JP3232137B2 - Active oxygen scavenger - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an active oxygen scavenger which can be used in the fields of medicine, food, cosmetics and the like.

[0002]

BACKGROUND OF THE INVENTION A active oxygen means normally activated than oxygen oxygen and related compounds, particularly associated with biological, superoxide (O ₂ ^-), hydrogen peroxide (H ₂
O _2), hydroxy radical (HO ·), and the four singlet oxygen ⁽¹ O ₂₎ is referred to as active oxygen ( "active oxygen and free radicals", Volume 1, No. 1, page 38).

[0003] Oxygen taken into the living system from the atmosphere in the lungs is transmitted by blood to all parts of the body, and is involved in the production of adenosine triphosphate (ATP) as an energy source. Are converted to active oxygen such as superoxide by cell lines such as erythrocytes and macrophages, intracellular granules such as mitochondria and microsomes, enzyme systems such as xanthine oxidase and NAD (P) H oxidase, and proteins such as hemoglobin and myoglobin. You. It is known that the generated active oxygen acts as an important protective factor in vivo.
For example, when bacteria, viruses, foreign substances, and the like enter the living body, phagocytes such as neutrophils, monocytes, and macrophages are activated, and dynamic functions such as migration ability and phagocytosis are expressed. As a result, lysosomal enzymes and Active oxygen is produced and secreted. They are directly or indirectly involved in phagocytic melting and disinfection, protecting the body from external enemies.

However, in recent years, it has also been found that an excessive amount of active oxygen in a living body causes various tissue disorders. Usually, superoxide produced in a living body and serving as a starting material for other active oxygen is superoxide dismutase (SO2) contained in cells.
D) is sequentially erased by the catalytic action of D). However, when the production of superoxide is excessive or when the action of SOD is reduced as in the case of an elderly person, the erasure is insufficient and the superoxide is insufficient. Oxide levels increase, which causes tissue damage such as rheumatoid arthritis and Behcet's disease, myocardial infarction, stroke, cataracts, spots, freckles, wrinkles, diabetes, arteriosclerosis, stiff shoulders, and coldness. Even if you are not an old man, the skin is directly affected by environmental factors such as ultraviolet rays, so it is an organ that is particularly easy to produce superoxide.Therefore, it is easy for the superoxide concentration to increase and the resulting lipid peroxide to occur, which is the cause. Are liable to cause troubles such as formation of melanin pigments, spots and fine wrinkles.

[0005] In order to prevent or treat the above-mentioned various disorders caused by excessive active oxygen, attempts have been made to use superoxide dismutase as a drug or to add it to cosmetics, foods and the like and to take it daily. However, since this enzyme is unstable to heat and easily deactivated, it is difficult to add and process it in cosmetics and foods.

An attempt to find a substance having an active oxygen scavenging action other than superoxide dismutase has been disclosed in
No. -50877, which utilizes baicalein in a gougon (Scarabae). However, baicalein is very expensive because it is contained only slightly in the gogon. If a substance having an active oxygen scavenging action is added to food or drink and taken orally, it should not adversely affect the properties and flavor of the food or drink, so it may be soluble in addition to heat stability. And taste and quality matters.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide an active substance comprising a stable and inexpensive substance which is not unstable like superoxide dismutase and can be easily incorporated into pharmaceuticals, cosmetics, foods and the like. It is to provide an oxygen scavenger.

[0008]

The active oxygen scavenger which has been successfully provided by the present invention contains astilbine as an active ingredient. The present invention also relates to extracts from plants containing astilbin, that is, an organic solvent having an intermediate polarity, obtained by extraction of Engelhardtia genus plants, Ki杞 in the lower alcohol or water, extracting solvent extraction The present invention provides an active oxygen scavenger comprising a substance.

Has a common basic skeleton with astilbin
(+)-Taxifolin was known to scavenge active oxygen
However, the active oxygen scavenging effect of astilbin is
Was not known. Astilbin and Taxifolin
The difference is that the former is the latter glycoside.
R is the (+) - For taxifolin are hydrogen atoms, the case of astilbin is alpha-L-rhamnose residues.

[0010]

Embedded image

Astilbine is a plant of the genus Engelhardtia, such as E. chrysolepis HANCE, and Lyonia o.
valifolia), Chryanthus glber, Smilax glabra, Astilb
e microphylla knoll), Triangular pear (Astilbe od)
onlophylla Miq.).

