JP2013253071A - Oph activity enhancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve such a problem that although prevention of diseases and ageing can be expected by promoting activity of an oxidized protein hydrolase (OPH), there is a small number of substances known as active substances promoting the OPH activity.SOLUTION: An OPH activity enhancer comprises a combination including one or more extracts of burdock, oolong tea, roasted tea, lemongrass, Aloe arborescens, Sasa veitchii, pepper, Horduem vulgare, Gynostemma pentaphyllum, Gymnema sylvestre, Senna obtusifolia, Carthamus tinctorius, mulberry, Ganoderma lucidum, pu-erh tea, coarse tea, Houttuynia cordata, Coix lacryma-jobi var. frumentacea, and Senna occidentalis; and activates the oxidation proteolytic activity of oxidation proteolytic hydrolase (OPH) by 1.2 times or more.

Description

  The present invention relates to an OPH activity enhancer that enhances the activity of oxidized protein hydrolase (OPH), which is an in vivo enzyme that degrades oxidized protein, and foods and beverages and pharmaceuticals containing the OPH activity enhancer. About.

  It is known that an oxidation reaction in a living body adversely affects cells and tissues. For example, aging changes such as tissue collagen, Alzheimer's disease, cataract, skin aging, and other diseases and aging.

  Oxidized protein is decomposed and removed by OPH. OPH is a kind of serine protease that is widely distributed in living tissues and preferentially degrades oxidized proteins. Acylamino-acid-releasing enzyme: an enzyme that releases N-terminal acylated amino acids of proteins. AARE). It is known that the activity of OPH decreases with age.

  It has been reported that OPH activity is significantly increased in the serum of diabetic rats, and that the amount of carbonyl-modified protein is low in serum with increased OPH activity, and that OPH may be related to the suppression of diabetes and aging progression. Has been.

  Although it is expected to prevent diseases and aging by promoting the activity of OPH, only dodami, hawthorn, roman chamomile, and grape are known as agents that promote OPH activity. (Patent Document 1).

International Publication Number WO2011 / 004733

  In view of the above circumstances, an object of the present invention is to provide an OPH activity enhancer that enhances the activity of OPH.

  As means for solving the above problems, the following inventions and the like are provided. That is, as the first invention, the extract of burdock, oolong tea, roasted tea, lemongrass, yellow tea aloe vera, pepper, barley, barley, macaques, gymnema, shrimp, safflower, mulberry, litchi, pu-erh tea, bancha, dokudami, pearl barley, hawthorn And an OPH activity enhancer that activates the oxidized proteolytic activity of oxidized proteolytic enzyme (OPH) by a factor of 1.2 or more.

  As a second invention, there is provided an OPH activity enhancer comprising a combination containing at least one extract of burdock, oolong tea, and hoji tea and activating OPH activity three times or more.

  The third invention consists of a combination of one or more extracts of oolong tea, roasted tea, lemongrass, bancha, pu-eru, safflower, which activates the oxidized proteolytic activity of OPH by 1.2 times or more and decomposes AGEs of OPH Provided is an OPH activity enhancer that activates.

  As 4th invention, the food / beverage products, health food, food additive, pharmaceutical, cosmetics, quasi-drug containing the combination containing 1 or more types of OPH activity enhancer as described in 1st invention from 3rd invention are included. I will provide a.

  According to the present invention, an OPH activity enhancer that enhances the oxidized proteolytic activity of OPH can be provided.

The figure which shows transition of OPH activity enhancement action of 17 kinds of samples The figure which shows the OPH activity enhancement rate 90 minutes after 19 types of samples The figure which shows each measurement result of fluorescent AGEs, pentosidine and CML The figure which shows the measurement result where AGEs degradation activity enhancement rate became plus

Embodiments of the present invention will be described below. In addition, this invention should not be limited to these embodiments at all, and can be implemented in various modes without departing from the gist thereof.
<Embodiment 1>
<Summary of Embodiment 1>

In this embodiment, OPH activity enhancing action of OPH, which is an oxidized protein degrading enzyme (hereinafter simply referred to as OPH activity), was confirmed by tests compared to control (no sample added). The present invention also relates to an OPH activity enhancer comprising 17 kinds of plant extracts.
<Configuration of Embodiment 1>

  The OPH activity enhancer according to the present embodiment includes burdock, oolong tea, roasted tea, lemongrass, yellow tea aloe vera, kumazasa, chili pepper, barley, macaque, gymnema, shrimp, safflower, mulberry, litchi, pu-erh tea, bancha, dokudami, pearl barley, hawthorn It consists of a combination containing one or more types of extracts. The 17 types of samples listed here are more than 1.2 times more OPH than the control to which no sample is added, among the over 100 types of samples that have been subjected to the OPH oxidation proteolytic activity enhancement test described later. An action of enhancing oxidized protein degradation activity was observed.

