JPH11196849A - Extraction of antimelanin substance-containing material and fractionation and purification of antimelanin substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for purifying and fractionating antimelanin substances T1 and T2 utilizable for a bathing agent and a cosmetic and contained in Sake (a Japanese fermented, mildly alcoholic beverage made from rice) lees for aiming at effective utilization of the Sake lees which are industrial wastes. SOLUTION: A sample is prepared from a Sake and Sake lees and a tyrosine- containing reactional solution containing the sample therein is then reacted with a tyrosinase derived from a potato to measure the absorbance. Thereby, it is confirmed that the same antimelanin substances are obtained from the Sake and Sake lees. A crude Sake lees extract dried powder is produced from the Sake lees and antimelanin substances T1 and T2 are fractionated and purified from the resultant crude Sake lees extract dried powder.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for extracting an anti-melanin substance-containing substance in the form of a crude sake extract extracted dry powder obtained from sake lees in order to obtain an anti-melanin substance contained in sake and sake lees. The present invention relates to a method for fractionating and purifying an anti-melanin substance from an extracted dry powder.

[0002]

2. Description of the Related Art Sake lees obtained as a by-product of sake production is used only as a part of food materials with limited uses, such as amazake, lees soup, pickled lees, and buns. Most of them are disposed of as industrial waste due to the food situation. In view of the current situation of sake lees being discarded in large quantities, the inventor of the present application has conducted intensive research experiments on the development of products using sake lees as a raw material in line with social demand from the viewpoint of effective use of resources. Finally, a method for extracting an anti-melanin substance-containing substance having a whitening effect and a method for fractionating and purifying the anti-melanin substance have been completed.

[0003] That is, it has been said that sake has a whitening effect for whitening skin, and if the sake component contains such a substance, sake lees after squeezing the mash after sake fermentation. It is presumed that similar components are also contained in. When an enzymatic reaction is carried out by allowing potato-derived plant tyrosinase to act on the L-tyrosine-containing reaction solution, dopachrome, which is a precursor of a melanin pigment, is produced.
There is a report that the addition of a sake lees extract in this reaction suppressed the production of dopachrome. Cosmetics containing kojic acid derived from koji used in the sake brewing process are already on sale for the purpose of suppressing the production of melanin pigment, which is the causative substance of spots and freckles. However, since sake does not contain kojic acid, it was not clear which component of sake exerts an anti-melanin effect.

[0004]

Sake exists as a valuable product by itself, while sake lees are mostly waste, except for some food ingredients. Sake lees contain a large amount of water and are heavy, so that they are expensive to transport, dry, and extensively dispose of, and are expensive to dispose of. Therefore, aiming at the effective use of waste sake lees, fractionation and purification of an anti-melanin substance having a whitening effect from sake lees, and extraction and drying of coarse sake lees to develop cosmetics and bath agents using the anti-melanin substance The purpose is to extract powder and fractionate and purify anti-melanin substances. Also,
It is an object of the present invention to obtain a useful anti-melanin substance from sake lees by proving that the anti-melanin substances contained in sake and sake lees are the same substance, even if the substance is not obtained from sake.

[0005]

A sample is prepared by concentrating the alcohol extract of sake and sake lees under reduced pressure. The tyrosine-containing reaction solution containing the sample is reacted with potato-derived tyrosinase, the change in absorbance is measured, and the presence of the anti-melanin substance is confirmed by comparing the amount of dopachrome produced with the sample-free reaction solution. An anti-melanin substance with a low yield is defined as T1, and an anti-melanin substance with a higher yield than T1 is defined as T2. Then, it is confirmed that T1 and T2 are contained in the sample derived from sake and the sample derived from sake lees. After it has been proved that sake and sake lees contain the same T1 and T2, the anti-melanin substance T1 is produced using sake lees as waste.
And T2 are fractionated and purified.

[0006] First, the sake cake extract is concentrated and dried to obtain a crude sake cake extract dry powder containing an anti-melanin substance, and then the crude sake cake extract dry powder is dissolved in distilled water, filtered, defatted, reduced in pressure and concentrated. . Next, the concentrated solution is dissolved in ethanol, passed through a glass filter filled with alumina, the anti-melanin substance is adsorbed on the alumina, eluted with ethanol from the adsorbed alumina, and the anti-melanin substance T2 is reduced and concentrated. And an anti-melanin substance T1 is obtained by eluting from alumina with distilled water.

[0007]

Next, a method for confirming the presence of anti-melanin substances T1 and T2 according to the present invention will be described. After concentrating 500 ml of the sake under reduced pressure using an evaporator, dissolving 500 ml of distilled water in 500 ml of distilled water, defatting three times with a separating funnel using an equal volume of n-hexane, and concentrating the concentrated sake again using an evaporator under reduced pressure. A sample was used. Also, after 500 g of sake lees was immersed in 3 liters of 75% ethanol overnight, the filtrate was concentrated under reduced pressure by an evaporator, dissolved in 500 ml of distilled water, and the same amount of n
A liquor extract derived from liquor was defatted three times with a separating funnel using hexane and concentrated under reduced pressure with an evaporator to obtain a sample derived from sake lees (see FIG. 1).

