JP6949431B2 - Antioxidant composition - Google Patents

Description

The present invention relates to antioxidant compositions.

Ampelopsin is a flavonol compound contained in wisteria tea and is also called dihydromylicetin.
Wisteria tea, a plant containing ampelopsin, is classified into the genus Ampelopsis of the Grapes family, which is mainly produced in China, and its Chinese name is Akito snake grape. The scientific name is Ampelopsis grossedentata. This plant is mainly distributed in provinces and autonomous regions such as Guangxi, Guangxi, Yunnan, Guizhou, Hunan, Hubei, Gangxi, Fujian, China. Beverages made from these stems and leaves are commonly used as tea by the Zhuang and Yao people in provinces and autonomous regions such as Guangxi and Hunan in China (this is called "Fujicha"), and colds and throats. It is also used for pain. Amperopsin has various physiological and pharmacological activities exhibited by Fujicha, such as therapeutic action for liver diseases (Patent Document 1), lipase inhibitory action (Patent Document 2), matrix metalloproteinase inhibitory action (Patent Document 3), and antibacterial action (Patent Document 3). It is specified as the main body of action and activity such as Document 4), fragrance deterioration preventing action (Patent Document 5), and dye fading preventing action (Patent Document 6).

Purified ampelopsin is known to have a strong antioxidant effect. In Non-Patent Document 1, dihydromylicetin having a purity of 98% suppresses the production of malondialdehyde (MDA) in the myocardium, liver, and brain tissue homogenates of rats, and the higher the purification purity, the more the experimental strain. It reduces the DPPH free radicals in it, and dihydromilcetin suppresses the production of MDA in fats and oils, and as the purity (60% to 90%) increases, the antioxidant effect becomes stronger, and strong antioxidant in animal oils and vegetable oils. It is described that it has an effect.

Japanese Unexamined Patent Publication No. 2003-026584 Japanese Unexamined Patent Publication No. 2003-012536 JP-A-2002-173424 JP-A-2002-159566 Japanese Unexamined Patent Publication No. 2004-018756 Japanese Unexamined Patent Publication No. 2002-065201

http://saitouboueki.com/new/fujicha/characteristic.html

The present inventors have found that ampelopsin synergistically enhances the singlet oxygen scavenging ability of ascorbic acid.
The present invention has been made based on this finding, and an object of the present invention is to provide a new antioxidant composition containing ascorbic acid and ampelopsin.

The main configuration of the present invention is as follows.
(1) An antioxidant composition containing ascorbic acid and ampelopsin.
(2) The antioxidant composition according to (1), which contains 0.5 to 10 parts by mass of ampelopsin with respect to 90 parts by mass of ascorbic acid.
(3) The antioxidant composition according to (1) or (2), wherein ampelopsin is derived from a wisteria tea extract.
(4) The antioxidant composition according to any one of (1) to (3), wherein the antioxidant action is a singlet oxygen scavenging action.

INDUSTRIAL APPLICABILITY The present invention provides a new antioxidant composition. This antioxidant composition enhances the singlet oxygen scavenging action of ascorbic acid and exerts an antioxidant action. Further, since the antioxidant effect is enhanced by containing ampelopsin, the amount of ascorbic acid used can be reduced.

It is a schematic diagram of the plot for obtaining the approximate expression for obtaining the singlet oxygen scavenging effective concentration from the ESR measurement result.

The present invention relates to an antioxidant composition containing ascorbic acid and ampelopsin.

Ampelopsin is expressed by the following formula.

The ampelopsis used in the present invention is, for example, Fujicha (Ampelopsis glossedenta), Ampelopsis megalophylla, Cantonese snake grape (Ampelopsis cantoniensis), Hovenia dulcis (Hovenia dulcis). , Erythrophilum africanum and katsura (Cercidiphyllum japonicum) can be isolated and purified from plant extracts selected. Among these, wisteria tea is preferable.

