JPH03251514A - Melanin decomposing substance - Google Patents

Abstract

PURPOSE:To obtain a melanin decomposing substance prepared by extracting algae marine such as brown algae, green algae or red algae and a cosmetic having high safety, an agent of preventing fishes and shellfishes from blackening, an agent of keeping vegetables and fruits from blackening, containing the substance. CONSTITUTION:Especially brown algae (e.g. WAKAME seaweed, sea tangle, sea trumpet, Nemacystus decipiens or gulfweed) are roughly extracted with an organic solvent (e.g. lower alcohols or ketones) having low selectivity to raw material marine algae, then with an organic solvent having high selectivity to inorganic substances, the objective components is subjected to partition (water or organic solvent) extraction with an organic solvent (e.g. hexane or chloroform) having high selectivity, treated by column chromatography and impurities are removed to produce a melanin decomposing substance. The melanin decomposing substance suppresses melanogenesis, not only decomposes formed melanin, decolors am but also removes color, smell, viscosity substance, etc.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a melanin-degrading substance extracted from seaweed, a cosmetic containing the melanin-degrading substance, which has an excellent skin whitening effect and is highly safe, and the melanin-degrading substance. A highly safe blackening prevention agent for seafood that can be mixed with seafood feed or used by contacting (spraying or immersing) seafood, and a melanin-degrading substance that can be used on fruits and vegetables. This invention relates to a highly safe agent for preventing browning of fruits and vegetables that can be used by contacting (spraying or dipping) with

[Technical background] Spots, freckles, etc. that occur on human skin are mainly caused by the formation of melanin in tissues caused by sunburn, etc.

Skin whitening agents are one of the additives added to cosmetics, and they prevent the formation of melanin, which causes spots and freckles.Consumers are very interested in cosmetics containing this agent. It's increasing.

Although various skin whitening additives have been incorporated into cosmetics, they are not necessarily perfect in terms of effectiveness, safety, and commercial value. Skin disorders thought to be caused by these additives have occurred, and some creams may discolor when used as cosmetics, and some are said to be mutagenic. The development of skin whitening additives is desired.

In addition, the blackening of seafood is also caused by the formation of melanin, and various measures are currently being taken to maintain the freshness of seafood and prevent blackening, but the equipment and measures required are extremely costly. Therefore, producers are very interested in safe and low-cost methods of preventing blackening. Therefore,
It can be said that there is a great need for an inexpensive and highly safe method for preventing blackening in the fisheries field.

Melanin formation inhibitors can be used as such skin whitening agents or as blackening prevention agents for fish and shellfish.Therefore, it can be said that there is a very high need for melanin formation inhibitors in the cosmetics and fishery fields, and no research has been done so far. Various melanin formation inhibitors have also been reported.

In view of the above-mentioned demand for melanin formation inhibitors, the present inventors were investigating the tyrosinase inhibitory effect of extracts of various seaweeds, and discovered that the extraction of various seaweeds using several stages of organic solvents The above-mentioned target components are richly concentrated in the extracted components obtained by the method, and the components not only inhibit the formation of melanin, but also have the activity of decomposing the melanin produced, making it colorless, and further improving color and odor. They also discovered that impurities such as viscous substances can also be removed, thereby completing the present invention.

The invention came about when the inventors happened to place floating seaweed on top of the fish they had caught while fishing in the sea. He discovered the difference in the degree of blackening between fish and fish that had not been touched, and thought that there must be medicinal ingredients in seaweed that prevent blackening. As a result of research, it was discovered that extracts of many seaweeds have outstanding effects and also have a melanin-degrading effect, leading to the present invention.

Since these seaweeds are edible natural products, they are extremely safe.

The melanin-degrading substance of the present invention can be used as an agent for preventing blackening caused by melanin formation, in addition to the above-mentioned cosmetics and as an agent for preventing blackening of seafood, such as for preventing blackening caused by melanin formation. It can also be used as an anti-blackening agent.

[Structure of the invention] The melanin-degrading substance of the present invention consists of a seaweed extract.

Examples of seaweeds to be used in the present invention include brown algae (wakame, kelp, kashime, aran, obamoku, mozuku, sargassum, etc.), green algae (ulva, aonori, miru, etc.), red algae (seaweed, hirafusa, fukuro nori, etc.). can be mentioned. Above all, the activity of brown algae is extremely high.

The melanin-degrading substance of the present invention can be obtained by appropriately cutting the above seaweed, leaving it untreated, and extracting it by a combination of solvent extraction with several types of organic solvents and column chromatography. The extraction method, temperature, etc. are not particularly limited, but from the viewpoint of extraction efficiency, it is preferable to heat and reflux the solvent together with the seaweed.

Although the reflux temperature varies depending on the solvent used, the activity of the melaninolytic substances of the present invention is not impaired even if relatively high temperatures are used.

