JP2020029529A - Antioxidant - Google Patents

Abstract

To provide a highly-safe and easily-ingestable antioxidant showing high antioxidant activity in a body, concerning an antioxidant containing S-1-propenyl cysteine.SOLUTION: Since being excellent in active oxygen cancellation capability, and showing high antioxidative effect, an antioxidant containing S-1-propenyl cysteine which is one of sulfur-containing amino acids in an onion extract can be included in a food and drink such as a health food or a health drink, or livestock feed, and further can be blended into an agent for preventing or improving various symptoms affected by oxygen.SELECTED DRAWING: None

Description

  The present invention relates to antioxidants containing S-1-propenylcysteine.

  Normally, the amount of active oxygen produced and the amount of active oxygen reduced by active oxygen scavenging enzymes are balanced in the human or animal body, and the amount of active oxygen in the body is kept constant. However, it has been pointed out that active oxygen in the body increases due to the influence of the living environment such as aging, stress, and unbalanced eating. The presence of unnecessary active oxygen in the body becomes an oxidative disorder, so-called oxidative stress, and damages various tissues and organs in the body, adversely affecting aging, increasing the risk of lifestyle-related diseases and increasing the risk of carcinogenesis.

  Specifically, for example, arteriosclerosis, myocardial infarction, cerebral infarction, cerebral hemorrhage and other cardiovascular and cerebral nervous system diseases and rheumatism, collagen disease, inflammatory bowel disease, autoinflammatory disease and other inflammatory diseases Has been known to be related to active oxygen in the body, but recently it has been linked to the development of cancer, lifestyle-related diseases such as diabetes and hyperlipidemia, as well as sleep disorders and cognition. It is said that active oxygen is also associated with the disease.

  For these reasons, in recent years, attention has been focused on supplementing the antioxidant function in the body with an antioxidant that has the effect of eliminating active oxygen, and certain antioxidants are used in diseases involving active oxygen in the body. It is incorporated into pharmaceutical preparations used for the purpose of preventing or treating arteriosclerosis, heart diseases such as cerebral infarction and myocardial infarction, lifestyle diseases such as cancer and diabetes, and dementia associated with aging. ing. For example, probucol, an antioxidant, is used to treat hyperlipidemia, ubidecarenone is used to treat congestive heart failure, and edaravone is used as a cerebroprotective agent to improve neurological symptoms associated with the acute phase of cerebral infarction, impaired daily living, and dysfunction. It is known to be used, and further, antioxidants such as α-tocopherol and ascorbic acid have been incorporated into various pharmaceutical preparations.

  It has been known that allium genus plants have antioxidant activity. In particular, several studies have been made on the antioxidant activity of onion and garlic-derived components. For example, a food composition having an antioxidant effect containing an onion extract (Patent Document 1), an antioxidant containing a ginger garlic extract as an active ingredient (Patent Document 2), garlic sprout fine powder having an antioxidant activity A substance (Patent Document 3) and a method for producing a crushed garlic having an active oxygen scavenging ability (Patent Document 4) are already known.

  Also, Allium plants include S-allyl-L-cysteine, S-allyl-L-cysteine sulfoxide, S-methyl-L-cysteine, S-methyl-L-cysteine sulfoxide, and S-1-propenyl-L-. It contains amino acids having a sulfur molecule such as cysteine sulfoxide and analogs thereof, and there have been several reports on the antioxidant activity of such sulfur-containing amino acids derived from Allium plants. For example, LDL oxidation inhibitory activity of various sulfur-containing compounds derived from Allium plants such as S-allyl-L-cysteine (Non-Patent Document 1) and S-alkyl (alkenyl) -L-cysteine sulfoxides derived from Allium plants The antioxidant activity (Non-patent document 2) and the DPPH radical scavenging activity of red onion-derived S-3-pentenylcysteine sulfoxide (Non-patent document 3) have been reported.

