JP3590908B2 - Enzyme inhibitor having α-amylase inhibitor activity or tyrosinase inhibitor activity and method for producing the same - Google Patents

Description

以上に見られるようにカリン抽出溶液にはαアミラーゼインヒビター活性が認められた。実施例２で調整したマルメロ抽出液についても上記と同様に試験した結果、試験管Ｎｏ．０〜２を除いて、すべてヨード澱粉反応陽性であった。
【００１４】
実施例３
生カリン（１９９４年新潟県中魚沼郡津南町にて収穫）４ｋｇの皮および芯を除去して得られたカリン素材３ｋｇにエタノール４．５ｌを加え、ミキサーでよく粉砕し、のち室温にて一週間放置する。この濾液（約６．５ｌ）を減圧下に濃縮、乾固すれば黄褐色のシラップ２８３ｇが得られる。このシラップを一回あたり４００ｍｌのエタノールで３回加熱抽出し、その抽出液を合併し減圧濃縮すれば黄褐色のシラップが４３ｇ得られる。本シラップをエタノール１００ｍｌに加温溶解し乳糖４００ｇを加えよく混和し、のち温風にて乾燥、粉砕すればαーアミラーゼインヒビター活性の強いサンプル４４０ｇが得られる。
本サンプルのαーアミラーゼインヒビター活性は試験例３に示した方法で測定すると次のようであった。

【００１５】
試験例３ サンプルのαーアミラーゼインヒビター活性（２）
αーアミラーゼの阻害反応
０．１％サンプル水溶液１ｍｌに０．５％パンクレアチン（日本薬局方）水溶液および精製水３ｍｌを加えよく混和後、３７℃、１０分間阻害反応を行う。対照としてサンプルに代わり精製水を用い同様に反応を行う。
糖化反応
１％可溶性澱粉液３ｍｌに上記阻害反応液１ｍｌを加え、よく混ぜた後３７℃、１５分間糖化反応を行う。反応終了後、この液２ｍｌをとりソモギーの変法（食品分析ハンドブック：建帛社）にて生成ぶどう糖の分析を行う。
阻害率の算出
対照区の生成ぶどう糖量・・・・・Ａｍｇ
実験区の生成ぶどう糖量・・・・・Ｂｍｇ
阻害率（％）＝１００ー【Ｂ／Ａ】ｘ１００
【００１６】
実施例４
美白クリームの調整
実施例１で得られたエキス５ｍｌをとり、減圧下に溶媒を留去してのち、次の処方で美白クリームを調整した。
カリン抽出エキスる ５ｍｌ相当分
ステアリルアルコール ６．０ｍｌ
ステアリン酸 １．０
水添ラノリン ５．０
オクチルドデシルアルコール １０．０
ポリオキシエチレン（２０モル）
モノセチルアルコールエーテル ４．０
グリセリンモノステアリン酸エステル ２．０
プロピレングリコール ５．０
精製水 ６６．０
香料 適量
防腐剤 適量
【００１７】
実施例５
ダイエット用クッキーの調整
ショートニング３００ｇ，牛乳２０ｇ，砂糖５０，アスパラテーム５ｇを泡立て器でよく混合する。これに卵６０ｇを少しずつ加えてさらによく混ぜ合わせる。別に小麦粉３００ｇ，ベーキングパウダー１ｇおよび実施例２で得られたマルメロエキス５０ｍｌ相当分の溶媒を減圧下完全に溜去したものをよく練り合わせる。これを先の混ぜ合わせたものとよく混合したのち、冷蔵庫で３０分放置する。次にこれを適当な型に成型し、約１７０℃で２０分間焼成して、目的のクッキー約１，０００ｇを得る。
【００１８】
【発明の効果】
本発明のカリンおよびマルメロの抽出エキスは優れたチロシナーゼインヒビターならびにαーアミラーゼインヒビター活性を有し、化粧品あるいはダイエット食品用などの添加物として有用である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an enzyme inhibitor obtained by extracting from a fruit of a Rosaceae plant, and more particularly, to an enzyme inhibitor derived from a fruit of a Rosaceae plant having tyrosinase inhibitor and α-amylase inhibitor activity. .