Although the melanin-decomposing substance of the present invention is a water-soluble component, it is soluble in organic solvents with low selectivity such as lower alcohols. Therefore, in order to produce the melanin-degrading substance of the present invention from seaweed, raw material seaweed is crudely extracted using an organic solvent with low selectivity such as a lower alcohol, and the resulting extract is extracted with a highly selective solvent such as hexane. Preferably, the extraction is carried out by partition extraction. It is also preferable that the extraction includes a step of removing impurities using a highly selective solvent.

Therefore, methods for purifying the target product include, for example: ■ Crude extraction of raw seaweed with an organic solvent with low selectivity ■ Extraction removal of impurities with an organic solvent with high selectivity ■ Extraction removal of inorganic substances with an organic solvent with high selectivity ■ Selectivity It consists of partitioning of the target component with a high organic solvent (water, organic solvent) extraction ■ Column chromatography 〇 Removal of impurities.

Solvents with low selectivity used for the crude extraction include alcohols (methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol, amyl alcohol, etc.) and ketones (acetone, diethyl ketone, methyl propyl ketone, etc.) that are compatible with Kasagi. Examples include organic solvents with properties. The extraction conditions for crude extraction can be selected arbitrarily, but for example, it can be carried out by using several parts by weight to several tens of parts of a solvent for 1 part by weight of raw seaweed, and allowing it to stand at room temperature for 2 to 3 days. can.

Further, highly selective organic solvents used for impurity removal and partition extraction include hydrocarbons such as hexane and toluene, and halogenated compounds of aliphatic hydrocarbons such as chloroform and carbon tetrachloride. Also, ethers (ethyl ether, isopropyl ether, dimethyl dioxane, calpitol, cellosolve, etc.) and esters (ethyl acetate, propyl acetate, butyl acetate, methyl propionate, amyl formate, butyl formate, methyl lactate, methyl acrylate, acrylic acid) Ethyl, etc.) may also be used, but those with a low boiling point are preferred. Although extraction with these solvents can be carried out under any conditions suitable for the purpose, it is particularly preferable to carry out extraction at the reflux temperature of the solvent used to separate the target substance.

On the other hand, column chromatography uses silica gel, alumina, zeolite, activated carbon, etc., and its eluent is used in combination with the above-mentioned organic solvents.

[Example] The present invention will be explained below with reference to Examples.

Example 1 [Method for collecting melanin-decomposed substances] 5 kg of brown algae Arame (collected from the Miura coast in Kanayo prefecture) was washed with water, thoroughly drained, cut into pieces, and then added to 15 L of methanol at room temperature for 30 minutes.
After soaking day and night and distilling off the methanol from the soaking liquid, 1500ml of hexane was added to the residue, shaken, and filtered to remove impurities.The residue was dissolved in 200ml of methanol to remove insoluble matter such as salt. , methanol is distilled off.

Next, the residue was dissolved in 100 ml of water and 100 ml of ethyl acetate was added.
Distributed extraction is performed at m1. Similar extraction is repeated twice, the extracts are combined and the solvent is distilled off, followed by silica gel column chromatography (eluent: ethyl acetate), a green fraction followed by a yellow fraction is collected, and the solvent is distilled off. Then, the residue is dissolved in a small amount of water, insoluble matter is removed, and the water is distilled off under reduced pressure to obtain a residue of 200 μm. This is the target melanin-degrading substance. When a portion of this was dissolved in a small amount of water and subjected to TLC (silica gel, developing solvent: ethyl acetate, coloring: iodine), a clear spot near Rf = 0.6 was observed along with some minor spots.

This spot was colored by silver nitrate and exhibited ultraviolet absorption at a wavelength of 254I. Further, elemental analysis showed that it did not contain nitrogen, and a test using Dragendorff's reagent was negative.

Example 2 [Evaluation of melanin degrading substances] Solani 2 degrading activity 0.1 ml of the 0.01% solution of the melanin degrading substances obtained in Example 1 was mixed with 4.9 ml of a melanin suspension formed as shown below. When the mixture was shaken at 37°C, the melanin was completely decomposed in 5 hours and a clear liquid was obtained.

On the other hand, no decomposition of melanin was observed in the control 0.01% aqueous solution of kojic acid, which is well known as a melanin formation inhibitor.

Add 2 ml of L-tyrosine solution (0.3 μ/ml) to the melanin suspension test tube.
, add 2 ml of pine kilvain buffer (pif6.8) and tyrosinase (manufactured by Sigma, derived from pine mushroom) solution (1,25 μ/ml) at O, la + 1, stir well, and incubate for 10 minutes in a thermostatic water bath at 37°C. After that, it is left for one day and night to form melanin.

20 ml of tyrosinase inhibitory L-tyrosine solution (0,3*/ml)
, put 20 ml of pine kilvain buffer (pH 6, 8) and 0.6 g of agar in a flask, stir, heat to boiling, and then cool to 45°C. When the tyrosinase solution (
Add 0.2 ml of 1.25 μ/ml), stir, and transfer to a petri dish to make an agar plate. A paper with a diameter of 1 G+m impregnated with a 0.01% aqueous solution of the melaninolytic substance obtained in Sample Example 1. When the tyrosinase inhibitory activity was determined by placing a disk and measuring the diameter of the inhibition circle centered on the disk, a clear halo of 16 m+ was obtained.
A similar test using 0.01% kojic acid as a control sample yielded only 12 halos.

