JPH05310590A - Active oxygen elimination agent - Google Patents

Abstract

PURPOSE:To provide an active oxygen eliminating agent safely usable in a wide field such as pharmaceuticals, foods and cosmetics. CONSTITUTION:Residue of alcoholic fermentation drink prepared from cereals is extracted with water or an organic solvent and the obtained extract or the distillation residue of the alcohol-fermented cereal is used as an active oxygen eliminating agent or as a component of the agent.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention comprises an extract of a fermentation residue or a distillation residual liquid, which is produced when an alcoholic fermented beverage made from cereals such as sake, beer and shochu is produced, or contains the same. Thus, the present invention relates to an active oxygen scavenger that is safe and can be used in a wide range of fields such as medicine, foods, and cosmetics.

[0002]

2. Description of the Related Art When human beings maintain a healthy body, SOD which is an active oxygen scavenging enzyme in the living body and active oxygen in the body
(Superoxide dismutase) is always balanced and the concentration of active oxygen is kept almost constant. However, currently, SOD production is decreased due to imbalance of dietary habits, excessive stress, and aging, and on the other hand, active oxygen is increased due to smoking, air pollution and the like.

As a result, active oxygen is excessively present in the living body, causing various tissue disorders. Particularly in the elderly, the SOD activity decreases and the active oxygen concentration increases, causing disorders such as rheumatoid arthritis and Pechet's disease. In addition, lipid peroxides generated by active oxygen, myocardial infarction, stroke, cataract, spots, freckles, wrinkles,
It is a leading cause of modern diseases such as diabetes, arteriosclerosis, stiff shoulders, and coldness.

In addition, even if not aged, active oxygen is particularly likely to be generated at a site such as the skin which is directly stimulated by environmental factors such as ultraviolet rays, so that the melanin pigment increases with the increase of the active oxygen concentration. It is easy to cause problems such as the generation of blemishes, stains, and wrinkles.

Therefore, SODs that eliminate excess active oxygen, which causes various disorders as described above, have attracted attention, and in order to prevent or treat these disorders, SODs are used as medicines, cosmetics and foods. Attempts have been made to add and use.

However, since SOD is heat-labile, deactivates by oral administration, and is extremely expensive, elimination of active oxygen by SOD has not been successful.

[0007] In view of the above circumstances, studies on active oxygen scavengers (including antioxidants that act similarly to SOD enzymes) have been conducted, and active oxygen scavengers using crude drug extract and the like have also been developed. At present, not only is it expensive due to a special raw material, but it is also difficult to supply a stable material.

On the other hand, sake lees, which are the production residue of sake, can only be used for dipping, lees, shochu, amazake, etc., and it is no exaggeration to say that distilling is the center. For this reason, the price falls sharply due to the inability of sardines and the like, which not only puts pressure on the management of the brewery, but also has no takeover.
Disposal is a problem, and it is a major environmental problem. In addition, the production residues of beer and shochu are currently used as feed or discarded.

[0009]

Since various obstacles caused by active oxygen have been recognized as described above, various studies have been actively conducted to eliminate active oxygen in the living body. In addition, nowadays, it is desired to live in a healthier and older life with the aging society. On the other hand, active oxygen scavengers are also attracting attention from the viewpoint of beauty. Therefore, there is a demand for the development of an active oxygen scavenger that is safe and inexpensive for the human body, has an excellent effect of scavenging active oxygen that causes various disorders, and that can be easily manufactured and can be stably supplied.

[0010]

[Means for Solving the Problems] From the viewpoint of animal and plant mixing, the present inventors have been conducting research on various plant components centering on cereals, and at present, sake lees, which are difficult to treat, We have attempted research and development on the use of the residue of alcohol-fermented beverages made from grains such as beer lees and shochu lees. In the process, the active oxygen scavenging effect of this residue extract was measured, and it was found that there was a very remarkable active oxygen scavenging effect, and the present invention was completed.

That is, the present invention is characterized in that an alcohol-fermented beverage production residue obtained from a grain as a raw material is extracted with water or an organic solvent, or a distillation residual liquid of an alcohol-fermented grain is contained as it is or is contained. It is possible to obtain a very excellent active oxygen scavenger exhibiting the above-mentioned effect, which is simple and inexpensive, and is completely safe.