In order to confirm the presence of a mixture of the anti-melanin substances T1 and T2 contained in the sample derived from sake and the sample derived from sake lees, a tyrosinase derived from potato was reacted with a tyrosine-containing reaction solution at 37 ° C. for 12 hours to inhibit tyrosinase activity. The effect of dopachrome formation was compared between when the sake-derived sample and the sake lees-derived sample were added and when they were not added (see FIG. 2).

When a sample derived from sake or sake lees was added during the reaction, the production of dopachrome showing the maximum wavelength at an absorbance of 475 nm was clearly reduced as compared with the case without addition. From this, it was possible to confirm the presence of a mixture of anti-melanin substances T1 and T2 having a tyrosinase activity inhibitory effect in a sample derived from sake or a sample derived from sake lees (Table 1,
See Table 2).

[Table 1]

[Table 2]

[0010] Next, in order to confirm the chemical properties of the fractions of the anti-melanin substances T1 and T2 contained in the sample derived from sake and the sample derived from sake lees, each sample was developed and separated by thin layer chromatography. Was sprayed with 10% sulfuric acid and heated to develop a color for comparison. The spots detected by the 10% sulfuric acid spray heating method from the sample derived from sake and the sample derived from sake lees were almost the same, and several types of organic compound component spots were detected. The components separated based on this result
A potato-derived tyrosinase was added to the tyrosine-containing reaction solution, which was recovered from the thin-layer support with 0% methanol, and each of the samples was added and allowed to act when the enzyme reaction was carried out. This did not inhibit the fraction inhibiting dopachrome formation. You can find the fraction. Using this inhibition reaction as an index, a method for fractionating and purifying a mixture of antimelanin substances T1 and T2 from a sample derived from sake lees was established.

Next, a method of separating and fractionating the mixture of the anti-melanin substances T1 and T2 by thin-layer chromatography using various developing solutions was examined. Furthermore, by using several detection reagents after development, it was confirmed whether the fractions of the anti-melanin substances T1 and T2 obtained from sake and sake lees were the same substance. As a result, it was found that there were two types of anti-melanin substances having a tyrosinase activity inhibitory effect obtained from sake and sake lees samples, respectively, and there was no difference depending on the sample, and both were the same substances T1 and T2. That is, Silica gel 6
Using a thin support plate of 0-TLC, n-BuOH: CH ₃ COOH: H ₂ O = 10: 10:
When developed at 3, the anti-melanin substance T1 developed at Rf 0.40 (0.32 to 0.46) and Rf 0.71 (0.64 to 1.0) near the development front
(See Tables 1 and 2).

Next, the anti-melanin substance T1 contained in sake lees
In addition, a method for fractionating and purifying T2 will be described. Anti-melanin substance T1 contained in sake and sake lees (Rf 0.40)
Can be used for chloroform in thin layer chromatography:
When redeployed with methanol = 3: 1, it is adsorbed by the support and stays at the origin, utilizing the fact that it hardly moves.
2 (Rf 0.71) developed a fractionation purification method using the fact that it migrates to Rf 0.42 by redevelopment. 75 g of 500 g of sake lees
After immersing overnight in 3 liters of 3% ethanol, the filtrate is concentrated under reduced pressure by an evaporator and dried to obtain a dry powder extracted from crude sake cake (see FIG. 3 (A)). In order to fractionate and purify a mixture of the anti-melanin substances T1 and T2 from the crude sake lees extracted dry powder, as shown in FIG. 3B, the mixture was dissolved in 500 ml of distilled water, filtered, and an equal amount of n-hexane was added to the solution. The mixture was degreased three times with a separating funnel using an evaporator, and concentrated under reduced pressure with an evaporator. Then, the concentrated solution was dissolved in 20 ml of water to obtain fluidity, and a mixture of the anti-melanin substances T1 and T2 was adsorbed on 200 g of alumina. , Alumina 5
Refill on a glass filter (diameter 10 cm) filled with 00 g. Pour ethanol evenly from the top,
Alumina and anti-melanin substance T2 having low adsorptivity are fractionated. The yield of the anti-melanin substance T2 was about 12.5%. The anti-melanin substance T1 is adsorbed on alumina.
Then, 2 liters of distilled water was flowed to lower the activity of alumina, so that the adsorbed anti-melanin substance T
Collect 1. The fraction of the collected sake lees was identified as anti-melanin substances T2 and T1 contained in the sake lees by component analysis by thin layer chromatography and by inhibiting potato-derived tyrosinase activity. The yield of the anti-melanin substance T1 from sake lees by the fractionation purification method was about 2.
5%. According to Table 2, although the yield of T1 was lower than that of T2, the tyrosinase activity inhibition rate was as high as 55.2% with a small yield. Therefore, the anti-melanin substance T1 had a stronger tyrosinase activity inhibitory effect than T2.

