JPH023495A - Antioxidant - Google Patents

Abstract

PURPOSE:To obtain an antioxidant having a superoxide dismutase-like oxidation inhibiting activity by employing as an active ingredient an org. solvent extract from a hot water extract of roasted vegetable fibers. CONSTITUTION:A roasted vegetable fiber extract is obtd. by roasting vegetable fibers, such as those of soybean, bran, wheat, rice, tea leaves, etc., at 170-230 deg.C for 5min-1hr, adding 10-20 times as much water, boiling for 5-80min, filtering to separate an extract, and concentrating the extract in a vacuum to a concn. ratio of 0.1-0.3, if desired, followed by dry spraying until the water content reaches about 4%. The extract is then extracted with 5-10 times as much org. solvent (e.g., 95% ethanol), and filtered so as to remove residue, followed by removal of the org. solvent in a vacuum. Thus, an antioxidant is obtd.

Description

[Detailed Description of the Invention] 11! The present invention relates to an antioxidant and cosmetics containing the same.

i-Suichigo-Go-Sake Oxygen is an essential substance for living organisms to maintain their life in terms of energy production, metabolism, etc., and this is caused by reactions in energy production systems, enzyme reactions, reactions by ultraviolet rays, radiation, etc. etc., oxygen anion radical (02-′″; 5up
eroxide), hydroxyl radical (HO-),
- Heavy ring oxygen ('02: singletoxygen
), hydrogen peroxide (H2O2>, etc.) is known to become so-called active oxygen.However, active oxygen has a bactericidal effect on white blood cells, that is, an action to decompose foreign substances eaten by phagocytes. On the other hand, if the active enzyme is produced in excess, it may be secreted outside the phagocytic cells and cause damage to the living tissue itself, or it may cause damage to oleic acid, linoleic acid, and arachidone, which are abundant in the living body. It promotes the peroxidation of unsaturated fatty acids that form the phospholipids of biological membranes, such as acids, and induces the production of inflammatory factors such as lipid peroxides.It also promotes the generation of alkoxy radicals and hydroxyl radicals along with this lipid peroxide. These substances may attack biological membranes, leading to membrane damage and deactivation of various useful enzymes [Metabolism, (10).

(See 1978 Special Feature on Active Oxygen).

On the other hand, enzymes involved in the metabolic deactivation of the above-mentioned active oxygen, such as superoxide dismutase, are present in living organisms.
peroxide dismutase, 3Q[)
), catalase, glutathione peroxidase, etc., and various vitamins with antioxidant ability, such as α-tocopherol (vitamin E), etc.
Normal biological maintenance is normally achieved through these actions.

In particular, the above-mentioned SOD is an enzyme that reduces active oxygen 02- in the living body, and is present in a wide range of organisms from plants to animals, and decomposes the excess active oxygen that causes tissue damage, etc., and protects tissues It is well known as having the effect of

However, in the environment surrounding living organisms, there are a large number of various chemical substances that promote the in vivo production of active oxygen, such as herbicides, insecticides, detergents, pharmaceuticals, and various food additives, which are considered secondary chemicals. The influence of substances often causes the generation of active oxygen, the production and accumulation of lipid peroxides, which exceed the capabilities of the appropriate defense mechanisms such as the enzymes and vitamins, and the deficiencies of the defense mechanisms. Furthermore, the SOD secretion ability in humans decreases with age, and in particular, in the prime of life after 40 years of age, a considerable decrease in in vivo SOD activity is observed. When abnormal generation of active oxygen occurs, the peroxidation reaction progresses in a chain reaction, causing serious damage to the living body, such as various inflammations, liver damage, kidney damage, gastric damage, arteriosclerosis, hemolysis, and aging. Or senile dementia, retinopathy, liver damage, drug-induced mental and! 1
iiiIS! Various pathological conditions such as vascular disease and ischemic vascular disease occur. In addition, the above-mentioned active oxygen is involved in many diseases such as Behcet's disease and Kawasaki disease, as well as malignant rheumatoid arthritis, ulcerative colitis, Crohn's disease, progressive systemic sclerosis, polymyositis, and atopic dermatitis. is said to be involved.