[0002]
Problems to be solved by the prior art and the invention
Melanin is a brown to black pigment formed by the oxidation of L-tyrosine by tyrosinase in melanocytes and through several intermediates. It is considered that suppression of melanin formation is preferable not only from a cosmetic viewpoint but also from a medical viewpoint such as the appearance of skin cancer, and various sunscreen agents have been provided. However, there is still a need for the development of safe and effective sunscreen actives. Obesity is highly associated with adult diseases such as diabetes, and it is pointed out that obesity needs to be prevented or eliminated. At present, various dieting methods have been proposed, including dietary foods. Here, however, the development of active ingredients for safe and effective dietary foods is required.
[0003]
As one means for preventing the above-mentioned melanin formation and obesity, a substance that inhibits the activity of an enzyme involved in an in vivo reaction leading to them is employed. Melanin is produced by an enzymatic reaction involving the oxidation of L-tyrosine by tyrosine, while obesity originates, for example, from the digestion of starch by α-amylase. Therefore, it is considered that the above object can be achieved by using tyrosinase inhibitors and α-amylase inhibitors that inhibit the activities of these enzymes. Specifically, as an example of the former, substances such as kojic acid and alptin are used as cosmetic additives (functional cosmetics, pp. 213 to 223, edited by Japan Cosmetic Chemistry Research Association, CMC Publishing Co., Ltd.). As an example of the latter, an extract from wheat is shown as an anti-obesity diet food (Shiraishi, Chemistry and Biology, Vol. 27, p. 491, 1989). However, the development of more effective and safe enzyme inhibitors has been awaited.
[0004]
[Means for Solving the Problems]
The present inventors have conducted intensive studies with the aim of obtaining effective and safe tyrosinase inhibitors and α-amylase inhibitors, and as a result, aromatic components were distilled off from extracts of fruits of Rosaceae plants. It was found for the first time that the tyrosinase inhibitor and α-amylase inhibitor activities were present in the residue, and the present invention was completed. That is, the present invention provides an enzyme inhibitor obtained by extracting from fruits of a Rosaceae plant.
[0005]
Specifically, the enzyme inhibitor of the present invention is an enzyme-containing syrup extracted from the fruit of a Rosaceae plant. For the purposes of the present invention, the enzyme inhibitors of the present invention include extracts from the fruits of any Rosaceae plants that contain enzyme inhibitory activity. Quince and karin are preferred as examples of such Rosaceae plants. In the fall, yellow ripe quince and quince fruits have a unique fragrance, and they have been used as antitussives by decocting dried fruit pieces from the old days. Recently, it has been known that essential oils contained therein have an effect of removing bad breath (Yoshioka et al., JP-A-63-135176). On the other hand, it has been pointed out that the viscous substance contained in quince seeds has an excellent moisturizing effect and is useful as an additive for kincare cosmetics (Komazaki et al., JP-A-59-16312). However, it was not known before the present invention that it contained enzyme-inhibiting active ingredients, especially tyrosinase inhibitors and α-amylase inhibitors.
[0006]
BEST MODE FOR CARRYING OUT THE INVENTION
In order to obtain the enzyme inhibitor of the present invention, fruits of a Rosaceae plant such as quince or quince are used as they are, or pulp obtained by removing the skin, core and seeds. All are cut into small pieces and extracted with a suitable solvent. For the purposes of the present invention, either dried fruits or fresh fruits after harvest can be used. Further, in order to improve the extraction efficiency, the material may be pulverized using a mixer or the like. Examples of the extraction solvent include alcohols such as methanol, ethanol, propyl alcohol, and isopropyl alcohol.Ethanol is preferred in consideration of extraction efficiency and safety to the human body. . In addition, a mixture thereof or a mixed solvent of these and water can also be used.