[0013]

The anti-melanin substance T contained in sake lees
Animal test and mutagenicity test revealed that 1 and T2 were non-toxic and non-mutagenic. By producing cosmetics and bath additives to which a high concentration of anti-melanin substance fractionated and refined from sake lees was added,
An even higher whitening effect can be expected. Also, by adding the sake lees dry powder (see FIG. 4) described in the examples, a moisturizing effect can be expected. Thus, the whitening-effective anti-melanin substances T1 and T2 can be fractionated and purified from the sake lees, which is a waste, and by using it, the usable range is widened. It has become possible to provide effective cosmetics and bath additives.

[0014]

Example 1 Next, a method for producing an anti-melanin substance-containing bath agent obtained from sake lees will be described. Sake lees as a by-product at the time of sake production contain about 8% of alcohol, and it is difficult to use as it is as a cosmetic or bath additive. Therefore, as shown in FIG. 4, the sake lees is first heated at 60 ° C. to remove water until the content becomes 10%. At this time, a dry powder containing almost no alcohol can be obtained. The dry powder is pulverized to obtain a sake cake dry powder having a diameter of 0.1 mm or less (see FIG. 4). 10 g of this sake lees dry powder, 8 g of sodium sulfate, and sodium hydrogen carbonate 3.
2 g, and 1.6 g of citric acid were evenly mixed, and 100 mg of the anti-melanin substance T1 or 1 g of crude sake lees extracted and dried powder containing T1 and T2 (see FIG. 3 (A)) was added as a bath agent. Used for 200 liters of hot water around 42 ° C. Since the anti-melanin substance-added bath agent has a tyrosinase activity inhibitory action, a strong whitening effect can be expected.

Example 2 Next, a method for producing an anti-melanin-containing cosmetic (paste peel-off type pack) obtained from sake lees will be described. 150 g of vinyl acetate resin emulsion, 80 g of polyvinyl alcohol, 50 ml of ethanol, 30 ml of olive oil, 50 g of sorbitol, 70 g of kaolin, 30 g of sodium carboxymethyl, 80 g of titanium oxide, an appropriate amount of preservative, and dried sake lees (see FIG. 4).
0 g, a dry powder extracted from crude sake lees containing T1 and T2 (Fig. 3
(A) is dissolved in 450 ml of purified water to produce a paste-like peel-off type pack. Since the sake lees pack containing the anti-melanin substance has a tyrosinase activity inhibitory action, a strong whitening effect can be expected.

Example 3 Next, a method for producing an anti-melanin-containing cosmetic (jelly-like peel-off type pack) obtained from sake lees will be described.
150 g of polyvinyl alcohol, 50 g of sodium carboxymethyl cellulose, propylene glycol 30
g, 100 ml of ethanol, an appropriate amount of preservative, 20 g of dried sake lees, 3 g of anti-melanin substance T1 or 15 g of anti-melanin substance T2, and 600 ml of purified water to produce a jelly-like peel-off pack. Since the sake lees pack containing the anti-melanin substance has a tyrosinase activity inhibitory action, a strong whitening effect can be expected.

[Brief description of the drawings]

FIG. 1 shows a method for producing a sample derived from sake and sake lees.

FIG. 2 shows a method for confirming an anti-melanin substance contained in a sample derived from sake and sake lees.

FIG. 3 (A), (B) and (A) show a method for obtaining a dry powder extracted from sake lees from sake lees, and FIG. 3 (B) shows a method for fractionating and purifying anti-melanin substances T1 and T2 from the dried powder extracted from coarse sake lees. is there.

FIG. 4 is a method for obtaining a sake lees dry powder from sake lees.

Claims (2)

[Claims] 1. A method for extracting an anti-melanin-containing substance, which is a crude sake lees extracted and dried powder obtained by immersing sake lees in ethanol, and filtering, depressurizing, concentrating and drying the immersion liquid. 2. A sake lees is immersed in ethanol, and the immersion liquid is filtered, decompressed, concentrated, and dried. The crude dry cake extracted and dried powder is dissolved in distilled water, filtered, defatted, decompressed, concentrated, and then concentrated. Dissolve the solution in ethanol, pass it through a glass filter filled with alumina, adsorb the anti-melanin substance on the alumina, elute from the adsorbed alumina with ethanol, elute with decompressed and concentrated anti-melanin substance and distilled water from alumina A method for fractionating and purifying an anti-melanin substance obtained by purifying an anti-melanin substance obtained by the above method.