Various studies have been conducted on compounds that have the effect of removing active oxygen, which is considered to be the main cause of the various disorders and diseases mentioned above, and preventing or reducing the production and accumulation of lipid peroxides in the body. , especially various enzyme agents including the above-mentioned SOD (Superoxide and Medicine, Yoshihiko Oyanagi, 1981, Kyoritsu Shuppansha, Nos. 137-141)
In particular, SOD has been attracting attention in the medical field, for example in suppressing rejection after kidney transplantation and in preventing tissue damage that occurs due to blood flow recovery in ischemic heart disease. disease, malignant rheumatism, Crohn's disease,
Its effectiveness has also been confirmed in the treatment of inflammatory diseases such as ulcerative colitis.

However, since SOD is a high molecular weight protein, it is destroyed in the stomach and intestines when taken internally, making it impossible to administer orally, which is a fatal drawback.Even when administered intravenously, it is degraded in a very short time. This has the disadvantage of being

In recent years, in place of SOD, various substances with similar enzymatic activity (SOD-like antioxidant activity) have been introduced not only in the pharmaceutical field, but also in the fields of Chinese medicine, natural foods, health foods, etc.
Although it is being actively researched, no product has yet been developed that is satisfactory in terms of production, raw materials, and effects such as antioxidant power.

Problems to be Solved by the Invention: 1. The applicant has been conducting research on the deodorization of foods, and in the process developed a roasted vegetable fiber material as a deodorizer for meat.
Starting with Japanese Patent Publication No. 58-32579), we have succeeded in developing various deodorizing agents containing various substances related to the roasted vegetable fiber material as active ingredients (Japanese Patent Publication No. 59-7427, Japanese Patent Publication No. 62-1982). No. 9301, etc.] However, the active ingredient compounds of deodorizers related to each of the above patents have not yet been elucidated, and in the process of researching and elucidating these compounds, in particular, the hot water extract of the above-mentioned roasted plant fiber material, Furthermore, we have discovered the surprising fact that the extract obtained by extraction and purification with an organic solvent has an excellent SOD-like antioxidant effect, regardless of its excellent deodorizing effect.

In addition, we have obtained the knowledge that this extract is highly safe for the human body, can be taken orally, and that its excellent SOD-like antioxidant activity does not substantially change even after oral intake. also found that by incorporating this into cosmetics, it is possible to provide cosmetics with SOD-like antioxidant effects.

The present invention was completed as a result of further research based on these new findings.

In order to solve the problems, the present invention provides an antioxidant characterized by containing an organic solvent extract of a hot water extract of roasted plant fiber material as an active ingredient, which can be taken orally in the form of food or medicine. The present invention relates to the above-mentioned antioxidant and cosmetics containing the above-mentioned antioxidant.

The antioxidant of the present invention has the essential requirement of containing the above-mentioned specific extract as an active ingredient, and by applying it to living organisms in various forms that can be taken orally and in the form of cosmetics, it is possible to obtain the above-mentioned effects. The components exhibit SOD-like effects, that is, the effects of removing active oxygen and preventing or reducing the in vivo production of lipid peroxides, thereby exerting the desired antioxidant effect.

Therefore, the antioxidant of the present invention, in the form of food and medicine, can be used to treat various disorders and diseases caused by excessive generation of active oxygen, bioaccumulation of lipid peroxides, or lack of anti-RAM mechanisms. It can be highly effective in the prevention and treatment of.

More specifically, the above-mentioned disorders and diseases that can be prevented or treated by the antioxidant of the present invention include Behchit's disease, Kawasaki disease, autoimmune diseases (malignant rheumatoid arthritis, ulcerative colitis, etc.), Crohn's disease, and progressive systemic disease. sexual sclerosis, polymyositis,
Incurable diseases such as Raynaud's disease; neuralgia, rheumatism, myalgia, cerebral thrombosis, heart disease (ischemic heart disease), hypertension, hypotension, diabetes,
General diseases such as liver disease, atopic dermatitis, skin diseases, hemorrhoids, and alveolar S leakage; gastrointestinal ulcers in the stomach, duodenum, etc., liver diseases such as hepatitis and cirrhosis, kidney diseases such as nephritis, pancreatitis, etc. Examples include various tissue cell disorders such as pancreatic disease.