However, it is yellowish that it is most easily used as a raw material of the active oxygen scavenger of the present invention. Huang Yai is a subtropical plant distributed in Guangdong, Guangxi and southern Fujian provinces of China, and its leaves have a subtle sweetness.It has been called sweet tea for a long time and has been consumed for the purpose of reducing fever and reducing obesity. There is no problem in terms of taste and safety.

[0013] Astilbin is a plant containing it,
Preferably, the leaves are extracted by extracting the leaves with an organic solvent having an intermediate polarity, a lower alcohol or water. The extraction treatment is performed, for example, by immersing the raw material plant in about 5 to 15 times the amount of the extraction solvent at normal temperature or under reflux.
Specific examples of preferred extraction solvents include ethyl acetate, acetone, methanol, ethanol, isopropanol, water and the like. These organic solvents and water may be used as a mixture. The obtained astilbin-containing extract can be used as it is as an active oxygen scavenger, but the astilbin content was increased by purifying it by liquid-liquid distribution extraction, chromatography, ion exchange resin treatment, membrane separation treatment, etc. Those can be used as active oxygen scavengers that show stronger activity.

[0014]

The active oxygen scavenger of the present invention can be used not only as a pharmaceutical, but also in cosmetics (including quasi-drugs), foods, etc. to prevent various disorders caused by the excessive in vivo active oxygen. Wide range of use is possible. Pharmaceuticals include, for example, tablets, capsules, lozenges, powders,
It can be used in the form of oral preparations such as solutions and syrups, and parenteral preparations such as injections, external preparations and suppositories. These preparations may optionally contain excipients, binders, disintegrants,
It can be produced by any method using an auxiliary agent such as a lubricant, a stabilizer, and a flavor / odorant.

For use in food, there are a method of kneading, coating or spraying the food or its raw material, and a method of dipping the food or its raw material in a solution. Astilbin, which is easily soluble in water and has a weak sweetness, is particularly advantageous as an active oxygen scavenger added to foods and drinks. For use in cosmetics, it may be appropriately mixed with a cosmetic base at any stage of the manufacturing process. Suitable amount of the active oxygen scavenging agents of the present invention varies depending on the addition target, if the food or cosmetics, about 0.001 to 3.0% by weight with Asuchirubi down, preferably about 0.01. 0
% By weight.

[0017]

Example 1 200 g of dried and ground leaves of yellow fir were immersed in 2 liters of methanol and heated under reflux for 2 hours. After filtration, the residue was treated again with 2 liters of methanol. The extracts obtained by the two treatments were combined and concentrated under reduced pressure to obtain 34 g of a methanol extract.
It was then suspended in 200 ml of water and extracted three times with 200 ml of chloroform, followed by three times with 200 ml of ethyl acetate. Each extract was concentrated to obtain 12 g of a chloroform layer extract and 8.5 g of an ethyl acetate layer extract, and the aqueous layer after the above extraction was further concentrated to obtain 13 g of an aqueous layer extract. As a result of analysis by silica gel thin-layer chromatography, it was confirmed that the ethyl acetate layer extract contained astilbin and other dihydroflavonol glycosides.

Example 2 200 g of dried and ground yellow leaf was dried with 2 liters of hot water.
The extract is concentrated for 3 hours under reduced pressure.
2.0 g of hot water extract was obtained. This was suspended in 200 ml of water, extracted three times with 200 ml of ethyl acetate, and the extracts were combined and concentrated to obtain 5.2 g of an ethyl acetate layer extract. The aqueous layer after the above extraction was further concentrated to obtain 27.0 g of an aqueous layer extract. As a result of analysis by silica gel thin-layer chromatography, it was confirmed that the ethyl acetate layer extract contained astilbin and other dihydroflavonol glycosides.

The active oxygen scavenging action of the extracts according to the above two examples was examined. The test method (NBT method) is as follows. (1) Reagent 0.05 M Na ₂ CO ₃ buffer (pH 10.2) 3 mM xanthine solution: Dissolve 45.64 mg of xanthine in the buffer to make 100 ml. 3 mM EDTA solution: Dissolve 111.7 mg of EDTA · 2Na in distilled water to make 100 ml. BSA solution: Dissolve 15 mg of Bobvin serum albumin (Sigma) in distilled water to make 10 ml. 0.75 mM NBT solution: 61.32 mg of NBT (nitroblue tetrazolium) is dissolved in distilled water to make 100 ml. Xanthine oxidase solution: dilute xanthine oxidase with distilled water. The concentration is such that the absorbance in the blank test in the operation (2) described below falls within the range of 0.20 to 0.23. 6 mM CuCl ₂ solution: Dissolve 102.29 mg of CuCl ₂ .2H ₂ O in distilled water to make 100 ml.