Specifically, ampelopsin can be obtained from Ampelopsis planta, which is a plant belonging to the genus Ampelopsis, as follows.
That is, the extract obtained by extracting the branches and leaves of dried wisteria tea with hydrous ethanol is concentrated, subjected to column chromatography using, for example, a porous resin (DIAION HP-20), and eluted with 80% by volume hydrous methanol. Amperopsin is obtained in the picture. This can be further purified by reverse phase silica gel column chromatography or recrystallization. The purified ampelopsin is also sold as a reagent, and this can also be used.

To obtain ampelopsin from wisteria tea, perform the following operations.
Using crushed dried wisteria tea leaves or stems or powder as an extraction raw material, it is put into water or a hydrophilic organic solvent or a mixed solvent thereof, and extracted using an arbitrary device at room temperature or a temperature below the boiling point of the solvent. Can be obtained by

Examples of the organic solvent used for extraction include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene glycol, propylene glycol and iso. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as propylene glycol and glycerin.
Further, a mixed solvent of these hydrophilic organic solvents and water can be used. When a mixed solvent of water and a hydrophilic organic solvent is used, the amount of lower alcohol is 30 to 90 parts by mass with respect to 10 parts by mass of water, and the amount of lower aliphatic ketone is 10 parts by mass of water. On the other hand, it is preferable to add 10 to 40 parts by mass, and in the case of polyhydric alcohol, 10 to 90 parts by mass with respect to 10 parts by mass of water.

A dried and crushed product of wisteria tea is put into a treatment tank filled with an extraction solvent, and allowed to stand for 30 minutes to 2 hours with occasional stirring as necessary to elute soluble components, and then filtered to form a solid product. Is removed, the extraction solvent is distilled off from this extract, and the extract is dried to obtain an extract. The amount of the extraction solvent is preferably 5 to 15 times the amount (mass ratio) of the extraction raw material, and the extraction conditions are usually about 1 to 4 hours at 50 to 95 ° C. when water is used as the extraction solvent. Is. When a mixed solvent of water and ethanol is used as the extraction solvent, it is usually about 30 minutes to 4 hours at 40 to 80 ° C.

When the extraction solvent is distilled off from the obtained extract, a paste-like concentrate is obtained. Further drying gives a solid extract. In the present invention, as long as the content of ampelopsin is 10% by mass or more, preferably 20% by mass or more, the above extract or a concentrated solution thereof may be used. These may be used after being purified by a method such as activated carbon treatment, adsorption resin treatment, ion exchange resin, or liquid-liquid countercurrent distribution.
Therefore, an extract extracted from the above-mentioned wisteria tea and having an increased concentration of ampelopsin can also be used as the composition of the present invention.

The content of ampelopsin in the composition can be analyzed by a known analytical method such as HPLC. The outline of the quantification method is as follows.
-Preparation of sample solution Before filtration with a membrane filter adjusted by HPLC method, the sample solution (24 μg / ml) was diluted exactly 5-fold with 30% methanol (containing 0.5% ascorbic acid) to 0.2 μm. The standard solution is filtered through a membrane filter.

-HPLC method sample preparation Approximately 30 mg of the sample is precisely weighed, 30% methanol (containing 0.5% ascorbic acid) is added, and the sample is dissolved by ultrasonic waves to make exactly 50 mL. 1 mL of this solution is accurately diluted to 25 mL with 30% methanol (containing 0.5% ascorbic acid) and filtered through a 0.45 μm membrane filter to obtain a sample solution (24 μg / ml).

-Preparation of standard solution The 8 μg / ml standard solution prepared by the HPLC method is diluted exactly 5-fold with 30% methanol (containing 0.5% ascorbic acid) to prepare a 1.6 μg / ml standard solution. This is diluted exactly 5-fold with 30% methanol (containing 0.5% ascorbic acid) to prepare a 0.32 μg / ml standard solution. The 8 μg / ml standard solution, 1.6 μg / ml standard solution, and 0.32 μg / ml standard solution are filtered through a 0.2 μm membrane filter to obtain a standard solution.