In addition, the antioxidant of the present invention can be orally ingested in the form of food or medicine to treat, for example, sensitivity to cold, stiff shoulders, hangovers, ginseng, obesity, constipation, insomnia, drug damage, harm caused by alcohol and cigarettes, whiplash, etc. It is also effective for increasing energy, preventing fatigue and shortness of breath, waking up feeling refreshed, maintaining and recovering the firmness and youthfulness of the voice, preventing cancer, and preventing aging.

Furthermore, when the antioxidant of the present invention is applied in the form of a cosmetic product, it can restore and maintain the firmness, luster, and smoothness of the skin, remove stains, prevent sunburn, improve adhesiveness, and make hair shiny. It is also very effective in maintaining the recovery of suppleness, preventing gray hair, etc.

In the antioxidant of the present invention, it is essential that an organic solvent extract of a hot water extract of a roasted plant fiber substance is used as an active ingredient. The roasted plant fiber substance extract used as the raw material for the active ingredient is obtained by extracting roasted plant fibers such as soybeans, bran, bran, wheat, rice, tea confectionery, etc. with water and then concentrating as necessary. and is itself publicly known (Okamura et al., Nutrition Guide, 106-117 (197B>, Osaka City Institute of Environmental Science I), Edited by Establishment College of Nutrition, 129-136 (ibid.)
1978), New 7L/-Bar Vo1.15. N
o.

4.25-35 (1981), Special Publication No. 58-32579
See Japanese Patent Publication No. 62-9301, etc.]. Particularly preferable extracts of roasted plant fiber substances include defatted soybeans, defatted bran, etc. at about 170 to 230'C
Roast for 1 minute to 1 hour (light roast), then about 10 to 20 minutes (8
1 of water was added and boiled for about 5 to 80 minutes, and the extract obtained by filtration separation was concentrated under reduced pressure to a concentration ratio of 0.1 to 0.3, and the above concentrated extract had a water content of about 4. An example is one obtained by spray drying to about %.

The active ingredient of the antioxidant of the present invention is prepared by extracting the above-mentioned hot water extract of the roasted plant fiber substance with an organic solvent. Examples of the organic solvent used here include lower alcohols such as methanol, ethanol, and isopropyl alcohol, benzene, chloroform, n-hexane, and carbon tetrachloride; among these, methanol,
Lower alcohols such as ethanol are preferred. A particularly preferred organic solvent extraction operation is, for example, extracting the water extract (spray-dried product) with about 5 to 10 times the amount of 95% ethanol, filtering the residue, and then removing the ethanol under reduced pressure. An example is a method in which ethanol is removed and then ethanol is added to the concentrate. The details are as shown in the examples.

The active ingredient of the antioxidant of the present invention may also be obtained by, for example, extracting an extract of the roasted vegetable fiber material previously extracted with an organic solvent, extracting it with hot water, and then extracting it again with an organic solvent.

The antioxidant of the present invention, which has as an active ingredient the organic solvent extract of the hot water extract of the roasted plant fiber material obtained as described above, can be used alone or in combination with various carriers, diluents, and others as necessary. They are prepared in various forms depending on the purpose by adding and blending agents as desired, and are applied to living organisms. The above-mentioned form may be any of the various forms normally adopted depending on foods, medicines and cosmetics, and preparation into various forms such as secondary forms can also be carried out according to conventional methods.

For example, when preparing the antioxidant of the present invention in a pharmaceutical form, the pharmaceutical preparation is prepared in the form of a suitable pharmaceutical composition using an effective amount of the antioxidant of the present invention together with a conventional pharmaceutical carrier, and is administered orally to humans or animals. administered.

The above preparation can be prepared in the same manner as conventional pharmaceutical preparations for oral administration, such as tablets, granules, capsules, oral liquid preparations, and the like. The various forms of formulations described above are prepared according to conventional methods using various commonly used carriers. For example, tablets are formed by mixing the antioxidant of the present invention as an active ingredient with excipients such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic, and the like. Capsules are prepared by filling hard gelatin capsules, soft capsules, etc. with a mixture of the active ingredient and an inert filler or diluent. Liquid preparations for oral administration are prepared by diluting the active ingredient in a diluent such as distilled water or physiological saline. The various forms of preparations described above may contain coloring agents, preservatives, perfumes, flavoring agents, sweeteners, MvM agents, solubilizing agents, and other active pharmaceutical ingredient compounds, if necessary.