(2) Procedure Transfer 2.4 ml of Na ₂ CO ₃ buffer solution into a test tube, and add 0.1 ml each of a xanthine solution, an EDTA solution, a BSA solution and an NBT solution. Sample solution (80% ethanol solution)
Add 0.1 ml, leave at 25 ° C for 10 minutes, add 0.1 ml of xanthine oxidase solution, stir quickly, and incubate at 25 ° C. After 20 minutes, 0.1 ml of CuCl ₂ solution is added to stop the reaction, and the absorbance is measured at 560 nm. Separately, a blank test is performed using distilled water instead of the sample solution. The test results are shown in Table 1 as IC ₅₀ (50% inhibitory concentration).

[0021]

TABLE 1 active oxygen scavenging action specimen IC ₅₀ (μg / ml) Example 1 undistributed methanol extract 14.75 chloroform layer extract 35.75 Ethyl acetate layer extract 5.00 aqueous layer extract 27.50 Example 2 Undistributed hot water extract 19.75 Ethyl acetate extract 15.50 Aqueous layer extract 30.50 From the results shown in Table 1, both methanol extract and hot water extract of yellow leaf are effective in eliminating active oxygen. It was found that astilbin, which is the active ingredient, migrated to the ethyl acetate layer by re-extraction.

Example 3 The ethyl acetate layer extract according to Example 1 was subjected to silica gel chromatography and eluted with methylene chloride-methanol-water (70: 10: 1 → 40: 10: 1).
The main fraction obtained was treated with methanol-0.1% TFA.
Purification by reverse phase high performance liquid chromatography (column: TSKgel ODS-120T, 21.5 mm × 30 cm) with (1: 1)
Astilbin was obtained from the original leaves in a yield of about 1%. Further, a part of the astilbin obtained above was treated with crude hesperidinase and hydrolyzed to obtain (+)-taxifolin.
(Used as a reference example) .

The chemical structures of the obtained astilbin and (+)-taxifolin are based on the measured values of ¹ H- and ¹³ C-NMR, specific rotation, melting point, and the like of each sample.
arm.Bull., 36, 4167 (1988)]. For the above-mentioned astilbin and (+)-taxifolin, the active oxygen scavenging action was examined by the NBT method. Table 2 shows the results. In addition, the measurement example about quercitrin and baicalein which are known to have an active oxygen elimination action is also shown in the same table.

[0024]

TABLE 2 active oxygen scavenging action specimen IC ₅₀ (μg / ml) astilbin 4.60 (+) - taxifolin 1.80 baicalein 12.50 quercitrin 5.50

Example 4 Luminol Dependence and FCLA Using Astilbin and (+)-Taxifolin Using Animal Intraperitoneal Exudate Cells
The active oxygen scavenging effect was investigated using various stimulants by dependent chemiluminescence. OPZ was used as a phagocytic stimulant, and f-ML was used as a neutrophil receptor-mediated stimulant.
P, PM as a stimulant acting on protein kinase C
A, A-23 as a stimulator of calcium ion uptake
187 was used. The test method is as follows.

(1) Reagent Luminol reagent: After adding 40 μl of triethylamine to 10 mg of luminol, the total volume was adjusted to 10 ml with phosphate buffered saline (PBS) having a pH of 7.4. FCLA reagent: 7 mg of FCLA was adjusted to a total volume of 5 ml with phosphate buffered saline (PBS) having a pH of 7.4. m'HBSS: Hank's Balanced Salt Solution Powd
er (GIBCO) 9.476 g and HEPES (Dojindo) 10 mM were dissolved in water to make the total volume 1 liter, and the pH was adjusted to 7 with NaHCO ₃ .
And 2. OPZ reagent: Opsonize Zymosan with serum, PB
S was adjusted to 50 mg / ml. f-MLP reagent: N-Formyl-Met-Leu-Phe-OH (BACHEM)
Into a 10% to 40 nM 10% DMSO solution. A-23187 reagent: calcium ionophor A-23187 0.5 μm
M was used as a 10% DMSO solution.