Example 3 Melaninolytic substances were extracted from seaweed shown in Table 1 below in the same manner as in Example 1 above. The yield is also shown in Table 1.

Example 4 The melanin-degrading substances from various seaweeds obtained in Example 3 were tested for melanin-degrading activity and tyrosinase inhibitory activity in the same manner as in Example 2. The results are shown in Table 2.

Table 2 Note 1. 0.01% Kojic Acid Note 2. Add 0.0ml to 4.1ml of the same melanin solution as in Example 2.
0.9 ml of an aqueous solution of each melanin-degrading substance at 0.01% was added and the determination was made in the same manner.

Judgment criteria: +++ = Melanin completely decomposed and became clear within 24 hours = Melanin decomposition not observed even after 72 hours Note 3. Using a 0.01% aqueous solution of each melanin decomposition substance,
The test was carried out in the same manner as in Example 2.

Judgment criteria: +++ = Diameter of inhibition circle of 5 m or more + = Diameter of inhibition circle of 10 m or more = Inhibition cannot be achieved The following examples of the cosmetics of the present invention will be listed to further explain the present invention. Although explained in detail, the present invention is not limited to these prescription examples.

In addition, A in the following examples indicates the melanin-degrading substance of the present invention obtained in Example 1.

Example 5 Facial cleansing cream glycerin 2.0g stearic acid
20.0g myristic acid
10.0g lauric acid 5
．． 0g polyoxyethylene lauryl 1.0g ether A
10 ■Fragrance and preservative Appropriate amount Mix these ingredients, heat and dissolve at about 75℃, add about 75℃
Potassium hydroxide 5.5g water heated to ℃
56.0g
After adding the solution with stirring, the mixture was cooled to obtain a facial cleansing cream having a skin whitening effect.

Example 6 Back polyvinyl alcohol 15.0g Preservative
Agent 0.1g water
? 5.
OgA
Glycerin 4.0g Ethanol
5.0g fragrance
Appropriate amounts were mixed and dissolved uniformly to obtain a bag.

Example 7 Oil-based cream Beeswax paraffin wax Lanolin Isoprobyl myristes squalane Liquid paraffin 10.0g 6.0g 3.0g 6.0g 8.0g 25.0g 20■ Polyoxyethylene sorbitan monostearate 1.8g Sorbitan monostear Rate 4.2g preservative
Mix appropriate amounts of these ingredients, heat and dissolve at about 75℃, and add about 75℃ to this mixture.
Propylene glycol 2.0g water heated to ℃
sand 0.
7g urea
5.0g water
26.0 g of the mixture was added with stirring, and after cooling, an appropriate amount of fragrance was added at 55 °C, and the mixture was heated to 45 °C with stirring.
The mixture was cooled to ℃ and allowed to stand to obtain an oily cream.

Example 8 Lotion (oil-based) Sorbitol (70%) 30g Glycerin 5.0g A1. Og water 7
Mix and dissolve 0.0g of these ingredients. To this, a mixed solution of 0.1 g of allantoin, 0.5 g of polyoxyethylene hydrogenated castor oil derivative, 20.0 g of ethanol, and an appropriate amount of fragrance was added with stirring to obtain a homogeneous solution, thereby obtaining a lotion having a skin whitening effect.

Example 9 Lotion (drying) Ethylene glycol 2.5g Propylene glycol 5.0g Silicone
0.0001 gA
5.0g water
Mix and dissolve 65.0g of these ingredients. In addition, urea
1.0g polyoxyethylene sorbitan monolaurate 1.2g ethanol 15, Ogε-aminocaproic acid 0.1g fragrance
An appropriate amount of the mixed solution was added while stirring to make a homogeneous solution, and a lotion with skin whitening effect was obtained.

Example 10 A live fish (red sea bream) was brought into contact with a solution of A 5■ in IQ 00 ml of seawater for 5 minutes, and 48 hours later a control group (
When compared with fish caught at the same time and not exposed to the blackening prevention agent of the present invention, there was a clear difference in the degree of blackening between the two. It was found to suppress black discoloration.

Example 11 A 1■ was dissolved in 500 ml of water, and a peeled apple was brought into contact with the solution for 2 minutes. Control group (no contact)
Although the apples were brought into contact with the fruit and vegetable browning prevention agent of the present invention, no discoloration was observed even after 60 minutes.

Claims (5)

[Claims] (1) Melanin-degrading substance extracted from seaweed. (2) The melanin-degrading substance according to claim 1, wherein the seaweed is brown algae, green algae, or red algae. (3) A cosmetic containing the melanin-degrading substance according to claim 1 or 2. (4) An agent for preventing blackening of seafood, comprising the melanin-degrading substance according to claim 1 or 2. (5) An agent for preventing browning of fruits and vegetables, comprising the melanin-degrading substance according to claim 1 or 2.