JP6515427B2 - OPH activity enhancer - Google Patents

Description

  The present invention relates to OPH activity enhancers that enhance the activity of oxidized protein hydrolase (OPH), which is an in vivo enzyme that degrades oxidized proteins, and food and drink products, pharmaceuticals, etc. that contain the OPH activity enhancers. About.

  It is known that oxidation reactions in vivo adversely affect cells and tissues. For example, it becomes one of the causes of aging and various diseases such as age-related changes such as tissue collagen, Alzheimer's disease, cataract, skin aging and the like.

  Oxidized protein is degraded and removed by OPH. OPH is widely distributed in living tissues, and is a type of serine protease that preferentially degrades oxidized proteins, and is an enzyme that releases the N-terminal acylated amino acid of proteins (Acylamino-acid-releasing enzyme: Known as AARE). This OPH is known to decrease its activity with age.

  It has been reported that OPH activity is significantly elevated in the serum of diabetic rats, and serum with elevated OPH activity is reported to have a low amount of carbonyl-modified protein, and it is possible that OPH may be involved in the suppression of diabetes and aging progression. It is done.

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

International Publication Number WO 2011/040733

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

  The following invention etc. are provided as a means for solving the said subject. That is, as the first invention, extracts of burdock, oolong tea, roasted tea, lemon grass, bitter gourd, pepper, barley, barley, amacha-zuru, gimmunema, ebisugusa, safflower, mulberry, reishi, puyar tea, bancha, dokdami, mistletoe, ginseng extract The present invention provides an OPH activity enhancer that activates the oxidative protein degradation activity of oxidative proteolytic enzyme (OPH) by 1.2 times or more.

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

  The third invention comprises a combination of one or more extracts of oolong tea, roasted tea, lemon grass, bancha, pueal and safflower, and activates OPH's oxidative protein degradation activity by 1.2 times or more and OPH's AGES degradation Provide an OPH activity enhancer that activates

  As a fourth invention, food and drink containing a combination containing one or more of the OPH activity enhancer according to the first invention to the third invention, health food, food additive, medicine, cosmetics, quasi-drug I will provide a.

  According to the present invention, it is possible to provide an OPH activity enhancer that enhances the oxidative proteolytic activity of OPH.

Figure showing transition of OPH activity enhancement effect of 17 samples Figure showing OPH activity enhancement ratio after 90 minutes of 19 samples Figure showing measurement results of fluorescent AGEs, pentosidine and CML The figure which shows the measurement result in which the AGE degradation activity enhancement rate became positive.

Hereinafter, embodiments of the present invention will be described. The present invention should not be limited to these embodiments at all, and can be implemented in various modes without departing from the scope of the invention.
First Embodiment
Embodiment 1 Outline

In the present embodiment, OPH activity enhancing action is recognized by test with respect to the enhancing action of the oxidative proteolysis activity (hereinafter simply referred to as OPH activity) of OPH which is a degradation enzyme of oxidative protein, as compared with the control (no sample added). The present invention relates to OPH activity enhancers comprising extracts of 17 kinds of plants.
Embodiment 1 Configuration

  The OPH activity enhancer according to the present embodiment includes burdock, oolong tea, roasted green tea, lemon grass, lemongrass, blackberry, red pepper, barley, mackerel, gymnema, ebisugusa, safflower, mulberry, reishi, puyar tea, bancha, dokdami, hatomi, And a combination comprising one or more extracts of The 17 samples listed here are 1.2 times or more OPH compared with the control to which no sample is added among the 100 or more samples subjected to the OPH oxidative protein degradation activity enhancement test described later. The oxidative protein degradation activity enhancing action was recognized.

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

  The "burdock" Arctium lappa is a perennial plant of the genus Asteraceae.

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

  "Hojicha" is a tea made with bancha.

  "Lemongrass" Cymbopogon citratus is a perennial plant of the grass genus Ogarukaya.

  "Kidachiaroe" Aloe arborescens is a succulent plant of the genus Aloe.

  "Kazazasa" Sasa veitchii is a plant of the genus Sasa.