  The plant extract in this embodiment may be extracted from any part of the plant, for example, extracted from whole grass, flowers, seeds, fruits, branches, stems, bark, roots, etc. Good. Moreover, the property of the extract is not limited. Below, the plant used by this embodiment is demonstrated.

  The "burdock" Arctium lappa is a perennial plant belonging to the genus Burdock.

  "Oolong tea" is a tea that is semi-fermented by heating tea leaves (Camellia sinensis leaves) during fermentation to stop fermentation.

  "Hojicha" is a tea roasted from bancha.

  “Lemongrass” Cymbopogon citratus is a perennial plant belonging to the genus Ogarkaya.

  "Kidachi aloe" Aloe arborescens is a succulent plant of the genus Aloe.

  "Kumazasa" Sasa veitchii is a plant belonging to the genus Sasa genus.

  “Capsicum” Capsicum annuum is a plant belonging to the genus Capsicum.

  "Barley" Hordeum vulgare is a plant of the genus Barley.

  “Amateur” Gynostemma pentaphyllum is a plant belonging to the genus Acuchar.

  "Gymnema" Gymnema sylvestre is a plant belonging to the genus Houraiaokazura.

  "Ebisugusa" Senna obtusifolia is a plant belonging to the genus Senna genus Jackaceae.

  “Safflower” Carthamus tinctorius is a plant belonging to the genus Asteraceae.

  "Mulberry" Morus lhou, M. bombycis, M. alba, M. nigra, M. serrata, M. laevigata, M. tiliaefolia, M. boninensis are plants of the genus Mulberry family.

  “Reishi” Ganoderma lucidum is a fungus of the genus Mannentake.

  “Puer tea” is a post-fermented tea made by oxidative fermentation of tea leaves (Camellia sinensis leaves) by heating and aging with microorganisms such as strawberries.

  “Bancha” is made by harvesting hard tea leaves (leaf of Camellia sinensis) after 2nd tea, which are picked after summer, together with stems.

  "Dokudami" Houttuynia cordata is a plant of the genus Dokudami family.

  "Betley" Coix lacryma-jobi var. Frumentacea is a plant belonging to the genus Giusedama.

  “Husou” Senna occidentalis is a plant belonging to the genus Senna genus.

The OPH activity enhancer comprising one or a combination of two or more of the 19 kinds of plant extracts described above is applied as a food / beverage product, health food, food additive, pharmaceutical product, cosmetic product, quasi drug, etc. containing the same. (The same applies to the second embodiment).
<Embodiment 1 test>

The 19 plant extracts mentioned above were confirmed to have an effect of enhancing OPH activity in the following tests.
<Test 1>

  (1) Sample extraction

  3.75 g of each dried sample was added to 150 mL of distilled water heated to 80 ° C. in a constant temperature water bath and incubated for 1 hour. Thereafter, the extract was centrifuged at 4500 rpm for 15 minutes, and the supernatant was collected to obtain a sample extract.

  (2) Preparation of reaction reagent

A tube containing 0.5 U of acylamino acid releasing enzyme (AARE; Takara Bio) as OPH was diluted by adding 1 mL of 50 mmol / L sodium phosphate buffer. Further, it was diluted 10-fold with 200 mmol / L Tris-HCl (pH 7.4) to obtain 0.05 U / mL OPH. Next, 50 mmol / L N-acetyl-L-alanine p-nitroanilide (AAPA) substrate (Funakoshi) 50% ethanol aqueous solution was prepared. Reaction reagents A to D were prepared by mixing the prepared reaction solutions as shown in Table 1 below.

  (3) Measurement of OPH activity enhancing action

  The prepared reaction reagent (excluding AAPA substrate) was placed in each well of a 96-well microplate, and then AAPA substrate was added to initiate the reaction. The set temperature of the microplate reader at the time of measurement was 37 ° C., the absorbance at a measurement wavelength of 405 nm was measured every 10 minutes, and the measurement was continued for 90 minutes.

For the OPH activity enhancing action, first, the absorbance of each of the reaction reagents A to D was determined. The value obtained by applying the obtained absorbance of each reaction reagent to the following formula was defined as the OPH activity enhancement rate. A sample with an OPH activity enhancement rate exceeding 100 indicates that the OPH activity is enhanced, and a sample below 100 indicates that the OPH activity is decreased.
(Formula 1) OPH activity enhancement rate = (reaction reagent B−C) / (reaction reagent A−D) × 100

  (4) Results

  FIG. 1 is a graph showing the transition of the OPH activity enhancement rate after 30 minutes, 60 minutes, and 90 minutes after the start of measurement for 17 types of 19 samples excluding pearl barley and yellowtail.

