as an active ingredient is further used by using a known method to provide a food, food additive, pharmaceutical product, quasi-drug containing the protein cross-link degrading agent. Products, cosmetics, etc.

  For example, in the case of a pharmaceutical product, the protein cross-linking agent of the present embodiment is powdered or granulated and filled into a capsule, or an excipient, a binder, a disintegrant, etc. are added to a tableting machine, etc. Can be used. Moreover, when setting it as a foodstuff, it can provide by simmering each plant suitably with a hot water such as drying or crushing. Moreover, you may provide in the form like a capsule or a tablet like a pharmaceutical, and can also provide in the aspect which added the collagen crosslinking agent to various foodstuffs, such as another drink, a seasoning, and confectionery.

Moreover, it can also be set as cosmetics, such as a cosmetic liquid, cream, and lotion. For example, in the case of a cosmetic liquid, in addition to the collagen cross-linking agent of this embodiment, water, rice bran oil, pentylene glycol, glycerin, squalane, cetyl palmitate, dimer dilinoleic acid and the like as main components, hyaluronic acid Additives include Na, hydrogenated rapeseed oil alcohol, carbomer, xanthan gum, hydroxylated K, dimethicone, polysorbate-60, glyceryl stearate, hydrogenated castor oil, phenoxyethanol, urea, arginine, arbutin, citric acid and the like. Then, after each component is dissolved in a water-soluble raw material and an oil-soluble raw material, they are heated and mixed and emulsified. While cooling this, an additive such as an extract is blended, and a highly volatile substance such as essential oil or fragrance is added when the temperature becomes lower. Thereafter, a predetermined safety test (bacteria, pH, temperature stability, viscosity, etc.) is performed, and the product can be provided as a product after being filled into a bottle.
<Test>

  In this test, N-phenacylthiazolium bromide (N-phenacylthiazolium bromide), which is known to have protein cross-linking degradation activity and α-dicarbonyl compound degrading activity, using an extract obtained by extracting each plant as described above with water as a sample. Hereafter, it shows as a relative value with respect to PTB). The measurement method is based on the method of Vasan s et al. (Nature, Vol. 382, p. 275-278, 1996), and in 1-phenyl-1,2-propione dione (PPD), which is a model compound for AGEs crosslinking. Benzoic acid produced when the C—C bond of the α-diketone structure is decomposed is measured by HPLC (high performance liquid chromatography).

  The following samples were selected as samples for this test, which are relatively well known as healthy teas and are not difficult to obtain. In addition to the above-mentioned strawberry tea, cypress (bamboo leaf tea), kumazasa (kumazasa tea), banaba (banaba tea), roasted tea, jasmine tea, pu-erh tea, dokudami tea, rooibos tea, hawthorn tea, hamax Tea, Salacia tea, eucommia tea, oolong tea, rosehip tea and green tea were also used. Moreover, each sample used what was manufactured as tea leaves of tea brewed by Hikawa Co., Ltd. Sample preparation and test methods, results, etc. are described in detail below. In addition, tea includes so-called tea outside tea brewed from leaves, buds, flowers, bark, roots, and the like of plants other than tea tree.

(1) Preparation of sample 2 g of each tea leaf was added to 40 mL of distilled water heated to 80 ° C., and extracted for 1 hour in a water bath set at 80 ° C.

(2) Measurement of AGEs crosslinking degradation action The measurement of AGEs crosslinking degradation action was based on the method of Vasan et al. As a reaction substrate for the AGEs cross-linking model, 1-phenyl-1,2-propione dione (PPD) dissolved in 50% acetonitrile was used. When 1 mol of PPD is decomposed, 1 mol of benzoic acid is liberated. For measurement of the AGEs cross-linking degradation action, 500 μL of a sample, 100 μL of 10 mmol / L PPD, and 400 μL of 0.2 mol / L phosphate buffer were mixed and reacted at 37 ° C. for 8 hours. Thereafter, 200 μL of 2 mol / L HCl was added to the reaction solution to stop the reaction, and the amount of benzoic acid in the supernatant centrifuged at 10,000 rpm (9,170 g) for 2 minutes was measured by HPLC.

  The HPLC was used with a Cadenza CD-C18 75 × 4.6 mm ID (Imtakt,) connected to a Shimadzu LC-10A system (Shimadzu Corporation). The analysis conditions were as follows: eluent: 2 mmol / L ethylenediamine-N, N, N ′, N′-tetraacetic acid, disodium salt, dihydrate (EDTA-2NA) containing 0.2% acetic acid / acetonitril (70/30), flow rate: 1.0 mL / min, column temperature: 40 ° C., detection wavelength: UV 270 nm, injection amount: 50 μL.