In the present invention, when extracting a residue for producing an alcoholic fermented beverage using cereals as raw material (hereinafter referred to as fermentation residue), extraction with water (including acid and alkali), or
Alternatively, it is extracted with an organic solvent such as alcohol. The fermentation residue of the present invention is mainly sake lees, beer lees and shochu lees, but in the case of shochu, water extraction is performed at the time of distillation without the need for separate extraction, so the distillation residue is separated. It is enough, and it is more efficient and effective. The distillation method in this case may be atmospheric distillation or vacuum distillation. When the fermentation residue is extracted, the amount of water added is 1 to 5 times and the amount of water is efficiently extracted, but it is appropriately selected depending on the yield, workability, end use purpose and the like. After that, the mixture is heated and the extraction is completed at the time of boiling.
Note that hot water may be added from the beginning to perform extraction.

Although the active ingredient in the extract has not been clarified, it is heat-stable, so the extraction temperature for water extraction is high at high temperature, but the same at low temperature for a long time. is there. However, at a low temperature of 40 ° C. or lower, it is necessary to carry out some kind of preservative such as making the pH acidic or alkaline. The extraction time may be several minutes in the case of boiling extraction, but several hours to one day are required in the case of moderate temperature below that. If the temperature is low, several days to one month is required. However, even in this case, it is more effective to heat at the end as much as possible.

In order to facilitate the extraction, it is more effective to use a method such as enzymatic reaction or hydrochloric acid decomposition.
Regarding enzymes, those treated with amylase, cellulase, etc. that react with the fermentation residue are all more effective than those obtained by water extraction alone. That is, the more effective the extraction, the more effective it is. Extraction with an organic solvent may be carried out by a conventional method using a common one such as alcohol.
Further, the obtained extract may be subjected to organic acid fermentation such as alcoholic fermentation and lactic acid fermentation.

The effect of eliminating the active oxygen of the product of the present invention will be described below. First, the effect of the product of the present invention as a superoxide scavenger was examined. The test method was the NBT method.

Preparation of Reagents 0.05M Na ₂ CO ₃ buffer (pH 10.2) 3 mM xanthine solution; 45.64 mg of xanthine is dissolved in the buffer to make 100 ml. 3 mM EDTA solution; EDTA.2Na 11
Dissolve 1.7 mg with distilled water to make 100 ml. BSA solution; Bovine Serum Albumin (Sigma) 1
Dissolve 5 mg in distilled water to make 10 ml. 0.75 mM NBT solution; NBT (nitroblue tetrazolium) 61.32 mg was dissolved in distilled water to obtain 10
Make up to 0 ml. Xanthine oxidase solution: Dilute xanthine oxidase with distilled water and adjust it so that the absorbance in the blank test of the operation method (analysis method) described below is in the range of 0.2 to 0.23. 6 mM CuCl ₂ solution; CuCl ₂ · 2H ₂ O
Dissolve 102.29 mg in distilled water to make 100 ml.

Method of Operation Take 2.4 ml of Na ₂ CO ₃ buffer solution into a test tube and add it to a xanthine solution, EDTA solution, BSA solution, NBT.
Add 0.1 ml of each solution. Then, 0.1 ml of the sample solution is added, and after standing at 25 ° C. for 10 minutes, 0.1 ml of the xanthine oxidase solution is added, and the mixture is quickly stirred and incubated at 25 ° C. After 20 minutes, 0.1 ml of CuCl ₂ solution was added to stop the reaction, and the absorbance was measured at 560 nm. For comparison, instead of the sample, superoxide dismutase (Cu, Zn type SOD, activity 3000 to
4000 unit / mg Wako Pure Chemical Industries, Ltd.) The same procedure was performed for 0.1 ml of the aqueous solution, and this value was used as the superoxide elimination rate 1
00. In addition, distilled water is used instead of the sample, and the same procedure is performed to obtain a blank. The measurement results are shown in Table 1.

[0018]

[Table 1] Note 1 Water extract was obtained according to Example 1. Note 2 Alcohol extract was obtained according to Example 2. Note 3 Distillation residual liquid was obtained according to Example 3.