  FIG. 2 shows the OPH activity enhancement rates 90 minutes after the start of measurement for 19 samples, arranged from the top.

As described above, all of the 19 plant extracts according to the present embodiment were observed to have an effect of enhancing OPH activity by 1.2 times or more compared to the control without addition. In particular, the measurement results of 599% for burdock, 457% for oolong tea, and 326% for houji tea were obtained, and in all cases, the effect of enhancing OPH activity by 3 times or more was observed compared to the control without addition.
<Embodiment 1 effect>

The plant extract according to this embodiment has an OPH activity enhancing effect of 1.2 times or more as compared with the control without any addition, and in particular, burdock, oolong tea, and hoji tea have 3 times or more OPH activity. A potentiating effect was observed.
<Embodiment 2>
<Overview of Embodiment 2>

  In this embodiment, first, it is verified whether OPH, which is an enzyme that degrades oxidized protein, has a degradation action on glycated proteins and glycated end products (AGEs). And, when OPH has a degrading action, can the 19 plant extracts according to Embodiment 1 in which the OPH activity enhancing action is recognized be able to further activate the degrading action of OPH on glycated proteins and AGEs? It is shown whether or not.

  Glycated proteins and AGEs are produced by the reaction of in vivo cells and tissues with carbohydrates essential for life support. It is well known that glycated proteins and AGEs have adverse effects such as skin aging, dementia, cancer, hypertension, arteriosclerosis and the like.

  By the way, a proteasome is known as an enzyme that degrades oxidized proteins, but this proteasome cannot degrade glycated proteins and AGEs. Therefore, as long as life activity is carried out by ingesting oxygen and sugar, the proteasome cannot be sought for the effect of suppressing both protein oxidation and saccharification reactions that are unavoidable. However, if OPH can also degrade glycated proteins, it is possible to efficiently suppress both oxidation and saccharification reactions by ingesting OPH.

Furthermore, if a substance that activates the degradation action of OPH on oxidized proteins can also activate the degradation action of OPH on glycated proteins and AGEs, the effect of OPH can be further increased, and humans are healthy and young. It is expected to make a great contribution to being able to live.
<Embodiment 2 test>
<Test 1>

  In this test, a measurement test is conducted to determine whether OPH has a degrading action on glycated proteins and AGEs. In addition, the sample used by this test is the same as the sample extracted from 19 types of plants which concern on Embodiment 1. FIG.

  (1) Adjustment of AGEs

  100 mmol / L phosphate buffer (pH 7.4) 500 μL, 2 mol / L glucose (Glc) 100 μL, 40 mg / mL human serum albumin (HSA) 200 μL, distilled water 200 μL, mixed with Glc and HSA (Glc- HSA G +) was prepared. Next, 500 μL of 100 mmol / L phosphate buffer, 200 μL of 40 mg / mL HSA, and 300 μL of distilled water were mixed to prepare a solution containing no Glc (Glc-HSA G-). Each mixture was incubated at 60 ° C. for 40 hours.

  (2) Reaction between OPH and glycated protein

Using Glc-HSA G + and Glc-HSA G- after the incubation, reaction solutions A to E in Table 2 below were prepared and incubated at 37 ° C. for 90 minutes. To the reaction solution after incubation, 100 μL of PCA was added and centrifuged to precipitate the protein and remove the supernatant. Furthermore, 350 μL of Tris-HCl (pH 7.4) was added to the precipitate and redissolved.

  (3) Measurement of fluorescent AGEs

  250 μL of the re-dissolved reaction solutions A to E were added to a 96-well microplate, and fluorescence at an excitation wavelength of 370 nm and a detection wavelength of 440 nm was measured with a microplate reader.

  (4) Pentosidine measurement

  1 mL of 6N hydrochloric acid was added to the reaction reagents A to E after redissolving, followed by hydrolysis with a block incubator at 100 ° C. for 18 hours. Thereafter, the reaction solution sample was dried and dissolved with 1 mL of distilled water. Next, 500 μL of each reaction solution sample was injected into an ion exchange column (Oasis MCX), and after passing 3 mL of 0.1N hydrochloric acid, pentosidine was eluted with 4.5 mL of 7% NH 3 aqueous solution.

  The pentosidine eluate was evaporated to dryness, and 300 μL of 0.2% HFBA containing 10% acetonitrile (ACN) was added and dissolved therein. The sample was then analyzed by reverse phase HPLC.