The amount of the active ingredient to be contained in the above-mentioned pharmaceutical preparation is not particularly limited, but it is preferably contained in an amount of about 1 to 70% by weight in the entire composition. The effective amount is orally administered depending on the various forms mentioned above, conditions such as patient age, degree of disease, etc. The dosage is
The amount of active ingredients is approximately 0.5-10m per 1kO of body weight per day.
The amount can be increased or decreased as appropriate using the amount of approximately G as a guide.

Furthermore, when prepared in the form of food, the antioxidant of the present invention can be added and blended into various forms of processed foods and food raw materials thereof. Furthermore, the antioxidant of the present invention can be applied to various cooked foods, either alone or in the same manner as various seasonings, and can also be used as a food additive. The above-mentioned food forms are not limited as long as they can be taken orally, and include processed starch products such as noodles and breads, processed oil products such as various edible oils and mayonnaise, processed soybean products such as tofu and miso, and ham. processed meat products, seafood products, dairy products,
Vegetables, fruit processed products, confectionery, drinks, etc., and other cooked products may be used. Specific examples of particularly preferred food forms include various confectioneries and drinks.

Preparation into these forms and other ingredients used at that time can also be the same as those used in ordinary second-class foods. The amount of the antioxidant of the present invention used when preparing the above food form can be determined as appropriate and is not limited, but
Usually, the amount is preferably about 0.1 to 20% by weight, more preferably about 1% by weight. The antioxidant of the present invention in food form thus obtained can exert desired effects by eating or drinking it.

Furthermore, the antioxidant of the present invention can be formulated into a cosmetic product, and the present invention also provides such a cosmetic product. The cosmetics can be prepared in the same manner as conventional cosmetics except for incorporating the antioxidant of the present invention, and their form is not particularly different from conventional cosmetics.

Examples of the cosmetics prepared by applying the antioxidant of the present invention include creams such as vanishing cream, cleansing cream, neutral cream, and cold cream;
Basic cosmetics such as emulsions and lotions, makeup cosmetics such as foundation, white powder, and lipstick, medicinal cosmetics such as sunscreen, deodorization, and acne treatment, shampoo, hair treatment, hair styling agents, cold wave lotion, etc. Hair cosmetics, other oral cosmetics, bath cosmetics, fragrances, aerosol products, etc. are included, and due to their appearance, these products come in various forms such as creams, milky lotions, lotions, lotions, shampoos, aerosols, and toilet soaps. It can be anything.

The blending ratio of the antioxidant of the present invention in each cosmetic product varies depending on the type, form, use, etc. of the cosmetic product, and can be appropriately selected from a wide range without being particularly limited. It is reasonable to set the amount to about 1 to 5% by weight.

To explain in detail the manufacturing method of creams as a typical example of the cosmetics mentioned above, first, aqueous raw materials such as glycerin (moisturizer), preservatives, alcohol, fragrance, etc. are added to purified water and heated to approximately 70°C. Prepare an aqueous phase kept at a temperature of
Separately, a surfactant is blended with oily raw materials such as mineral oil, vegetable oil, lanolin, waxes, oils, beeswax, etc., and heated and melted to prepare an oil phase maintained at a temperature of about 70'C. The antioxidant of the present invention is prepared by gradually adding the oil phase, then uniformly emulsifying it in an emulsifier and cooling it.
They can be added to both the oil phase and mixtures thereof.