As described above, it was found that all the fermentation residue and the distillation residual liquid have a superoxide scavenging effect.

Next, the thermal stability of the product of the present invention was examined. Various samples and SOD used in Table 1 were heat-treated at 90 ° C. for 5 minutes, and their superoxide scavenging ability was examined. The superoxide erasing rate was measured by the above method. The measurement results are shown in Table 2.

[0021]

[Table 2] Note: As in Table 1, each extract used was obtained according to each Example.

As described above, it was found that SOD is unstable with respect to heat, whereas all the products of the present invention have excellent thermal stability. Therefore, it can be said that the active ingredient for eliminating active oxygen in the product of the present invention has excellent stability against heat. Furthermore, the water extract of sake lees obtained in Example 1 was concentrated 5 times and the superoxide scavenging ability was examined. The superoxide scavenging ability was measured by the above method. The results are shown in Table 3.

[0023]

[Table 3] Note Concentration was performed with a rotary evaporator.

As described above, it was found that by concentrating, the product of the present invention has a superoxide scavenging effect which is almost the same as SOD. That is, SOD
May be low, but as animal feed,
It is a cheap and very effective means even when given in large quantities.

Since the product of the present invention exhibits a very remarkable effect of eliminating active oxygen and is safe, it can be used for medicines, cosmetics, foods and the like. Next, these applications will be described. As a medicine, it can be used as an anti-ulcer drug. The test methods for examining the anti-ulcer action of the product of the present invention and the results thereof are shown below.

The effect of oral administration of the product of the present invention on constrained water immersion stress ulcer was examined. The method was performed according to the method of Watanabe et al. That is, 8-week-old male ddY mice were fasted for 24 hours, orally the product of the present invention obtained in Example 1 was orally administered at 0.3 ml / mouse, and after 30 minutes, put into a stress gauge and put in a water at 15 ° C. The protrusions were dipped and restrained water immersion stress was applied. After 5 hours, the cervical vertebrae were dislocated and sacrificed, and the stomach was excised. After that, 1% formalin solution 1.5
The stomach tissue was lightly fixed by injecting ml into the stomach and further immersing in the same solution, and allowed to stand for 24 hours. After that, an incision was made along the greater curvature, and the length (mm) of the damage occurring in the glandular stomach was measured, and the total sum per animal was expressed as the ulcer index. As a control, the same amount of physiological saline was orally administered 30 minutes before being put into a stress gauge. Fifteen mice each were used. The results are shown in Table 4.

[0027]

[Table 4]

As shown in Table 4, the average ulcer index in mice administered with physiological saline as a control was 65.
In contrast, the average ulcer index in the mice administered with the product of the present invention was 32.8, clearly showing that the product of the present invention is effective as an anti-ulcer agent against restraint water immersion stress ulcer by oral administration. It turned out to be. As a result, it was revealed that the product of the present invention directly acts from the gastrointestinal mucosa and exhibits an effective action as an anti-ulcer agent.

The product of the present invention can also be used as a skin treatment agent. The product of the present invention is applied daily to panelists of various skin diseases.
The results are shown in Table 5, which was applied twice a day to the affected area in the morning and continuously for 1 month.

[0030]

[Table 5]

As shown in Table 5 above, since this product has various skin-treating effects, it can be seen that it has a fibroblast-activating effect and an antibacterial effect.
Moreover, since it is also useful for xeroderma, acne and the like, it can be seen that it also has a moisturizing effect and an effect of appropriately suppressing the increase of sebum, but actually suppresses this moisturizing effect and the increase of sebum. The test methods for examining the action and the results are shown below.

First, in order to demonstrate the strength of the moisturizing effect of the product of the present invention, a single coating test was conducted using a moisture meter (SKICON200). An environment in which the room temperature was 20 ° C. and the relative humidity was 65% was set as a measurement condition, and the panelists were allowed to rest in the above environment for about 10 minutes before the measurement. The test site (both sides) was selected on the flexion side of the forearm where no rash was observed. The panelists were five people who were suffering from xeroderma. The average value of changes in the water content of the stratum corneum between the main test (using the product of the present invention obtained in Example 1) read by a moisture meter and the control test (using water) is shown in FIG. The measurement method of the one-time application test is as follows.