As sulfur-containing amino acids derived from Allium plants, S-1-propenylcysteine is also known, and this compound is γ-glutamyl-one of the γ-glutamyl peptides characteristically contained in Allium plants. Cutting and grinding the Allium plant into S-1-propenylcysteine;
Alternatively, it is a compound produced by the action of endogenous γ-glutamyl transpeptidase upon maturation.

  As for the physiological function of S-1-propenylcysteine, there have been reports on a blood pressure lowering effect (Patent Document 5), an immunomodulatory activity (Patent Document 6), and a testosterone production promoting effect (Patent Document 7).

JP 2006-510010 A JP 2008-231055 A JP 2014-217340 A JP-A-2014-13 International Publication WO2016 / 199885 International Publication WO0216 / 08892 JP 2015-196688 A

J. Agric. Food Chem. 2003, 51, 7208-14 J. Agric. Food Chem. 2002, 50, 2488-93 J. Nat. Prod. 2008, 71, 2036-7

  However, the antioxidant activity of S-1-propenylcysteine has never been reported before, and therefore, no comparison of the antioxidant activity with sulfur-containing amino acids having other similar structures has been made. .

  An object of the present invention is to provide an antioxidant which is highly safe, can be easily taken, and exhibits high antioxidant activity in the body.

  The present inventors examined the antioxidant activity of various sulfur-containing amino acids in an onion extract, and found that S-1-propenylcysteine had an excellent antioxidant activity in the body as compared with other sulfur-containing amino acids. This led to the completion of the present invention.

  Since the antioxidant of the present invention is excellent in active oxygen scavenging ability and exhibits a high antioxidant effect, it can be expected to improve various symptoms affected by active oxygen in the body. Since the antioxidant of the present invention can eliminate active oxygen in the body by ingesting it, arteriosclerosis, cerebral infarction, heart disease, cancer, and diabetes, which are diseases involving active oxygen in the body, It can be used as a compounding component of a pharmaceutical preparation for preventing and alleviating symptoms such as hyperlipidemia, sleep disorder, dementia, and inflammatory disease.

Embodiments of the present invention include the following aspects.
(1) An antioxidant containing S-1-propenylcysteine as an active ingredient.
(2) The antioxidant according to the above (1), comprising 1 to 10,000 mg of S-1-propenylcysteine as a daily intake.
(3) The antioxidant according to the above (1) or (2), wherein S-1-propenylcysteine is contained in an extract of Allium genus.
(4) The antioxidant according to any one of (1) to (3) above, wherein S-1-propenylcysteine is contained in the onion extract.
(5) A food or drink containing the antioxidant according to any one of (1) to (4).
(6) A health food containing the antioxidant according to any one of (1) to (4).
(7) A health drink containing the antioxidant according to any one of (1) to (4).
(8) A feed containing the antioxidant according to any one of (1) to (4).
(9) A pharmaceutical preparation containing the antioxidant according to any one of the above (1) to (4).
(10) The pharmaceutical preparation according to (9) above, which is an agent for preventing or ameliorating an inflammatory disease.
(11) The pharmaceutical preparation according to (9), which is an agent for preventing or improving inflammatory diseases such as arteriosclerosis, cerebral infarction, heart disease, cancer, diabetes, hyperlipidemia, sleep disorder, and dementia.

  S-1-propenylcysteine, which is an active ingredient of the present invention, is a cysteine derivative represented by the following formula (1).

  S-1-propenylcysteine has a cis or trans configuration, but in the present invention, it may be any one of them, and also has an optical isomer because of having an asymmetric carbon derived from cysteine. However, D-form and L-form may be any of racemic forms. Further, in the present invention, S-1-propenylcysteine can be used in the form of any of an acid addition salt and a base addition salt.

