JPH0381227A - Superoxide remover - Google Patents

Abstract

PURPOSE:To provide the title remover having stable effect over a wide pH range, with the effect retained even by heating, containing the components extracted from Glycyrrhiza glabra using an organic solvent having medium polarity or its mixture with a lower alcohol. CONSTITUTION:Glycyrrhiza glabra or the residue after extraction of glycyrrhizin from Glycyrrhiza glabra with water is extracted using an organic solvent having medium polarity (e.g. benzene, ethyl ether, chloroform, n-butyl acetate, methylene chloride, trichloroethylene) or its mixture with 10-50vol.% of a lower alcohol, the resulting liquid extract is filtered and the solvent is distilled off, thus obtaining a tasteless, brownish extract. This extract is either directly, or, if needed, after purification such as decoloration, used as the objective superoxide remover. The present remover can be used not only as a pharmaceutical, but also find a wide range of applications such as incorporation in cosmetics, foods, etc.

Description

[Detailed description of the invention] [Industrial application field] The present invention can be used in the fields of medicine, food, cosmetics, etc.
This invention relates to a superoxide scavenger.

[Conventional technology]

Superoxide, ie, the radical 02- generated by one-electron reduction of oxygen molecules, is known to act as an important defense factor in living organisms. For example, bacteria,
When viruses, foreign substances, etc. enter the body, neutrophils, monocytes,
Phagocytic cells such as macrophages are activated and express dynamic functions such as migratory ability and phagocytosis, resulting in leprosy.

Sosomal enzymes and superoxide are produced and secreted, and these are directly or indirectly involved in the melting and sterilization of poor food, and protect the body from foreign invaders.

On the other hand, superoxide causes various tissue damage when present in excess in a living body. Normally, superoxide produced in living organisms accounts for less than 1% of the oxygen absorbed through respiration, and this is gradually eliminated by the catalytic action of superoxide dismutase (SOD) contained within cells. In cases where enzyme activity is reduced, such as in the body of the elderly, superoxide concentration becomes high due to insufficient scavenging, which can lead to tissue disorders such as rheumatoid arthritis and Behcet's disease, as well as superoxide and the excess produced by it. Oxidized lipids can cause myocardial infarction, stroke, cataracts, age spots, freckles, wrinkles, diabetes, arteriosclerosis, stiff shoulders, and sensitivity to cold. Even if you are not an elderly person, the skin is an organ where superoxide is particularly susceptible to the growth of superoxide as it is directly stimulated by environmental factors such as ultraviolet rays. It is easy to cause problems such as melanin pigment production, age spots, and small wrinkles.

Attempts to use superoxide dismutase as a medicine or as an additive to cosmetics or food to prevent or treat the various disorders mentioned above due to excess superoxide have been unsuccessful because this enzyme is unstable and inactivated by heat. It has not been successful because it is easy to use and is extremely expensive.

An attempt to find a substance other than superoxide dismutase that has a superoxide scavenging effect was made in JP-A-64-
It is described in Japanese Patent No. 50877, in which the pyrite rain in yellow waste is utilized. However, only a small amount of pyriterein is contained in yolk, and even if it is extracted, it is extremely expensive.

[Problem to be solved by the invention]

An object of the present invention is to provide a superoxide scavenger made of a stable and inexpensively available substance that is not unstable like superoxide dismutase and can be easily incorporated into pharmaceuticals, cosmetics, foods, etc. .

[Means to solve the problem]

The superoxide scavenger successfully provided by the present invention contains a component extracted from licorice or a water-extracted residue of licorice using an organic solvent having intermediate polarity or a mixture of this and a lower alcohol as an extraction solvent. This is a characteristic feature.

The superoxide scavenger of the present invention is thought to scavenge superoxide through the reaction 0, -+ 2 H+→Hzoz+o*, but it has not yet been elucidated what component in the licorice extract scavenges superoxide. do not have.

Glycyrrhizin, which is extracted from licorice by water extraction, has been used for medicines, sweeteners, etc. for a long time, but since the active ingredient of the superoxide scavenger of the present invention is in the water-insoluble components of licorice, it can be used as a raw material. It is possible to use not only the licorice roots and stems, but also the residue after glycyrrhizin is extracted from them by water extraction.