Measurement method 1) A test site of 5 × 5 cm and a control site are set on the forearm flexion side of the panel. 2) Measure the water content of the stratum corneum at each site. 3) Measure the water content of the stratum corneum immediately after coating the sample and after 30, 60, 90 and 120 minutes.

From FIG. 1, it was confirmed that the water content of the stratum corneum of the product of the present invention increased about 10 times as much as that of the control, immediately after coating. In addition, regarding the period from 30 minutes to 120 minutes after the application, in the application site of the present invention, from the control 2 to 120 minutes.
It can be seen that the water content is tripled.

Next, in order to numerically demonstrate the therapeutic effect of the product of the present invention on xeroderma, a water load test was carried out using the moisture meter (SKICON200) before use of the product of the present invention and after use for 2 weeks. .. The panelists were the 5 persons used in FIG. 1, and the measurement conditions were the same as the one-time application test. A control (measurement at a site not coated with the product of the present invention) was always set so that the effect of the seasonal change in the water content of the living horny layer would not influence the effect determination. The stratum corneum water content was shown as the average value of 5 panelists. The result is shown in FIG. As the product of the present invention, the product obtained in Example 1 was used. The measuring method of the water load test is as follows.

Measuring Method 1) The water content of the stratum corneum of the test site is measured. 2) Place one drop of distilled water on the test site, and after 10 seconds, completely wipe off the water drop with dry gauze. 3) Immediately after wiping, the water content of the stratum corneum is measured after 30, 60, 90 and 120 seconds.

As shown in the graph of FIG. 2, by applying the product of the present invention, the water absorption capacity of the skin (the water content of the stratum corneum after 0 seconds of water loading minus the value of the moisture content of the stratum corneum before loading) ),
It can be seen that both the water retention capacity (the curve drawn by the water content of the stratum corneum from 0 to 120 seconds after water loading) is improved at the same time.

That is, the skin before use of the product of the present invention has a very low water content in the stratum corneum before water loading (average 5.1), and the water absorption capacity (average 45.0) is also considerably reduced. In addition, the stratum corneum of the skin of a normal person also has a water-retaining ability, and after 30 seconds of water loading, the stratum corneum of the skin of the normal person returns to the value before the water loading. These results show that the water absorption capacity, the water retention capacity, and the barrier function are all reduced in the measured morbid stratum corneum. On the other hand, the skin after use of the product of the present invention has a two- to three-fold increase in the water content of the stratum corneum and the water absorption capacity before water loading, and the water retention capacity is considerably improved to the same level as that of a normal person. I understood.

From the above, it can be said that the product of the present invention has an excellent action for improving the water content of the pathological stratum corneum and improving the barrier function. Further, when the product of the present invention is evaluated in combination with the moisturizing action obtained from the one-time application test, the product of the present invention increases the water absorption capacity and water retention capacity of the stratum corneum and absorbs a large amount of water from the outside world.
Furthermore, it can be said that it has a moisturizing action of giving the stratum corneum the property of not releasing the once absorbed water.

Further, in order to experimentally demonstrate the secretion inhibiting effect of the sebum amount of the product of the present invention, changes in the sebum amount after washing the face were measured. As panelists, five persons randomly selected from those used in Table 5 were used, and the average value of the change in the amount of sebum between the main test (after washing the face and applying the product of the present invention) and the control test (only the face washing) is shown in FIG. It was shown to. As the product of the present invention, the product obtained in Example 1 was used.

As shown in the graph of FIG. 3, it was found that the application of the product of the present invention significantly suppressed the increase in the amount of sebum. The inhibitory effect on the amount of sebum secretion of the product of the present invention also confirmed the preventive and therapeutic effect on acne. It was revealed from the following tests that, when the product of the present invention was applied to the skin as a cosmetic, it had the effects of making the skin smooth and having a fine texture.