  As S-1-propenylcysteine which is an active ingredient of the present invention, a synthetic product or a commercially available reagent may be used. As a method for synthesizing S-1-propenylcysteine, for example, removal of a reductive benzyl group with sodium in liquid ammonia of (E) -1- (benzylyl) -1-propene and addition reaction of 3-chloro-L-alanine are performed. Can be easily obtained. (Non-patent document: Bull. Korean Chem. Soc. 2011, 32, 319-320)

  Alternatively, a composition containing a sulfur-containing amino acid produced by extracting or purifying from natural products such as animals and plants containing the above-mentioned substances can be used. Preferred examples include S-extracted from allium genus plants such as onions. A composition containing 1-propenylcysteine can be mentioned. Allium plants containing S-1-propenylcysteine include onion, garlic, elephant garlic, leek, leek, and the like, and these plants may be used alone or in combination. In addition, as the above-mentioned allium plants, raw ones can be used as they are, or those obtained by removing the outer skin and cutting or shredding as necessary. Further, a product dried by freeze drying or hot air drying, or a product obtained by powdering them may be used.

As an extraction and purification method from an Allium plant containing S-1-propenylcysteine, extraction with a water-miscible solvent such as methanol or ethanol, or a mixed solvent of a water-miscible solvent and water is performed, followed by ion exchange chromatography and hydrophobic treatment. A purification method in combination with chromatography using a hydrophilic resin can be used.

  By the above method, a fraction containing high content of S-1-propenylcysteine can be obtained from a plant of the genus Allium. The obtained fraction can be dried as it is and contained in the antioxidant of the present invention. Further, if necessary, in addition to or in place of the above drying, treatments such as freeze-drying, solidification, granulation, powderization, granulation, tableting, and encapsulation may be performed.

  The content of S-1-propenylcysteine in the antioxidant of the present invention is preferably from 1 to 10,000 mg, more preferably from 10 to 5000 mg, and still more preferably from 100 to 1000 mg, based on the adult daily intake.

  Antioxidants of the present invention, arteriosclerosis is a disease associated with active oxygen, cerebral infarction, heart disease, cancer, diabetes, sleep disorders, dementia and the like, for relieving symptoms, medicines and health foods, It can be contained in foods and drinks such as health drinks and feeds. The antioxidant of the present invention is applied to humans or non-human mammals, and examples of non-human mammals include pets, livestock animals, and animals raised in breeding facilities.

  Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

(Production example) Purification method from onion Hokkaido onion (commercial product) 323 g of outer skin was removed, heated at 85 ° C for 30 minutes to inactivate the enzyme, and then 320 mL of 50% methanol (v / v) was added. And crushed and extracted with a juicer mixer. After filtration, the obtained solution was concentrated at 45 ° C. under reduced pressure and dried to obtain 18.5 g of an extract.
A solution obtained by dissolving this extract in 100 mL of 20 mM acetic acid was added to a column packed with 50 mL of Amberlite 120 (H ⁺ type, Sigma-Aldrich) previously equilibrated with 20 mM acetic acid, and after passing through, washed with 100 mL of water. 5% ammonia water (W / V)
The S-1-propenylcysteine-containing fraction was eluted with 200 mL. The eluate was adjusted to pH 4 with 1 M formic acid, and then concentrated under reduced pressure at 45 ° C until the solution volume reached 75 mL. The concentrate was added to Sep Pak C18 (filler 5 g, column volume 20 mL, Waters), and after passing through, 20 mL of 0.05% trifluoroacetic acid (TFA) 10 mL of 5% acetonitrile / 0.05% TFA 10% Elution was performed in the order of 10 mL of acetonitrile / 0.05% TFA. After concentrating and drying 10% acetonitrile / 0.05% TFA solution containing S-1-propenylcysteine, preparative HPLC (column: CAPSEL PAK C18 (20 mmφ × 250 mm, Shiseido), column temperature: 40 ° C., transfer Phase: 7% acetonitrile / 0.05% TFA, flow rate: 6 mL / min, detection: OD 230 nm), and the peak of S-1-propenylcysteine showing a retention time of 16.5 minutes was collected (yield 1. 0 mg, 95% purity).