(2) Operation: Male Japanese white rabbits (body weight: about 4 kg) were intraperitoneally administered with 500 ml of 0.2% glycogen / saline, and 16 hours later, 500 ml of saline was intraperitoneally administered again. Then, the peritoneal cavity was washed, and the intraperitoneal exudate cells were removed. 1000 rpm
Then, m'HBSS was added to the residue to obtain a cell suspension of 1 × 10 ⁶ cells / ml. Take 0.484 ml of this Cell Suspension and add Luminol or FCLA1
0 μl and 1 μl of each sample were added, and the mixture was added at 37 ° C. for 10 minutes.
Incubated. Then, 5 μl of a stimulant was added, and the production of active oxygen was measured using Belthod's Biolmat for 5 to 30 minutes, and the production inhibition rate was calculated based on the highest active oxygen concentration. Confirmed inhibition rate and 50% inhibitory concentration (ID ₅₀₎ are shown in Tables 3 to 9.

[0028]

[Table 3] Inhibitory effect of stimulant OPZ on luminol-dependent active oxygen production

[0029]

[Table 4] Inhibitory effect of stimulant f-MLP on luminol-dependent active oxygen production

[0030]

[Table 5] Inhibitory effect of stimulant f-MLP on FCLA-dependent active oxygen production

[0031]

[Table 6] Inhibitory effect of stimulant PMA on luminol-dependent active oxygen production

[0032]

[Table 7] Inhibitory effect of stimulant PMA on FCLA-dependent active oxygen production

[0033]

[Table 8] Inhibitory effect of stimulant A-23187 on luminol-dependent active oxygen production

[0034]

[Table 9] Suppressive effect of stimulant A-23187 on FCLA-dependent active oxygen production

[0035] For active oxygen production by stimulation of phagocytic activity of neutrophils, Asuchirubi down represents the ability production inhibitory at low concentrations, S
It showed an OD-like effect. Shows the effect of suppressing Asuchirubi Nwaru Minoru dependent superoxide production against reactive oxygen production by stimulating receptor-mediated neutrophil myeloperoxidase-like effect was observed. It did not show any inhibitory effect on FCLA-dependent active oxygen production, but rather enhanced it. From these, Asuchirubi down the production of other active oxygen superoxide production produced by neutrophils enhances Although the spillover from it became clear that to suppress.

[0036] Protein kinase C stimulation of neutrophils, or to act to stimulate superoxide production nonspecifically calcium ions by incorporating into the cell, Asuchi <br/> ruby down the neutrophil As in the case of the receptor-mediated stimulation, the compound exhibited an effect of suppressing luminol-dependent active oxygen production, and a myeloperoxidase-like effect was observed. In addition, it did not show an inhibitory effect on FCLA-dependent active oxygen production, but rather enhanced it, and enhanced the production of superoxide produced by neutrophils and the suppression of the production of other active oxygen which spread from it. . This indicates that the active oxygen scavenging agents of the present invention can be expected even if the anti-inflammatory agent.

[0037]

As described above, the active oxygen scavenger of the present invention can be easily produced from readily available plants by a simple extraction and purification operation, and it is also important in terms of safety, taste and solubility. But it is excellent. In addition, its active oxygen scavenging effect is not impaired by heating, so it is used as a drug for the prevention or treatment of the above-mentioned diseases caused by excess in vivo active oxygen, and it is added to foods and cosmetics to maintain health.・ It is a very useful thing that can be used for promotion.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI C07H 17/07 C07H 17/07 (72) Inventor Kokichi Tamura 14703-10 Mukahigashi-cho, Onomichi-shi, Hiroshima Examiner of Maruzen Pharmaceutical Co., Ltd. ▲ Takami Oka, Hiromi (56) References JP-A-63-267774 (JP, A) JP-A-3-93782 (JP, A) JP-A-55-54883 (JP, A) Biochemical Pharmacology, Vol. 37, No. 6, p. 989-995, 1988 Journal of Natural Products, Vol. 51, No. 2, pp. 345-348, 1988 (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 35/78 C07D 311/00-311/96 C07H 17/07 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. An active oxygen scavenger comprising astilbine. 2. An Engelhardtia genus plant / yellow using an organic solvent having intermediate polarity, a lower alcohol or water as an extraction solvent.
An active oxygen scavenger consisting of an extract obtained by extraction processing.