(3) Calculation of AGE cross-linking decomposition rate When one molecule of PPD is decomposed, one molecule of benzoic acid is generated. For this reason, the value obtained by dividing the amount of benzoic acid in the reaction solution measured by HPLC by the amount of PPD added to the reaction solution was defined as the AGE cross-linking decomposition rate. In addition, the amount of benzoic acid in the reaction solution was subtracted from the measured value by previously measuring the amount of benzoic acid in the sample by the same method. The AGEs crosslinking cleavage rate was calculated as a relative value when the AGEs crosslinking cleavage rate of 0.4 mmol / L PTB was 100.

(4) Results FIG. 2 shows the results of the above test, which are shown in descending order of decomposition rate. As shown in the figure, the highest decomposition rate was roasted tea, and the relative value with PTB was 179. In addition, it was tochu tea that had the lowest degradation rate, and its relative value to PTB was 17.

  Then, a plurality of blended teas prepared by combining any three to four of the samples were mixed, and the same test was performed on them. The sample was prepared by mixing equal amounts of individual samples constituting the blended tea to 500 μL.

Among the plurality of measured blended teas, the blended tea obtained by combining and mixing “bamboo tea, persimmon leaf tea, kumazasa tea, banaba tea” exhibited a remarkably excellent AGE cross-linking rate. The specific results of this blended tea are shown in Table 1 below.

  As shown in Table 1, the AGEs cross-linking rate of this blend tea was 74.78 ± 6.22 (%), and the relative value with PTB was 325. Since the PTB relative value of each sample constituting this blended tea was “boiled tea 140, kashiwanoha tea 92, kumazasa tea 73, banaba tea 23”, the cross-linking cutting action exhibited by the individual samples by combining them was much higher. It has been found that it is possible to produce an action exceeding that.

In addition, in the blend tea by the other combination measured this time, what showed the synergistic effect recognized by the said combination was not found.
<Effect>

As described above, it has been found that by combining “bamboo tea, persimmon leaf tea, kumazasa tea, banaba tea”, an excellent AGE cross-linking action far exceeding the individual AGE cross-linking action is achieved.
<Example 2>

As described above, it was found that the blended tea obtained by combining and mixing “bamboo tea, kashiwanoha tea, kumazasa tea, banaba tea” exerted an AGEs cross-linking action exceeding individual actions. Therefore, a test was also conducted on the AGEs production inhibitory action.
<Test>

The AGE generation inhibitory action and anti-glycation activity (IC ₅₀ ) of each tea and blend tea constituting the above-mentioned blend tea were measured. Specifically, the target is human serum albumin (HSA) and collagen (Col), which are biological proteins, and 3DG (3-deoxyglucosone), a saccharification reaction intermediate produced in the living body, is the final saccharification reaction product. The production inhibitory action of certain fluorescent AGEs, pentosidine and CML (carboxymethyllysine) was measured.

(1) Extraction of sample 3.75 g of tea leaves of each sample were added to 150 mL of distilled water heated to 80 ° C. in a constant temperature water bath and incubated for 1 hour. Thereafter, the mixture was centrifuged at 4,500 rpm for 15 minutes, and the supernatant was collected. 5 mL of the collected sample extract was placed in an aluminum tray and placed in an incubator heated to 120 ° C. for 1 hour to completely evaporate moisture, and then the solid content weight was measured.

(2) Sample preparation Each sample extract was prepared in three concentrations: stock solution, 10-fold diluted solution, and 100-fold diluted solution. Moreover, about the blend tea, the mixing ratio of each structure was prepared to the said 3 density | concentration by making the same ratio (1: 1: 1: 1). In addition, aminoguanidine (aminoguanidine hydrochloride manufactured by Wako Pure Chemical Industries, Ltd .: code 6328-26432, Lot. EPN0180), which is a known saccharification reaction inhibitor, was 10.0 mg / mL, 1 mg / mL, 0.1 mg. / ML aqueous solution was prepared.

(3) In vitro saccharification reaction 0.05 mol / L phosphate buffer (pH 7.4), 8 mg / mL human serum albumin (HSA) (Sigma-Aldrich Corporation) or 0.6 mg / mL collagen type I bovine dermis ( (Col) (Nippi Co., Ltd.), 0.2 mol / L Glucose reaction solution was added to each sample at a concentration of 1/10, and at 60 ° C for 40 hours for HSA, for collagen, Incubated for 10 days. As a negative control, a sample to which distilled water was added instead of the sample was used. Each reaction solution after incubation was used for measurement of various AGEs.