Examples of intermediate polar organic solvents that can be used to extract the active ingredients of superoxide scavengers from licorice include benzene, ethyl ether, chloroform,
n-butyl acetate, isobutyl acetate, n-propyl acetate,
Examples of such alcohols include ethyl acetate, methylene chloride, trichlene, and percrene, and examples of lower alcohols that can be used in combination with these include methanol and ethanol. When a lower alcohol is used in combination, the appropriate mixing ratio is about 10 to 50vol%; if the mixing ratio of the lower alcohol is increased beyond this, the extraction efficiency of the component having a superoxide scavenging effect will decrease.

No special extraction conditions are required except for the use of an extraction solvent as described above. Standard extraction conditions include immersing the raw material to be treated in about 3 times as much extraction solvent at room temperature to elute soluble components, and after an appropriate period of time, performing solid-liquid separation. These operations are repeated 2 to 3 times, the resulting extracts are combined, filtered, and the solvent is distilled off to obtain a tasteless, brown extract. In many cases, this extract can be used as it is in the superoxide scavenger of the present invention, but if necessary, it may be subjected to purification treatments such as decolorization to the extent that its efficacy is not impaired.

The obtained licorice extract or its purified product is used as the superoxide scavenger of the present invention, either as it is or dissolved in a medium such as an appropriate oil or fat, 1,3-butylene glycol, propylene glycol, or glycerin monostearate. be able to. Also, after dissolving in ethanol etc., Tween 20. If a solubilizing agent such as saponin is added to water in advance and stirred, a clear solubilized state can be obtained.

The toxicity of the superoxide scavenger of the present invention is determined by Litcb.
LD was investigated by intraperitoneal administration using ICR mice according to the field Wilcoxon method. The value was 1.9 g/kg or more.

The superoxide scavenger of the present invention can be used in a wide range of applications, such as being used as a medicine, as well as being incorporated into cosmetics (including quasi-drugs), foods, and the like. When incorporated into cosmetics, etc., the preferred amount of the superoxide scavenger of the present invention is about 0.0001 to 1.0% as a licorice extract.

〔Effect of the invention〕

As mentioned above, the superoxide scavenger of the present invention can be easily produced using licorice as a raw material and is therefore inexpensive, and also exhibits a stable superoxide scavenging effect over a wide pH range, and its effect is impaired even by heating. Therefore, in addition to being used as a medicine for the prevention or treatment of the above-mentioned diseases caused by excessive superoxide,
It is extremely useful and can be added to foods, cosmetics, etc. for health promotion and beauty.

〔Example〕

Production Example 1 2 kg of ground grass roots were soaked in ethyl acetate for 5 hours at room temperature to extract the ethyl acetate soluble components, and then filtered to separate the extract. Repeat the same operation two more times,
A total of 2611 extracts were obtained. The solvent of the extract was distilled off, and the remaining solid was dried under reduced pressure and ground to obtain 72 g of a light brown powder (this is referred to as Formulation A).

Production Example 2 Glycyrrhizin was extracted by soaking 1 kg of licorice in 1 (l) of 1% aqueous ammonia. After drying the residue, it was heated under reflux with 3α ethyl ether for 2 hours to extract the ethyl ether soluble components. The same operation was repeated again for the extract and the separated residue to obtain a total of 5.5a of extract.Then, the solvent was distilled off from the extract and further dried under reduced pressure to obtain a brown powder. 25 g was obtained (this is designated as formulation B).

Production Example 3 Glycyrrhizin was extracted by immersing Sesame root IM in 10Q of warm water. The residue was dried and mixed with 3rb of chloroform/methanol mixed solvent (mixing ratio 9:1).
The mixture was heated under reflux for an hour to extract the solvent-soluble components. The same operation was repeated for the residue separated from the extract to obtain a total of 5.51 extracts. Thereafter, the solvent of the extract was distilled off, and the extract was further dried under reduced pressure to obtain 30 g of brown powder (this is referred to as Formulation C).

Test Example 1 The preparations obtained in each of the above production examples were examined for superoxide scavenging activity using the NBT method described below.