[0007]
The water content of alcohols varies depending on the water content of the material. For example, when a dried product is used, it can be extracted with about 50 to 80% of alcohol. It is preferred to extract. The ratio of the extraction solvent to the material is about 300 ml to 1000 ml for 100 g of the material. The extraction temperature and time are not particularly limited, and are usually between room temperature and the boiling temperature of the extraction solvent. The time required for extraction also varies depending on the raw material, extraction temperature, type of solvent, amount used, amount of water contained in the solvent, etc., but at room temperature, usually about 1 to 30 days, at the boiling temperature of the solvent, Sufficient to several hours, usually about one hour, is sufficient. After the solution thus extracted is roughly filtered with gauze or the like, it is further filtered using a filter paper, a glass filter, and the like, and then the solvent is distilled off under reduced pressure, so that there is no fragrance peculiar to karin or quince. As a result, the desired yellow or tan syrup as an enzyme inhibitor is obtained. Usually, about 20 to 60 g of syrup can be obtained from one quince or quince (average about 100 to 500 g). The present syrup can be commercialized into foods, cosmetics and the like as it is or by blending with lactose or sugar alcohols (for example, sorbitol). However, if necessary, it can be purified by means such as chromatography.
[0008]
When the syrup obtained from the Rosaceae fruit of the present invention is added to a food such as confectionery, a diet food is obtained by its α-amylase inhibitor activity, which is added to a cosmetic cream, an emulsion, a lotion and the like. Then, a cosmetic for whitening is obtained by the tyrosinase inhibitor activity.
[0009]
【Example】
Example 1
Immediately after harvest, one piece of karin (from Tsunan-machi, Niigata Prefecture) (360 g) is immersed in a solution consisting of 300 ml of ethanol and 100 ml of water, lightly crushed with a mixer to crush the pulp portion, and then the pulp and seed portions are removed. Extraction is carried out at room temperature for one day. Next, after filtration with gauze, the filtrate is filtered under reduced pressure through Enagrass covered with a small amount of celite, and the obtained filtrate is concentrated under reduced pressure to obtain 50 ml of a yellow solution.
[0010]
Example 2
Ten quinces (2630 g from Tsunan-machi, Niigata Prefecture) after harvest are peeled, and the fruit portion is separated from the fruit core and the seed portion. Then, a mixed solution of 3 L of methanol and 1 L of water is added and crushed with a mixer. After the pulverization, the mixture was allowed to stand at room temperature for 2 days to perform extraction, and then treated in the same manner as in Example 1 to obtain 50 ml of quince extract.
[0011]
The extracts prepared in the above Examples were tested for tyrosinase inhibitor activity and α-amylase inhibitor activity.
Test Example 1 Tyrosinase Inhibitor Activity of Karin Extract L-Tyrosine Aqueous Solution (30 mg / 200 ml) 10 ml
Buffer (pH 6.8 McIlbaine buffer) 10ml
20 ml of distilled water
0.9 ml of the kalin solution obtained in Example 1
After heating the solution having the above composition at 37 ° C. for 10 minutes, 0.1 ml of an aqueous solution of tyrosinase (1 mg / ml, derived from mushroom, Wako Pure Chemical Industries) is added, and the enzyme reaction is performed for 30 minutes. As a control, an enzyme reaction is performed using the same amount of distilled water instead of the kalin solution. The amount of dopachrome produced by tyrosinase (the substance at the previous stage when melanin is produced) was determined by measuring the absorbance at 650 nm. Based on this, the tyrosinase inhibitor activity of the kalin solution was calculated by the following formula.
Inhibitor activity = [(AB) / A] 100
A: Absorbance of control solution (0.470)
B: Absorbance of the sample containing the karin extract (0.002)
As a result of the calculation, the inhibitor activity of the kalin extract obtained in Example 1 was 99.6%. The quince extract prepared in Example 2 was also tested in the same manner as described above, and the following results were obtained.