The thus obtained cosmetics of the present invention, based on the combination of the antioxidant of the present invention, protect the above-mentioned pharmacological effects, particularly the healing effects of diseases on the skin, hair, etc. to which the cosmetics are applied, and provide moisturizing and moisturizing effects. It can have the effect of imparting smoothness, tension, luster, etc. Furthermore, even when the above-mentioned antioxidant is added to the cosmetics of the present invention, their original performance as cosmetics is not substantially adversely affected. That is, the cosmetics of the present invention in the form of basic cosmetics, such as creams and emulsions, spread on the skin to protect the skin, provide moist and smooth skin, maintain cleanliness, and contain oils, moisturizing ingredients, and other ingredients suitable for the skin. At the same time, it effectively supplies moisture and improves the flexibility of the skin.
It has characteristics such as maintaining wettability. In addition, the cosmetics of the present invention have excellent cleanability, are easy to use and wash off when necessary, and have a beautiful appearance, as well as other required properties. It has no skin irritation and can be applied extremely safely to the human body.

Effects of the Invention According to the present invention, it is possible to provide an antioxidant that has SOD-like enzyme activity and can be advantageously applied to living bodies in the form of foods, medicines, etc. that can be ingested orally, and cosmetics that can exhibit the same activity. When applied to humans, they can prevent and treat various disorders and diseases caused by active oxygen based on their activity, and can also have excellent cosmetic effects on the skin, hair, etc.

Below, examples of manufacturing the active ingredient of the antioxidant of the present invention, examples of preparing various forms of the antioxidant of the present invention using the obtained active ingredient,
The present invention will be explained in more detail by giving test examples to clarify the SOD-like enzyme activity of the active ingredient and the various seed protection or treatment effects and cosmetic effects based on this.

Production Example of Active Ingredients Production Example 1 100 parts by weight of defatted rice bran was roasted (lightly roasted) at about 170'C for 40 minutes while rotating in a rotary manner.

The resulting roasted product is then poured into water that is 10 times the amount of the roasted product, and the mixture is heated to a boiling point. After 40 minutes (total heating time 60 to 90 minutes), the mixture was filtered and separated to obtain a hot water extract, which was concentrated under reduced pressure to a concentration ratio of 0.2.

The concentrated extract thus obtained was spray-dried until the water content was about 4%, extracted with 10 times the amount of 95% ethanol, the residue was filtered, and the ethanol was removed under reduced pressure and concentrated. Ethanol was added to the obtained concentrate to obtain the active ingredient of the antioxidant of the present invention.

This stuff has a solid content of 75-78% and a nitrogen content of 1.1%.
Met. Hereinafter, this will be referred to as "active ingredient 1."

Production Example 2 In the same manner as in Example 1 above, using defatted soybeans instead of defatted bran, the active ingredient of the antioxidant of the present invention was obtained through a concentrated extract of a hot water extract of roasted defatted soybeans.

This has a solid content of 75-78% and a nitrogen content of 1.52.
%Met. Hereinafter, this will be referred to as "active ingredient 2."

Production Example 3 In the same manner as in Example 1 above, using defatted wheat bran instead of defatted bran, the active ingredient of the antioxidant of the present invention was obtained through a concentrated extract of the hot water extract of roasted defatted wheat bran. Hereinafter, this will be referred to as "active ingredient 3."

Production Example 4 In the same manner as in Example 1 above, using tea confectionery instead of defatted bran, the active ingredient of the antioxidant of the present invention was obtained through a concentrated extract of roasted tea confectionery hot water extract. Below, this is referred to as "active ingredient 4".
”.

<Preparation example of food form> Preparation example 1 Preparation of granules Active ingredient 1 7.5% by weight Active ingredient 2 7.5%.

Vitamin C10/.

Skimmed milk powder 25〃Powdered sugar
5 Glucose 28 Lactose 1
7 u Natural fragrance Appropriate amount Natural coloring agent Mix each of the above ingredients,
The mixture was granulated to obtain the antioxidant of the present invention in the form of granules.

Approximately 3 g of this product can be consumed as one serving.

Preparation Example 2 Preparation of Drink Active Ingredient 1 0.5% by weight Active Ingredient 2 0.5.

Vitamin CO, 8, / Liquid sugar (70%) 14〃 Water 81.
2. Natural fragrance (appropriate amount) Dog bark coloring agent (appropriate amount) Each of the above ingredients was blended and 50 cc of each bottle was filled into a container to prepare the antioxidant of the present invention in the form of a drink.