-Preparation of HPLC method standard solution Weigh 10.00 mg of the standard product, add an appropriate amount of methanol (containing 0.5% ascorbic acid), sonicate and dissolve, and add methanol (containing 0.5% ascorbic acid). In addition, make it exactly 25 mL. Take 5 ml of the solution and add 12.5% methanol (containing 0.5% ascorbic acid) to make exactly 25 ml. Take 4 ml of the solution and add 30% methanol (containing 0.5% ascorbic acid) to make exactly 20 ml, and prepare a 16 μg / mL standard solution. Dilute exactly 2-fold to make an 8 μg / ml standard solution. This was diluted exactly 2-fold to a 4 μg / ml standard solution. The standard solution is filtered through a 0.45 μm membrane filter.

·Measurement condition
LC device: ACQUITY UPLC (Waters)
MS device: TOD (Waters)
Column: ACQUITY UPLC BEH C18 (1.7 μm, 100x2.1 mm id, Waters)
Column temperature: 40 ° C
Mobile phase: A) 0.1% formic acid / Ultra Pure Water,
B) 0.1% formic acid / MeCN
Isocratic analysis for 7 minutes in 10% B solution. Then, for column cleaning, linearly raise to 90% B solution over 2 minutes, linearly return to 10% B solution in 0.5 minutes, re-stabilize with 10% B solution for 4 minutes, and then perform the next analysis. ..
Flow rate: 0.4 mL / min
Injection volume: 2.0 μL
Detector: UV 290 nm, MS (Mw 319.0434)
MS condition: Polarity ES- (capillary voltage 1.8 kV, sampling cone 40 V, extraction cone 4.0 kV, cone Voltage: 20 V, source temp. 150 ℃, desolvation temp. 400 ℃, cone gas flow 50 L / h, desolvation gas flow 1000 L / h, Mass scan range: 100-1000 Da, Scan time: 0.2 sec-1.
MRM condition: precursor ion> production: 318.83> 192.90
Polarity ES- was used in terms of detection sensitivity.
Autosampler: 5 ℃

-Calculation method Calculate by correcting with the purity (HPLC purity / moisture value) described in the standard product report.

The antioxidant composition of the present invention contains ampelopsin and ascorbic acid as essential constituents. Ascorbic acid is preferably L-ascorbic acid. It may be a salt or derivative of L-ascorbic acid. The composition of the present invention can be prepared into tablets, capsules, liquids, or powders by adding excipients and other components. The composition formulated in this way can be used as a supplement or health food having a single-term oxygen scavenging effect generated in a living body.

In the composition of the present invention, 0.5 to 10 parts by mass of ampelopsin is blended with respect to 90 parts by mass of L-ascorbic acid, preferably 1 part by mass of ampelopsin with respect to 90 parts by mass of L-ascorbic acid. When partially blended, it efficiently enhances the singlet oxygen scavenging ability of ascorbic acid.

Singlet oxygen is oxygen in an excited state, and in the living body, when exposed to ultraviolet rays, the pigment in the body acts as a sensitizer to generate singlet oxygen. Therefore, it is important to efficiently eliminate singlet oxygen.

The present invention will be described below with reference to test examples.
1. 1. Test method Evaluation was performed by ESR (electron spin resonance) measurement. The test object is ampelopsin purified from ascorbic acid and wisteria tea.

(1) Equipment used <ESR measurement conditions>
ESR-JES-RE1X: Made by JEOL Ltd. was used.
The ESR spectrum shows the organic radicals in the sample.
The measurement conditions of ESR used are shown.
Resonance frequency: 9.40GH
Output: 4.0mW
Magnetic field modulation: 100kHz
Observation magnetic field: 335.5 ± 5 mT
Measurement time: 1.0 min
Modulation width: 0.1mT
Amplification rate: 500
Response time: 0.1 sec

<LED light generator>
SLL-4100S RGB (manufactured by Shimatec Co., Ltd.) was used.