  "Capsicum" Capsicum annuum is a plant of the Solanaceous Capsicum genus.

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

  "Amacha-zuru" Gynostemma pentaphyllum is a plant of the family Uriaceae Amacha-zuru.

  "Gymnema" Gymnema sylvestre is a plant of the genus Streptomyces.

  "Ebisugusa" Senna obtusifolia is a plant of the family Scorpionaceae.

  "Safflower" Carthamus tinctorius is a plant belonging to the family Asteraceae.

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

  "Reishi" Ganoderma lucidum is a fungus of the genus Mannidaceae.

  "Puor tea" is a post-fermented tea that is produced by stopping the oxidative fermentation of tea leaves (Camellia sinensis leaves) by heating and maturing with microorganisms such as koji.

  "Bancha" is a tea made by cutting together with stems hard tea leaves (Camellia sinensis leaves) from second tea after picking in summer.

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

  "Hamatogi" Coix lacryma-jobi var. Frumentacea is a plant belonging to the genus Poaceae.

  "Habassau" Senna occidentalis is a plant of the family Senna of the family Jaccarinaceae.

The OPH activity enhancer consisting of one or two or more of the 19 plant extracts mentioned above is applied as food and drink containing the same, health food, food additive, medicine, cosmetics, quasi-drug and the like (The same applies to the second embodiment).
<Embodiment 1 Test>

The 19 plant extracts described above were those in which the OPH activity enhancing action was recognized in the following test.
<Test 1>

  (1) Extraction of sample

  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 to recover the supernatant, which was used as a sample extract.

  (2) Preparation of reaction reagents

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

  (3) Measurement of OPH activity enhancing action

  The prepared reaction reagents (except for the AAPA substrate) were placed in each well of the 96-well microplate, and then the AAPA substrate was added to initiate the reaction. The set temperature of the microplate reader at the time of measurement was 37 ° C., and the absorbance at the 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, each absorbance of reaction reagents A to D was determined. Then, the value obtained by applying the obtained absorbance of each reaction reagent to the following equation was defined as the OPH activity enhancement rate. A sample with an OPH activity enhancement rate above 100 indicates that OPH activity was enhanced, and a sample below 100 indicates that OPH activity was decreased.
(Formula 1) OPH activity enhancement rate = (Reagent B-C) / (Reagent A-D) x 100

  (4) Results

  FIG. 1 is a graph showing the transition of the OPH activity enhancement ratio after 30 minutes, 60 minutes, and 90 minutes from the start of measurement for 17 types of samples out of 19 types, except for barley and barley.

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

As described above, in each of the 19 plant extracts according to the present embodiment, an OPH activity enhancing action of 1.2 times or more was observed as compared with the control without any addition. In particular, the measurement results were 599% for burdock, 457% for oolong tea, and 326% for hojicha, and all three times more OPH activity enhancing action was observed compared to the control without addition.
<Embodiment 1 Effect>

The plant extract according to the present embodiment exhibited an OPH activity enhancing action 1.2 times or more as compared with any of the control without any additive, and in particular, for burdock, oolong tea and roasted tea, OPH activity three times or more. An enhancing effect was observed.
Second Embodiment
Embodiment 2 Outline

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

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

  By the way, although proteasome (proteasome) is known as an enzyme which degrades an oxidation protein, this proteasome can not degrade a glycated protein or AGEs. Therefore, the proteasome can not be required to have an effect of suppressing any of the oxidation and glycation reactions of proteins which inevitably occurs as long as oxygen and carbohydrates are consumed to perform life activities. However, if OPH can also degrade glycated proteins, it is possible to efficiently suppress both oxidation and saccharification reactions by taking OPH.

Furthermore, if a substance that activates the degradative action of OPH on oxidized proteins can also activate the degradative action of OPH on glycated proteins and AGEs, the effect of OPH can be further enhanced, and human health and health It is expected that it will greatly contribute to being motivated.
<Embodiment 2 Test>
<Test 1>

  In this test, a measurement test is conducted to determine whether OPH has a degrading effect 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, and distilled water 200 μL, and 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 Glc-free solution (Glc-HSG-). Each mixture was then incubated at 60 ° C. for 40 hours.