The product of the present invention was applied to the right arm part of the panelist twice a day for one month, and the site of application of the product of the present invention was measured by a dynamic friction meter. The same part of the left arm was used as a control. The panelists were 6 people. The measurement conditions are as follows. Temperature 25 ℃ Humidity 60% Sensor used KES-SE Friction tester SE-2 type (0.5mm piano wire used) Friction static load 50gf Measuring speed 1mm / sec Measuring distance 30mm (Integral effective range 20mm)

The MMD (coefficient of variation) was 0.0174 in the part of the left arm to which the product of the present invention was not applied, but in the part of the right arm to which the product of the present invention was applied for one month, the MMD was
(Coefficient of variation) fell to 0.0071. The average values of 6 persons were almost the same. It is considered that this is because the fluctuation due to the unevenness of the surface was reduced, and from this, it became clear that the texture of the skin became finer and that the wrinkled skin became younger. When MIU (friction coefficient) was also examined at the same time, it was 0.120 before application, but after application for 1 month, the skin dropped to 0.082, which also has the effect of making the skin smooth and It has been found that it also has an anti-aging effect that makes it smooth like a young person.

Further, in order to exemplify the whitening effect of the present invention, the tyrosinase activity inhibitory effect was tested. As an operation method, 1 ml each of a substrate solution (0.04% tyrosine solution) and a buffer solution (McllvaineBuffer pH 6.8) was accurately placed in an absorption cell, and 1 ml each of water and the product of the present invention obtained in Example 1 were obtained. Put each one accurately, stir and mix, and 35 ℃
After 5 minutes, adjust the absorbance scale to the wavelength of 475 nm and perform zero correction, and then add exactly 0.02 ml of tyrosinase solution (5.3 mg of tyrosinase dissolved in 0.9% NaCl solution) and immediately Incubated with agitation. The absorbance at this time was measured over time (every 3 minutes) and is shown in Table 6.

[0045]

[Table 6]

From the measurement results shown in Table 6, it can be seen that the product of the present invention has a tyrosinase activity inhibitory action. From this, it can be said that the product of the present invention has a whitening effect.

Further, as described above, the product of the present invention also has a moisturizing action enough to be used as a medicine. Therefore, all the basic effects of cosmetics are satisfied, and they have a wide range of applications such as creams, emulsions, lotions, cleansing, packs, and soaps. Also, by drinking the product of the present invention, the same effects as described above can be obtained.

The product of the present invention can also be used as a food preservative, antioxidant, and freshness-retaining agent. As a representative of Gram-positive bacteria, Bacillus subtili causes spoilage of rice and bread.
s, Bacillus cereus, and Escherichia coli as a general indicator of contamination as a representative of Gram-negative bacteria
The antibacterial activity test of the product of the present invention against ia coli and the results thereof are as follows.

The medium used was 10 ml of ordinary agar medium to which 1 ml of the product of the present invention (obtained in Example 5) was added. As a control, 1 ml of water was added instead of the product of the present invention. Incubate at 35 ℃,
The growth state of each bacterium after 24, 48 and 72 hours was observed and shown in Tables 7-9.

[0050]

[Table 7]

[0051]

[Table 8]

[0052]

[Table 9] Note 1 Evaluation: −: No growth +: Little growth ++: Development +++: Large growth

As can be seen from Tables 7 to 9, in the medium to which water was added as a control, the growth was already large for all the bacteria at the time of 10-hour culture, whereas the product of the present invention was used. In the medium supplemented with, Bacillus subt
The growth of ilis and Bacillus cereus was completely inhibited even after 72 hours of culture. Also, Es
Growth was observed in cherichia coli by culturing for 48 hours or more, and in this case as well, a large antibacterial effect was recognized as compared with the control.

Next, the effect of suppressing the production of oxides by the product of the present invention was investigated by the rodan iron method. That is, the effect of inhibiting oxidation of linoleic acid, which is very easily oxidized by the product of the present invention, was investigated. The measuring method is as follows.