  The column used was YMC Triart C18 / S-5 μm / 12 nm 150 × 4.6 mm, and the analysis conditions were a flow rate of 1.0 ml / min, a column temperature of 40 ° C., and fluorescence detection (excitation wavelength 335 nm, fluorescence wavelength 385 nm). The injection volume was 50 μL, and eluent conditions were A) 0.2% HFBA, B) ACN, and 20% B (0-9 min), 100% B (9-17 min) gradient analysis.

  (5) CML measurement

  CML in the reaction reagents A to E after re-dissolution was measured using a CircuitLex CML / Nε- (carboxymethyl) Lysine ELISA Kit (Cyclex).

  (6) Measurement of AGE degradation by OPH

Each measured value of fluorescent AGEs, pentosidine, and CML was obtained from the following formula 2 (fluorescence value including OPH) and formula 3 (fluorescence value not including OPH).
(Formula 2) OPH + = Reaction solution A-C-E
(Formula 3) OPH- = Reaction solution BD

  (7) Results

FIG. 3 shows the measurement results of fluorescent AGEs, pentosidine, and CML. As shown in the figure, since OPH + containing OPH shows a lower fluorescence value than OPH- without OPH, OPH contains AGEs contained in Glc-HSA G +. Permitted to have been disassembled.
<Test 2>

  In Test 1 above, OPH was found to degrade AGEs. Thus, a test was conducted to determine whether the 19 plant extracts according to Embodiment 1 in which the OPH activity enhancing action was able to activate the degradation action of OPH on AGEs.

  (1) Measurement of AGEs degradation activity enhancing action

  100 mmol / L phosphate buffer (pH 7.4) 500 μL, 2 mol / L glucose (Glc) 100 μL, 40 mg / mL human serum albumin (HSA) 200 μL, distilled water 200 μL, mixed with Glc and HSA (Glc- HSA G +) was prepared. Similarly, a solution (Glc-HSA G-) to which Glc was not added was prepared.

Reaction reagents shown in Tables A to D below were prepared, including reaction liquids obtained by incubating each of the mixed liquids at 60 ° C. for 40 hours. The prepared reaction reagents A to D were incubated at 37 ° C. for 90 minutes, and then 100 μL of 70% PCA was added and centrifuged to precipitate the protein in the reaction solution.

The precipitated protein was dissolved and re-dissolved by adding 50 mmol / L Tris-HCl (pH 7.4), and then 250 μL of reaction reagents A to D were added to each 96-well microplate. AGEs-derived fluorescence at an excitation wavelength of 370 nm and a detection wavelength of 440 nm was measured with a microplate reader. The AGEs degradation activity enhancement rate was obtained from the fluorescence value by applying the reaction reagents A to D to the following formula 4.
(Formula 4) AGEs degradation activity enhancement rate (%) = 100− (reaction reagent BCD) / (reaction reagent ACD) × 100

  (2) Results

  FIG. 4 shows a sample from which a positive result was obtained among the measurement results of the AGEs degradation activity enhancement rate.

As shown in the figure, it was shown that bancha, lemongrass, roasted green tea, oolong tea, pu-eru and safflower are more active in decomposing OPH to AGEs. Therefore, it was shown that these six kinds of plant extracts not only activate the degradation action of oxidized protein of OPH but also activate the degradation action on AGEs.
<Embodiment 2 effect>

  According to this embodiment, it becomes possible to provide an OPH activity enhancer that also activates AGE decomposition of OPH by containing one or more extracts of oolong tea, hoji tea, lemongrass, bancha, pu-eru, safflower. .

Claims (9)

  Containing one or more extracts of burdock, oolong tea, roasted green tea, lemongrass, yellowfin aloe vera, capsicum, red pepper, barley, flaxseed, gimnema, shrimp, safflower, mulberry, litchi, pu-erh tea, bancha, dokudami, pearl barley, hawthorn An OPH activity enhancer that activates the oxidized proteolytic activity of oxidized proteolytic enzyme (OPH) more than 1.2 times.   An OPH activity enhancer consisting of a combination of one or more extracts of burdock, oolong tea, and hojicha, which activates the oxidized proteolytic activity of OPH three times or more.   OPH activity consisting of a combination of one or more extracts of oolong tea, roasted tea, lemongrass, bancha, pu-eru, safflower and activated OPH oxidative proteolytic activity more than 1.2 times and activated OPH AGEs degradation Enhancer.   A food or drink containing a combination comprising one or more OPH activity enhancers according to claim 1.   A health food containing a combination comprising one or more OPH activity enhancers according to claim 1.   A food additive containing a combination comprising one or more OPH activity enhancers according to claim 1.   A pharmaceutical comprising a combination comprising one or more OPH activity enhancers according to claim 1.   A cosmetic comprising a combination comprising one or more OPH activity enhancers according to claim 1.   A quasi-drug containing a combination comprising one or more OPH activity enhancers according to claims 1 to 3.

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