(4) Measurement of fluorescent AGEs production inhibitory action and anti-glycation activity Fluorescent AGEs were measured according to a predetermined method (Takahiro Kitano, Masayuki Yagi, Keitaro Enomoto, Mio Hori, Shigeichi Shono, Yoshikazu Yonei, Takaaki Hara, Hideo Hara. , Akitoshi Yamaji: Study on the inhibitory action of edible purple chrysanthemum protein glycation end products (AGEs), New Food Industry, 53 (6), 1-10 (2011)) Wavelength 370 nm, fluorescence wavelength 440 nm). The fluorescence value was calculated as a relative value when the fluorescence value of 5 μg / mL quinine sulfate 0.1N sulfuric acid aqueous solution was 1000.

  The AGEs-derived fluorescence production inhibition rate (%) is the reaction solution (A) to which the sample was added, the one to which distilled water was added instead of the glucose aqueous solution (B), and the one to which only the solution without the sample was added and incubated ( C) As a blank (D) with distilled water added instead of glucose, it was calculated according to the following formula.

  (Formula 1) Fluorescence AGEs production inhibition rate (%) = {1− (A−B) / (C−D)} × 100

IC ₅₀ (50% production inhibition concentration: per solid content concentration) was calculated for anti-glycation activity and displayed to the third decimal place. Here, the test substance concentration that suppresses the production of a substance by IC ₅₀ reaction by 50%, the smaller this value, the stronger the inhibitory activity.

(5) Measurement of 3DG production inhibitory activity and anti-3DG activity According to the method of Reference Document 1, 3DG produced in the sample reaction solution uses 2,3-pentane-dione as an internal standard substance. Quantification was performed by prelabeling HPLC method.

  For 3DG measurement, 300 μL of distilled water and 25 μL of 20 mg / mL 2,3-pentanedione (Wako Pure Chemical Industries, Ltd.) as an internal standard substance were added to 200 μL of each sample and mixed with stirring. Next, 500 μL of 6.0% perchloric acid (Wako Pure Chemical Industries, Ltd.) was added and stirred, and then centrifuged at 12,000 rpm for 10 minutes. After centrifugation, 800 μL of the supernatant was dispensed into another container, and 1000 μL of a saturated aqueous sodium hydrogen carbonate solution (Wako Pure Chemical Industries, Ltd.) was added and stirred. Then, 100 μL of 1.0 mg / mL 2.3-diaminonaphthalene (Dojindo Laboratories, Inc.) was added as a labeling agent, stirred, and placed at 25 ° C. for 1 day, and then introduced into HPLC under the following conditions. 3DG was measured.

As the column, YMC-PackCN150 × 4.6 mm ID (YMC Corporation) was used. Measurement conditions were as follows: eluent: 50 mmol / L phosphoric acid: acetonitrile: methanol = 70: 17: 13, flow rate 1.0 mL / min, column temperature 35 ° C., detection wavelength UV 268 nm. For the anti-3DG activity, IC ₅₀ (50% production inhibition concentration: per solid content concentration) was calculated and displayed to the third decimal place.

(6) Measurement of Pentosidine Production Suppressing Action and Anti-Pentocidin Activity Pentosidine produced in the sample reaction solution was quantified by ELISA method using FSK Pentosidine Kit (Fushimi Pharmaceutical Co., Ltd.) according to the method of Reference Document 1 above.

50 μL of each sample and 100 μL of pronase solution were mixed and incubated at 55 ° C. for 90 minutes, then heated in boiling water for 15 minutes to inactivate pronase, and 50 μL of the auxiliary solution attached to the kit was added. Thereafter, 50 μL of the sample or pentosidine standard solution and 50 μL of the anti-pentosidin monoclonal antibody solution attached to the kit were dispensed into each well of the microplate and reacted at 37 ° C. for 60 minutes. Next, each well was washed three times with 200 μL of the cleaning solution attached to the kit, and then 100 μL of a solution containing 3′5,5′-tetra-methylbenzidine (TMB) attached to the kit was dispensed into each well and allowed to react for 10 minutes. . Thereafter, 100 μL of the reaction stop solution attached to the kit was added, and the absorbance at 450 nm (main wavelength) / 630 nm (reference wavelength) was measured within 10 minutes. The pentosidine concentration in the sample was calculated from a calibration curve prepared with a pentosidine standard solution. The anti-pentosidin activity was calculated by IC ₅₀ (50% production inhibitory concentration: per solid content concentration) and displayed to the third decimal place.

(7) CML generated in the measurement sample reaction mixture in CML formation inhibitory action and anti-CML activity, according to the method of the reference ^{1, CircuLexCML / N ε - (} carboxymethyl) lysine ELISA by ELISA KIT (LTD cycle Rex) Quantified by the method.