(Example 1) DPPH radical scavenging activity test In order to evaluate the antioxidant power of the antioxidant of the present invention, the following test was performed.
The test substances described in Table 1 were dissolved in 50% ethanol, and 100 μL each was added so that the addition amount to each well of the 96-well plate was 3.9 to 250 μg. 50 μL of 200 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.0) was added, and 50 μL of 1 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH, WAKO) ethanol solution was added. Stirring was performed for 20 minutes in the dark. The absorbance at 520 nm of each well was measured using a plate reader (SPECTRA MAX 190, Molecular D).
devices).
As a control, a well to which only 50% ethanol was added was provided, and as a positive control, 0.1% was used to compare with Trolox used in biological or biochemical applications to reduce oxidative stress or damage. Wells were added to add 078-5 μg of Trolox (CALBIOCHEM). The DPPH radical scavenging rate was calculated by the following equation.

DPPH radical scavenging rate (%) = {(BA) / B} × 100

In the formula, A represents “absorbance of test substance added well”, and B represents “absorbance of control well”.

DPPH radical scavenging activity of each test substance was calculated and IC ₅₀ is the concentration that shows the Trolox and DPPH radical scavenging rate of 50% from the regression line of concentration and DPPH radical scavenging rate of each test substance, IC ₅₀ (Trolox) / IC The value of ₅₀ (test substance) was shown as Trolox equivalent (μM Trolox equivalent / mM).

  As shown in Table 1, among the tested alkyl (alkenyl) cysteine and alkyl (alkenyl) cysteine sulfoxide, the compounds showing activity at an addition amount of 250 μg or less are S-1-propenylcysteine and S-allyl. -L-cysteine and S-propenyl-L-cysteine sulfoxide, but the activity of S-allyl-L-cysteine and S-propenyl-L-cysteine sulfoxide was 8% or less of that of S-1-propenylcysteine. .

(Example 2) Intracellular reactive oxygen scavenging activity test in RAW 264.7 cells RAW 264.7 cells were obtained from DS Pharma Biomedical. The cells were subcultured and maintained in a Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, D5796) supplemented with 10% fetal calf serum in a 5% carbon dioxide atmosphere at 37 ° C. A cell suspension of 5 × 10 ⁵ cells / mL was prepared from cells in a semi-confluent state, and 100 μL of the cell suspension was seeded on a 96-well plate and cultured overnight. After removing the medium, the cells were washed with 100 μL of Dulbecco's modified Eagle's medium (Sigma-Aldrich, D5921) containing no phenol red, and 100 μL of assay medium containing various concentrations of the test substance was added, followed by culturing for 3 hours. Was. As a control, 100 μL of assay medium alone was added, and culturing was performed in the same manner. 2 ′, 7′-dichlorofluorescein diacetate (20 mM, DCFDA, ABCAM abb13851 kit) was diluted to 200 μM with an assay medium, 10 μL of the diluted solution was added to each well, and culturing was further performed for 1 hour. The fluorescence intensity (Ex 485 nm, Em 535 nm) of each well was measured using a fluorescence plate reader (Arvo X4, Perkin Elmer). The intracellular active oxygen elimination rate (%) was calculated by the following equation.

Active oxygen elimination rate (%) = {(BA) / B} × 100

In the formula, A represents “the fluorescence intensity of the test substance added well”, and B represents “the fluorescence intensity of the control well”.

Table 2 shows the intracellular active oxygen scavenging activity of each test substance on RAW 264.7 cells at 0.3 and 1.0 mg / mL.
The only compound that showed activity at 0.3 mg / mL was S-1-propenylcysteine. At 1.0 mg / mL, S-1-propenylcysteine and S-1-propenyl-L-cysteine sulfoxide showed activity. The activity intensity of S-1-propenylcysteine at 1.0 mg / mL was higher than that of S-1-propenyl-L-cysteine sulfoxide.
On the other hand, S-allyl-L-cysteine and S-allyl-L-cysteine sulfoxide showed no activity at 0.3 and 1.0 mg / mL.