<Preparation example of pharmaceutical form> Preparation example 1 Preparation of tablet Active ingredient 1 2.5% by weight starch 66//magnesium stearate 9 n lactose
22,5 n Using each of the above ingredients, 1 tablet 2
00m0 tablets having the above composition were manufactured.

Approximately 8 to 10 tablets of this drug can be administered orally at a time.

Preparation Example 2 Preparation of tablets Active ingredient 1 2.5 parts by weight Active ingredient 2 2.5 Milk'aI (
50 11 Cornstarch (Japanese Pharmacopoeia) 25/l Crystalline Cellulose (Japanese Pharmacopoeia) 25 Methylcellulose (Japanese Pharmacopoeia)
1.5 0MCl Stearate (Japanese Pharmacopoeia Crystal) 1
〃The amounts of each component to achieve the above composition are mixed, granulated, dried,
Oral tablets each containing 2.5 mg of active ingredient 1 and active ingredient 2 were produced by press molding.

Preparation Example 3 Preparation of Liquid Active Ingredient 1 Active Ingredient 2 Glucose 100 m (1 100 m) (2 250 m (1 Appropriate amount) Total volume 5 mQ After dissolving active ingredient 1, active ingredient 2, and glucose in distilled water,
Inject into ampoule No. 51112, replace with nitrogen, 121°
A solution for oral administration was obtained by autoclaving at C for 15 minutes.

<Preparation example of cosmetic form> Preparation example 1 Preparation of emulsion Active ingredient 1 Active ingredient 2 Active ingredient 3 Liquid paraffin stearate vaseline cetyl alcohol 1.0% by weight 1, OI/ 1, O 7゜5 〃 2.5〃 5, O〃 1.8〃 Triethanolamine 1. O Polyoxyethylene monooleate ester 2.1 Polyethylene glycol 2.8 Fragrance 0
．． 3〃Preservative 0.1〃Water
A milky lotion was obtained using each of the above components, with the total amount being 100 parts by weight.

Preparation example 2 Preparation of cold cream Active ingredient 1 Active ingredient 2 Active ingredient 3 Squalane Hydrogenated lanolin Beetroot monoglycerin Cetyl alcohol Propylene glycol 1.0% by weight 1.0 1, O 36 6 7 2 4 〃 5 〃 Polyoxyethylene sorbitan monolaurate 2 rt fragrance
0.3 Preservative 0.1 Water
A cold cream was obtained using each of the above ingredients while adjusting the total amount to 100 parts by weight.

Preparation example 3 Preparation of lotion Active ingredient 1 1.0% by weight Active ingredient 2 1.0 #Ethyl alcohol 10.0 / Propylene glycol 4. ．． 0,.

Polyoxyethylene oleyl alcohol ester 2.6 Polyethylene glycol 1500 1.5.

Sodium citrate 0.1 Flavoring
0.3 Preservative 0.1 Water
A lotion was obtained using each of the above ingredients whose total weight was 100 lbs.

Preparation example 4 Preparation of hair tonic Active ingredient 1 1.0% by weight Active ingredient 2 2.0 Ethyl alcohol 80.0 Glycerin
1. O Polyoxyethylene oleyl alcohol ester 2. O〃Fragrance
1.0! /Water A hair tonic was obtained using each of the above components in an amount based on 100 parts by weight of the total amount.

Preparation Example 5 Preparation of hair liquid Active ingredient 1 1.0% by weight Active ingredient 2 2.0 Ethyl alcohol 5Q Lanolin
2. O Polyoxypropylene butyl ethyl ia, on Fragrance O18// Preservative 0.11/Water
A hair liquid was obtained using each of the above components in amounts to make the total 100 parts by weight.

Preparation Example 6 Preparation of Shampoo Active Ingredient 1 1.5% by weight Active Ingredient 2 1.5 “Active Ingredient 4
1.5 #Coconut oil fatty acid jetanolamide 3.0 /1 triethanolamine lauryl ether sulfate 1B, 0! ! Sodium lauryl ether sulfate 40.0 #Polyoxyethylene lanolin 0.5 11 Citric acid 0.2 N Fragrance
0.8〃 Preservative o, i in water
A shampoo was obtained using each of the above ingredients in amounts making the total 100 parts by weight.