  (2) Reaction of OPH and glycated protein

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

  (3) Measurement of fluorescent AGEs

  250 μL of the reaction solutions A to E after re-dissolution 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) Measurement of pentosidine

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

  The pentosidine eluate was evaporated to dryness, and 300 μL of 0.2% HFBA containing 10% acetonitrile (ACN) was added thereto for dissolution. 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 flow rate 1.0 ml / min, column temperature 40 ° C., fluorescence detection (excitation wavelength 335 nm, fluorescence wavelength 385 nm). The injection amount was 50 μL, and the eluent conditions were gradient analysis of A) 0.2% HFBA, B) ACN, 20% B (0-9 min), 100% B (9-17 min).

  (5) Measurement of CML

  CML in reaction reagents A to E after reconstitution was measured using CircuLex CML / Nε- (carboxymethyl) Lysine ELISA Kit (cyclex).

  (6) Measurement of AGE decomposition by OPH

Each measured value of fluorescence AGEs, pentosidine, and CML was calculated | required from following formula 2 (fluorescence value containing OPH) and Formula 3 (fluorescence value which does not contain OPH).
(Formula 2) OPH + = reaction liquid A-C-E
(Formula 3) OPH-= reaction solution B-D

  (7) Results

FIG. 3 shows the measurement results of fluorescent AGEs, pentosidine and CML. As shown in the figure, OPH contains AGEs contained in Glc-HSAG + since it shows a lower fluorescence value in OPH + containing OPH than in any of OPH- which does not contain OPH. It is considered to be disintegrated.
<Test 2>

  In Test 1 above, OPH was found to degrade AGEs. Therefore, it was tested whether 19 plant extracts according to Embodiment 1 in which the OPH activity enhancing action was recognized can further activate the decomposing action of OPH on AGEs.

  (1) Measurement of AGEs decomposition activity enhancement 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, and distilled water 200 μL, and mixed with Glc and HSA (Glc- HSA G +) was prepared. Similarly, a solution to which Glc was not added (Glc-HSAG-) was prepared.

The respective mixtures were incubated at 60 ° C. for 40 hours to prepare reaction reagents of the following Tables A to D including the reaction solutions obtained. 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 proteins in the reaction solution.

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

  (2) Result

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

As shown, it has been shown that the decomposing effect of OPH on AGEs is further activated for bancha, lemongrass, roasted tea, oolong tea, pueal, safflower. Therefore, it was shown that these six plant extracts not only activate the degradative action of OPH on oxidized proteins, but also activate the degradative actions on AGEs.
Embodiment 2 Effects

  According to this embodiment, by including one or more extracts of oolong tea, roasted tea, lemon grass, bancha, pueal, safflower, it is possible to provide an OPH activity enhancer that also activates AGEs degradation of OPH. .

Claims (8)

Burdock, oolong tea, roasted tea, lemon grass, bitter gourd, pepper, barley, amacha-zuru, gimmenema, ebisugusa, safflower, mulberry, leish, poea tea, banya, brown tea, cotton oat extract and one or more extracts of oxidative protein degrading enzyme An OPH activity enhancer that activates the oxidative proteolytic activity of (OPH) by 1.2 times or more. An OPH activity enhancer that contains one or more extracts of burdock, oolong tea, and hoji tea, and activates the oxidative protein degradation activity of OPH three times or more. Food / beverage products for OPH activity enhancement containing the OPH activity enhancer according to claim 1 or 2. The health food for OPH activity enhancement containing the OPH activity enhancer according to claim 1 or 2. The food additive for OPH activity enhancement containing the OPH activity enhancer according to claim 1 or 2. OPH activity enhancing drugs containing OPH activity enhancer according to claim 1 or 2. An OPH activity enhancing cosmetic comprising the OPH activity enhancer according to claim 1 or 2. An OPH activity enhancing pharmaceutical quasi-drug containing the OPH activity enhancer according to claim 1 or 2.

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