(Example 3) Effect evaluation test on production of nitric oxide (NO) in RAW 264.7 cells Effect of the antioxidant of the present invention on nitric oxide (NO), which is a physiologically active substance involved in inflammation in vivo The following tests were performed to evaluate
The RAW 264.7 cells were cultured in a 96-well plate in the same manner as in Example 2. After culturing for 24 hours, the medium was removed, and 0.1 µg / mL of lipopolysaccharide (LPS) containing various concentrations of S-1-propenylcysteine and Dulbecco's modified Eagle's medium containing 10% fetal bovine serum were added for another 24 hours. Cultured for hours. NO in the culture medium was measured by the measuring kit using the Griess reagent _{(NO 2 / NO 3 Assay kit} -CII, DOJINDO).

  The effect of S-1-propenylcysteine on NO production in RAW 264.7 cells is shown in Table 3. No addition indicates that LPS and the test substance were not added, and control indicates that LPS was added but the test substance was not added. S-1-propenylcysteine suppressed NO production of RAW 264.7 cells in a concentration range of 0.3 and 1.0 mg / mL.

(Example 4) Test for evaluating the effect of RAW 264.7 cells on PGE2 production The effect of the antioxidant of the present invention on the production of prostaglandin E2 (PGE2), which is a physiologically active substance involved in inflammation in vivo, was evaluated. The following test was conducted in order to do so.
The RAW 264.7 cells were cultured in a 96-well plate in the same manner as in Example 2. After culturing for 24 hours, the medium was removed, and 0.1 μg / mL of LPS supplemented with various concentrations of S-1-propenylcysteine and Dulbecco's modified Eagle medium containing 10% fetal bovine serum were added, followed by further culturing for 24 hours. PGE2 in the culture solution was measured using an ELISA measurement kit (Cayman Chemical).

  The effect of S-1-propenylcysteine on PGE2 production in RAW 264.7 cells is shown in Table 4. No addition indicates that LPS and the test substance were not added, and control indicates that LPS was added but the test substance was not added. S-1-propenylcysteine suppressed PGE2 production of RAW 264.7 cells in a concentration range of 0.1 to 1.0 mg / mL.

  The above results indicate that the antioxidant of the present invention containing S-1-propenylcysteine as an active ingredient has extremely high antioxidant power and effectively inhibits NO production and PGE2 production even in cells. Is shown. Therefore, diseases related to oxidative stress caused by active oxygen in the body, such as arteriosclerosis, cardiovascular and cerebral nervous system diseases such as myocardial infarction and cerebral infarction and cerebral hemorrhage, and inflammatory diseases, cancer, diabetes, and hyperlipidemia It is expected to be effectively used in the prevention and treatment of lifestyle-related diseases, sleep disorders, dementia, and the like.

Claims (11)

  An antioxidant containing S-1-propenylcysteine as an active ingredient.   The antioxidant according to claim 1, which contains 1 to 10,000 mg of S-1-propenylcysteine as a daily intake.   The antioxidant according to claim 1, wherein the S-1-propenylcysteine is contained in an extract of Allium plant.   The antioxidant according to any one of claims 1 to 3, wherein S-1-propenylcysteine is contained in the onion extract.   A food or drink comprising the antioxidant according to any one of claims 1 to 4.   A health food containing the antioxidant according to claim 1.   A health drink containing the antioxidant according to any one of claims 1 to 4.   A feed containing the antioxidant according to any one of claims 1 to 4.   A pharmaceutical preparation comprising the antioxidant according to any one of claims 1 to 4.   The pharmaceutical preparation according to claim 9, which is an agent for preventing or ameliorating an inflammatory disease.   The pharmaceutical preparation according to claim 9, which is an agent for preventing or improving arteriosclerosis, cerebral infarction, heart disease, cancer, diabetes, hyperlipidemia, sleep disorder, and dementia.