  First, 450 mL of purified water was added to 50 mL of the concentrated cleaning solution attached to the measurement kit (10-fold dilution) to prepare 500 mL of cleaning solution, and 3 mL of purified water was further added to the anti-CML monoclonal antibody (primary antibody) attached to the kit. Stir and let stand for 10 minutes. Of this, 600 μL was taken out, 5.4 mL of purified water was added (diluted 10-fold), and a total of 6 mL of First Antibody working solution was made. In addition, 500 μL of purified water was added to CML-HSA Standard attached to the kit to prepare CML-HSA Master Standard (20 μg / mL), which was used to create a calibration curve.

For the measurement of CML, 90 μL of the sample dilution buffer attached to the kit was added to 30 μL of each sample, 120 μL of the first prepared antibody working solution was added and stirred, and 100 μL was dispensed into each well on the microplate. Then, it was made to react, stirring for 60 minutes at room temperature. Thereafter, the reaction solution in each well was discarded and washed 4 times with 200 μL of the prepared cleaning solution. Furthermore, 100 μL of HRP conjugated Detection Antibody (secondary antibody) attached to the kit was dispensed into each well and allowed to react at room temperature with stirring for 60 minutes. After completion of the reaction, the same washing operation as described above was performed. In each well, 100 μL of Substrate Reagent attached to the kit was dispensed and stirred for 1 minute, and then the plate was covered with aluminum foil, shielded from light, and left for 10 minutes. Thereafter, 100 μL of the Stop solution attached to the kit was dispensed into each well, stirred for 1 minute, and immediately measured at 450 nm (main wavelength) / 540 nm (reference wavelength) with a microplate reader. For the anti-CML activity, IC ₅₀ (50% production inhibition concentration: per solid content concentration) was calculated and displayed to the third decimal place.

(8) Results FIG. 3 shows a list of anti-glycation activity (HSA, Col), anti-CML activity, anti-pentosidine (Pent) activity and anti-3DG activity of each sample.

  From this result, it can be seen that the activity level of the blended tea generally reflects the activity level of the most active tea among the teas constituting the blended tea. For example, in anti-glycation (HSA), the activity level of blended tea (0.045) reflects the activity level of strawberry tea (0.046). In addition, in the anti-CML activity, the activity level of blended tea (0.0002) reflects the activity level of banaba tea (0.0003). In addition, in the anti-3DG activity, the activity level of the blended tea (0.020) reflects the activity level of the banaba tea (0.020). As for the anti-pentosidin activity, the activity level of blend tea (0.007) reflects the activity level of kashiwanoha tea (0.005).

  However, the present inventors were the first to obtain such a result for the anti-pentosidine activity. That is, according to the present inventors' previous knowledge, the anti-pentocidin activity is the same as that for blended tea even when individual teas constituting the blended tea include those having anti-pentocidin activity. In many cases, no activity was observed. Such an example is shown in FIG.

  FIG. 4 shows the results of measuring the anti-glycation activity and the like in individual teas and various combinations in making blended teas by combining koji tea, dokudami tea, kashiwanoha tea, and gaba tea.

  As shown in the figure, the anti-pentosidin activity was observed except for dokudami tea, but when two or more kinds were combined, the measurement result was “NC (uncalculated)” in many cases. That is, when two or more kinds are combined, the activity often does not occur even though each tea has activity.

In view of the specificity of such anti-pentocidin activity, it is peculiar that a blended tea composed of a combination of “banaba tea, strawberry tea, kumazasa tea, and kashiwanoha tea” has excellent anti-pentocidin activity (0.007). It is thought that it is a thing.
<Effect>

  Blended teas made by mixing banaba tea, camellia tea, kumazasa tea, and persimmon leaf tea not only have an inhibitory effect on the generation of AGEs, CML, and 3DG, but also reflect the individual inhibitory activity when combined with multiple teas. It was found to have effective production inhibitory activity even for difficult pentosidine. That is, it can be said that the blended tea by this combination has a broad production inhibitory effect on AGEs and the like.

Claims (6)

  A protein cross-linking and decomposing agent containing, as an active ingredient, a mixture containing each extract of Tenno-komushi, Oyster, Kumazasa and Banaba.   A pharmaceutical comprising the protein cross-linking degradation agent according to claim 1.   A quasi-drug containing the protein crosslinking agent according to claim 1.   A food containing the protein cross-linking agent according to claim 1.   A food additive comprising the protein crosslinking agent according to claim 1.   Cosmetics containing the protein crosslinking agent according to claim 1

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