<SOD-like enzyme activity test> The SOD-like activity of the antioxidants (active ingredients) of the present invention obtained in each of the above production examples was investigated by the following methods.

■, Measurement of 02-■-1) Xanthine-based 0.1M potassium phosphate buffer [pH 7, 8, 0, 1m
M EDTA (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.)] 2mQ, 50mM
EDTA (same as above >50tIQ, hypoxanthine (
Sigma) solution [hypoxanthine 13.5mM 50
After stirring the mQ saline with a stirrer for 30 minutes, it was heated to 4°C.
Bottom, supernatant obtained by centrifugation at 3000 ppm for 10 minutes, the same applies hereafter] 100 μQ and cytochrome C [manufactured by Sigma, type IL12, 4 m (If/1, 5 mG physiological saline] 5 C) were mixed to prepare the test solution. create.

Absorbance of each of the above test solution alone (control) and a solution to which the antioxidant of the present invention was added to a predetermined concentration (
OD55onm) using a spectrophotometer [UVV manufactured by Beckman
5260] (0 minute value).

Next, each of the above test solutions was taken out into a test tube, and 0.1
xanthine oxidase (in an amount resulting in a concentration of 2 U/m2)
Add >100 μQ manufactured by Sigma, stir immediately, and measure the absorbance after 30 minutes in the same manner as above (30 minute value).

The amount of 02- in each test solution is the reduction constant (K
=21.1>, it is calculated as follows.

02- (nmol/min) = (30 minute value - 0 minute value) /21. lX1000■-2)
Neutrophil stimulation system Prepare three test tubes (A, B and C), and in A and B, 2X suspended in 2ml of KRP solution prepared according to the following.
Add 900 μQ of 10' neutrophils to each cell, and add 900 μQ of KRP solution to C (control).

Preparation of KRP solution 1015 ml of 9% aqueous solution of NaCQ91.35C1,
KCGl, 11.5% aqueous solution of 6C1 4QmQ.

15ml of 12.2% aqueous solution of CaCQ2 ・2H201,83Q and fVtgsO4 ・7H203,82Cl
10 precepts of 38.2% aqueous solution were mixed to make solution A.Meanwhile, 26.808CI of NaHPO4.7H20 was dissolved in 1Q of distilled water, and about 20mQ of 1.0MH (.9) was mixed therein to adjust the pH to 7. Adjust to .4 to make liquid B, and use the above liquid A 9.
0mG, 810ml of distilled water with 1Q of gelatin dissolved in advance
Mix 2 and 100 mQ of the above B solution, add and dissolve glucose (glucose 5mM > 900.8 mg), and filter to prepare a KRP solution.

Each of the contents of test tubes A, B and C was heated to 37°C for 5 minutes.
After shaking and incubating for a minute, each test tube was taken out into water, and A had KR.
Add 100 μQ of solution P, and add zymogen A to B and C.
Add 100 μQ of Zymosan A (manufactured by Sigma) and then add cytochrome Cl0O to each tube.
Add tlQ one by one.

After mixing each sample, react by incubating with shaking at 37°C for 30 minutes, immediately immerse the test tube in water to stop the reaction, and centrifuge at 2000 m4°C for 5 minutes.

0.2ml of supernatant in each test tube was added to test tube A'B' containing 2.8tll12 of 0.1M potassium phosphate buffer in advance.
After adding (15-fold dilution) to each of and C′ and mixing,
The absorbance of each was measured in the same manner as in I-1) above,
02-Amount is calculated by the following formula.

A: (A'-C')/21.1x1000x15nHo
l/30m1n/2 xIPB: (B'-C')/21.1x1000x15n
Mol/30m1n/2 x 106 The same test above is repeated with test tubes A and B containing the antioxidant of the present invention at a predetermined degree.

Measurement of Il, H202 ■-1) 2 m of xanthine-based 0.1M potassium phosphate buffer (pt-17,8)
Q, 50mM EDTA501 and hypoxanthine solution [13.5mM 50mG A test solution was prepared by mixing 1100μQ of physiological saline, and 20μQ/m12 of scopoletin (SCOpOletine, manufactured by Sigma >1
00LIQ was added, transferred to a colorimetric cell, and colorimetrically measured using a fluorescence spectrophotometer (Hitachi MPF-4), and the measured value was determined on a chart.