Preparation of Reagents 0.2M Phosphate Buffer (pH 7.0) 2.6% Linoleic Acid Ethanol Solution 75% Ethanol Solution 30% Ammonium Thiocyanate 0.02M Ferric Chloride 35% Hydrochloric Acid Solution

Method of operation 0.2 ml of sample solution, 0.1 m of 0.2 M phosphate buffer
1, water 0.5 ml, 2.6% linoleic acid ethanol solution 0.2 ml are added and mixed well, and the mixture is left at 37 ° C. for 5 days. 50 μl of the oxidation treatment solution, 4.85 ml of 75% ethanol solution, 50 μ of 30% ammonium thiocyanate
1, 50 μl of a 0.02 M ferric chloride 35% hydrochloric acid solution is mixed, and after 5 minutes, the absorbance at 500 nm is measured. Further, distilled water is used instead of the sample in the same manner to make a blank. The results are shown in Table 10.

[0057]

[Table 10] Note Sake lees extract was obtained according to Example 1.

As is clear from Table 10, the product of the present invention is
It was found that it has an excellent antioxidant effect against linoleic acid, which is extremely susceptible to oxidation.

Furthermore, the product of the present invention is effective as a freshness-retaining agent for vegetables, fish and the like, and the results of tests conducted by spraying the product of the present invention on lettuce are as follows. When lettuce was sprayed with water and allowed to stand at room temperature, it began to grow after 12 hours, and the wound had browned on the second day. However, lettuce sprayed with the product of the present invention diluted 50 times was fresh until 16 hours and did not discolor until the third day.

As described above, the product of the present invention is safe for the human body and has an antibacterial effect against various bacteria, an effect of preventing browning, and an antioxidant effect. It can be widely used in foods.

[0061]

As is apparent from the above data,
Easily and completely safe removal of active oxygen by using an extract obtained by extracting the alcohol fermentation residue from grains with water or an organic solvent, or the distillation residual liquid of the alcohol-fermented grains as it is or by containing it. It was found that an active oxygen scavenger having excellent effects, stable to heat, and various effects can be obtained.

Grains have been the staple food so far, and their safety has been sufficiently demonstrated. In addition, the fermentation residue has not yet been developed for use, and it is sometimes difficult to dispose of it. In other words, it is very significant for the sake brewing industry that the added value of the fermentation residue, which is currently in trouble for disposal, can be increased and the market can be opened up in a completely different field from the past.

[0063]

Example 1 15 kg of sake lees (brown rice sake lees) were added to 30 liters of warm water at 60 ° C., well stirred to adjust to 45 ° C., and left at that temperature for 10 hours. Then boil to 90 ° C for better extraction, cool it to about 30 ° C,
Squeezing with a squeezing machine, 27 liters of the present invention and the remaining 16 kg
Got

Example 2 To 10 kg of beer lees, 20 liters of 60% ethanol was added, well stirred, and allowed to stand for 24 hours. After that, squeezing with a squeezing machine, the product of the present invention 19.2 liters and residue 10.5 kg
Got

Example 3 150 ml of lactic acid, 7 kg of koji rice and yeast were added to 30 liters of water. The next day, 15 kg of rice bran (50-90% of polishing rate) was steamed and added. 4 days later, 60 liters of water, 9 kg of koji rice,
30 kg of rice bran was steamed and added, and after 5 days, 60 kg of rice bran was steamed, 120 liters of water and 40 g of starch degrading enzyme were added. The saccharification and fermentation were performed while keeping the product temperature at 15 to 20 ° C. After 25 days, distillation was carried out by a distiller, and 130 liters of distilled alcohol having an alcohol content of 42% and a distillation residue 1
90 liters of ethanol. Thereafter, 190 liters of the distillation residue was squeezed with a squeezing machine to obtain 158 liters of the product of the present invention and 29 kg of a residue.

[Brief description of drawings]

FIG. 1 shows a moisture meter (S
It is a graph which shows the result of having performed the coating test once using KICON200).

FIG. 2 is a graph showing the results of a water load test before using the product of the present invention and after using it for 2 weeks.

FIG. 3 is a graph showing the results of testing the change in the amount of sebum when the product of the present invention is applied after washing the face and only when the face is washed.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location A61K 7/00 W 9165-4C

Claims (1)

[Claims] 1. An active oxygen containing an extract obtained by extracting a residue of an alcohol-fermented beverage produced from a grain as a raw material with water or an organic solvent, or a distillation residual liquid of an alcohol-fermented grain as it is or containing the same. Erasing agent.