(2) Measurement procedure of singlet oxygen scavenging ability The amount of reagent added in one sample is shown below. Each evaluation sample was appropriately diluted with 40% (v / v) DMF solvent to prepare an evaluation sample.
The reagent solutions were added in the order of 1) to 4) shown below.
1) Evaluation sample Solvent: 40% (v / v) N, N-dimethylformamide (DMF) 100 μL
2) Saline 20 μL
3) 4-OH TEMP solvent: 40% (v / v) DMF 100 mM, 40 μL (final concentration 20 mM)
4) Rose bengal solvent: 40% (v / v) DMF 100 μM, 40 μL (final concentration 20 μM)
N = 3 was set for each evaluation concentration. A total of 200 μl is dropped onto a 96-well plate, and singlet oxygen that reacts by the photosensitization reaction of rose bengal by irradiation with green light (λmax: 525 nm) and 4-OH TEMP (2,2,6) present in the solution. , 6-tetramethyl-4-piperidinol) and adduct (≈ adduct) [2,2,6,6-tetramethyl-4-hydoxy-piperidinhy (4-OH TEMPO) radical] ESR (electron spin resonance) measurement I did it. The light irradiation device uses SLL-4100S RGB (manufactured by Shimatec), which is an LED (Light Emitting Diode Green (ΙMAX = 530 nm) light source, and is singlet oxygen using the following formula from the ESR signal intensity (Sn) with an irradiation time of 20 min. The erasure rate was calculated.

Singlet oxygen scavenging rate (%) = 100-100 × (Sn (sample concentration X) / Sn (control)

(3) Data quantification Data acquisition of ESR spectrum and quantification of signal ratio Sn (signal ratio of organic radical / Mn marker) were performed using a free radical analysis system Win-RAD (manufactured by Radical Research).

(4) Data analysis method IC50 (50% erasure concentration) and IC30 (30% erasure concentration) were calculated based on the erasure ability% at each concentration of each sample.
[Antioxidant component concentration in each sample] vs [F / 1-F]
* F / (1-F) ... (F = (1/100) x Q, Q is the singlet oxygen scavenging rate)
x-axis → [antioxidant component concentration] y-axis → [F / 1-F] is plotted, and the IC50 (50% elimination) concentration is calculated from the x value (concentration) for y = 1, and x for y = 0.429. The IC30 (30% elimination) concentration was determined from the value (concentration). The concentration at one point was measured at n = 3, and the 50% erasure concentration and the 30% erasure concentration were calculated based on the approximate expression derived from the results at the minimum three points. FIG. 1 shows a calibration curve for determining the singlet oxygen scavenging ability of ascorbic acid.

2. Measurement results (1) IC30 values IC30 (30% elimination concentration) of ampelopsin and ascorbic acid showed the values in Table 1 below.

Both ampelopsin and ascorbic acid showed a strong singlet oxygen scavenging effect.

(2) Singlet oxygen scavenging ability by combined use of ascorbic acid and amperopsin Singlet oxygen when amperopsin is added at a concentration of 1/90 (0.0000163% by mass) with respect to 0.00147% by mass of ascorbic acid. The results of measuring the erasing ability are shown in Table 2 below.

As shown in Table 2, the addition of 0.0000163% by mass, which is a concentration that does not show singlet oxygen scavenging ability with ampelopsin alone, showed a high singlet oxygen scavenging ability of 68.40% with the combination of ampelopsin and ascorbic acid. .. This was considered to be an effect derived from the addition of ampelopsin. That is, it is clear that ampelopsin and ascorbic acid coexist and exhibit high antioxidant capacity that cannot be predicted when used alone. Therefore, when a small amount of ampelopsin is used in combination with ascorbic acid, the amount of ascorbic acid compounded as an antioxidant can be significantly reduced.

Claims (2)

An oral antioxidant composition for scavenging singlet oxygen produced by exposure to ultraviolet rays, which contains 0.5 to 10 parts by mass of ampelopsin with respect to 90 parts by mass of L-ascorbic acid. Antioxidant composition of claim 1 Symbol placement AMPelopsin is equal to or is derived from Fuji tea extract.

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