(1) Reagent ■0. O5M N i2c Os buffer (pH 10,2
)■3mM xanthine solution: xanthine 45.64mg
Dissolve it in the buffer solution of ■ and get 1011! Let it be I+.

■3+oMEDTA solution: EDTA・2Nx 111
．． Dissolve 7 mg in distilled water to make loOml.

■BSA solution: Lbvin serum album
in (Sigma) 15m (dissolved in distilled water and
The area shall be 11m1.

■0.75@M NBT solution: NBT and troblue tetrazolium) 61.32mg
Dissolve 10 (1 mg) in distilled water.

■Xanthine oxidase solution: xanthine oxidase was diluted with distilled water, and the absorbance in a blank test of the procedure (2) below was 0.20-0. Make sure it falls within the range of H.

■6@MCuCI2 solution: CuC1*・2Hz0 1
Dissolve 01Ha+g in distilled water to make 100ml.

(2) Take 2.4 mg of N s 1 C03 buffer into a test tube,
To this, xanthine solution, EDTA solution, BSA solution, N
Add 1 ml of each liter of BT solution. Add 0.1 mg of sample solution (80% ethanol solution), leave at 25 °C for 10 minutes, add 11.1 mg of xanthine oxidase solution, stir briefly, and incubate at 25 °C. After 620 minutes, CuC1, solution 0.1 mg The reaction is stopped by adding 560 nm, and the absorbance is measured at 560 nm. For comparison, superoxide dismutase (CII-2n type SOD,
Activity: 3000-4000 units/m (.

Similar measurements are made for 0.1 mg of Wako Pure Chemical Industries' aqueous solution. A separate blank test is conducted using distilled water instead of the sample.

The measurement results are shown in Table 1.

Table 1 Superoxide elimination rate (%) Test Example 2 In a test similar to Test Example 1, each sample solution was
The superoxide scavenging ability was measured for those heated for 10 minutes. The results are shown in Table 2.

Table 2 Superoxide scavenging rate (%, after heating) Test Example 3 The intracellular superoxide scavenging ability of Formulation A was measured by the following method.

Medium: pH at 37°C using Eagle MEMI [(Nippon Pharmaceutical) 9.4g/10% NaHCO> solution.
was adjusted so that the value was 7.2. This medium is used for 24 hours after the seedlings have matured.
Used within hours.

Preparation of polymorphonuclear leukocytes: 10 ml of liquid paraffin was intraperitoneally administered to male Wistar rats, and 12 hours later, 60 ml of Eagle's medium was injected into the peritoneal cavity to collect 80 g of ascites fluid. The collected ascitic fluid was centrifuged for 5 minutes, and the precipitate was suspended in Eagle's MEM medium containing 1% rat serum at a concentration of 1X10' cells/ml to prepare a PMN specimen. In each experiment, viability was confirmed by Trivan blue staining.

Chemiluminescence measurement: 0.5 ml of PMN suspension
10 μm of luminol test solution and 5 μl of DMSO solution of Formulation A were added to the solution, and after incubation at 37°C for 10 minutes,
2y+sos*n solution (0.5mg/ml) 10μ
m was added, and the luminol-dependent chemiluminescence generated was measured at 37°C using a 7-oton counter.

Formulation A slightly delayed the time to reach the peak luminescence, but the superoxide scavenging effect was evaluated using the count at the time of maximum luminescence.

Table 3 shows the measurement results of superoxide elimination rate at various addition levels. Formulation A strongly captured and eliminated superoxide generated by 2ymosan stimulation in a concentration-dependent manner.

Claims (2)

[Claims] (1) A superoxide scavenger characterized by containing a component extracted from licorice or a water-extracted residue of licorice using an organic solvent having intermediate polarity or a mixture of this and a lower alcohol as an extraction solvent. (2) Use benzene, ethyl ether, chloroform, n-butyl acetate, isobutyl acetate, n-propyl acetate, ethyl acetate, methylene chloride, trichrene, or percrene as an organic solvent with intermediate polarity, and methanol or ethanol as a lower alcohol. The superoxide scavenger according to claim 1, which contains a component extracted using the superoxide scavenger.