Next, close the shutter of the spectrometer and use 200 μQ
/mQ peroxidase (manufactured by Toyobo Co., Ltd.) 50tlQ was added, and 0.12U/mQ xanthine oxidase 1
After adding 00 μQ and lightly pipetting, the shutter is opened and measurement is continued for 20 minutes, the degree of decrease in the measured value is determined, and the degree of decrease per minute is calculated.

H2O21 required to consume 1 mole of scopoletin
Since is 1 mol, the degree of decrease in the measured value per minute is calculated as x 139 picomole/min.

In the above, a predetermined concentration of the antioxidant of the present invention is added to the test solution, and the same operation is repeated.

(2) Neutrophil stimulation system 2.5 x 106 neutrophils are suspended in 2 mQ of KRP solution and incubated in a test tube at 37°C for 5 minutes. Scopoletin (20μq/mQKRP>0.
Add 1 mQ, transfer to Cerny for colorimetry, measure colorimetrically with a fluorescence spectrophotometer, and obtain the measured value on a chart.

Next, close the shutter of the spectrometer for -1 second, and remove 200 μg of
/mQKRP-peroxidase 0.5- was added, and 11mM mQ of opsonized zymogen [0pus
onized 2ymosan, made by Sigma Co., Ltd. 0.2m
After adding 12 and lightly pipetting, the shutter is opened and measurement is continued for 20 minutes, the degree of decrease in the measured value is determined, and the degree of decrease per minute is calculated.

In the above, a predetermined concentration of the antioxidant of the present invention is added to the test solution, and the same operation is repeated.

1 [Measurement of 1,0H・■-1) Xanthine-based 0.1M potassium phosphate buffer [pH 7, 8, 0, 1m
Contains EDTA] 2 courses, 50mMEDTA 50μQ
1 hypoxanthine solution [13.5mM 50mG physiological saline] 100tlQ, a predetermined amount of the antioxidant of the present invention and 0.1hypoxanthine solution [13.5mM 50mG physiological saline]
A mixture of 100 μQ of 12 LI/mQ xanthine oxidase was placed in a Maruem tube, and 1 mM KM was added to it.
Solution B [2.8 liters of distilled water and 50 mc of α-keto-T-methokiol loop tiric acid (KMB, manufactured by Sigma)
+ dissolved] 20 μQ was injected and heated to 37°C for 1 hour.
Incubate for 1 hour for 0 minutes, then incubate 4 times for 30 minutes at 10 minute intervals.
Each time, 200 μQ of the gas in the tube was taken out and injected through the sample inlet of a gas chromatograph (Hitachi model 163), and the peak of the amount of ethylene (OH) was recorded on recording paper. The cumulative total of the four measurements is determined by multiplying this equivalent value by a constant of the standard value (51,97 pmol).

Note that the same test is conducted as a control using a test solution that does not contain the antioxidant of the present invention.

■-2) Neutrophil stimulation system 6 x 106 neutrophils in a Maruem tube with 2 mQ of K
Float with RP solution. Two of the above specimens were prepared, and one was
Inject 20 μQ of MB solution, cover with a rubber stopper, and incubate in a constant temperature bath at 37° C. with shaking. The other sample was added with 11mMm12 equivalent of 0.2m12 of zymogen.
Add 20μQ of KMB, cover with a rubber stopper, and incubate in the same manner.

After 10 minutes of incubating each sample, gas chromatography is performed in the same manner as in ①-1 above.

The above active ingredients 1 and 2 as antioxidants of the present invention
The test results of (1), (2) and (2) above obtained using the above are shown in Table 1 (xanthine system) and Table 2 (neutrophil stimulation system) below.

From the results shown in Tables 1 and 2, it is clear that the antioxidant of the present invention exhibits an excellent antioxidant effect.

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Claims (3)

[Claims] (1) An antioxidant characterized by containing an organic solvent extract of a hot water extract of roasted plant fiber material as an active ingredient. (2) The antioxidant according to claim (1), which is orally ingested in food form or pharmaceutical form. (3) A cosmetic containing the antioxidant according to claim (1).