A: 0.510; B 0.005
Tyrosinase inhibitor activity = 99.0%
[0012]
Test Example 2 α-amylase inhibitor activity of extract (1)
1 ml of water is put into each of the 9 test tubes, and 1 ml of α-amylase (0.5% aqueous solution, derived from bacteria, Wako Pure Chemical Industries) is added to the first test tube and mixed well. One ml of the mixture is placed in a second test tube and mixed, and 1 ml of the mixture is further added to a third test tube and mixed. This operation is sequentially repeated to form a dilution series up to the ninth test tube. Discard 1 ml of the ninth liquid. A test tube containing only 1 ml of the amylase solution without water was also prepared and used as the 0th tube. The above is cooled in ice water. Next, 5 ml of a 1% soluble starch solution is added to each test tube, and after all the additions have been completed, they are simultaneously placed in a constant temperature bath at 40 ° C. and subjected to an enzyme reaction for 1 hour. Next, the test tube is placed in ice water and cooled. After cooling, water is added to about 80% of the height of the test tube, and a drop of 0.1N iodine solution is added thereto, and the color (blue-violet to red) due to the iodine starch reaction is observed. The solution of 1 ml + 9 ml of the karin solution obtained in Example 1 was put into each test tube instead of 1 ml of the above-mentioned water, and the enzyme reaction was carried out in the same manner, and the coloration due to the iodine starch reaction was observed. The results were as shown in Table 1 below. In addition, ● represents iodine starch reaction positive, and ○ represents negative.
[0013]

As can be seen from the above, the α-amylase inhibitor activity was observed in the kalin extraction solution. The quince extract prepared in Example 2 was tested in the same manner as described above. Except for 0 to 2, all were positive for iodine starch reaction.
[0014]
Example 3
To 4 kg of raw karin (harvested in Tsunan-cho, Naka-Uonuma-gun, Niigata in 1994), 3 kg of the karin material obtained by removing the skin and core was added with 4.5 l of ethanol, pulverized well with a mixer, and then at room temperature for one week. put. The filtrate (about 6.5 l) is concentrated under reduced pressure and dried to obtain 283 g of a yellow-brown syrup. This syrup is heated and extracted three times with 400 ml of ethanol each time, and the extracts are combined and concentrated under reduced pressure to obtain 43 g of a yellow-brown syrup. This syrup is heated and dissolved in 100 ml of ethanol, 400 g of lactose is added and mixed well, and then dried and pulverized with warm air to obtain 440 g of a sample having a strong α-amylase inhibitor activity.
The α-amylase inhibitor activity of this sample was measured by the method shown in Test Example 3 and was as follows.

[0015]
Test Example 3 α-amylase inhibitor activity of sample (2)
Inhibition reaction of α-amylase To 1 ml of a 0.1% aqueous sample solution, add a 0.5% pancreatin (Japanese Pharmacopoeia) aqueous solution and 3 ml of purified water, mix well, and carry out an inhibition reaction at 37 ° C. for 10 minutes. The same reaction is carried out using purified water instead of the sample as a control.
1 ml of the above-mentioned inhibition reaction solution is added to 3 ml of a 1% soluble saccharification starch solution, mixed well, and then saccharification reaction is performed at 37 ° C. for 15 minutes. After completion of the reaction, 2 ml of this solution is taken, and the glucose produced is analyzed by a modified method of somogy (Food Analysis Handbook: Kenjusha).
Calculation of inhibition rate Amount of glucose produced in control plots ... Amg
The amount of glucose produced in the experimental plot: Bmg
Inhibition rate (%) = 100− [B / A] × 100
[0016]
Example 4
Preparation of whitening cream 5 ml of the extract obtained in Example 1 was taken, the solvent was distilled off under reduced pressure, and then a whitening cream was prepared according to the following formulation.
Karin extract 5ml equivalent stearyl alcohol 6.0ml
Stearic acid 1.0
Hydrogenated lanolin 5.0
Octyldodecyl alcohol 10.0
Polyoxyethylene (20 mol)
Monocetyl alcohol ether 4.0
Glycerin monostearate 2.0
Propylene glycol 5.0
Purified water 66.0
Appropriate amount of fragrance and preservative
Example 5
Adjustment of cookie for diet 300 g of shortening, 20 g of milk, 50 of sugar and 5 g of aspartame are mixed well with a whisk. 60 g of eggs are added little by little to this and mix well. Separately, 300 g of flour, 1 g of baking powder, and a solvent equivalent to 50 ml of the quince extract obtained in Example 2 are completely distilled off under reduced pressure, and kneaded well. This is mixed well with the previously mixed one, and then left in a refrigerator for 30 minutes. Next, this is molded into an appropriate mold and baked at about 170 ° C. for 20 minutes to obtain about 1,000 g of the desired cookie.
[0018]
【The invention's effect】
The extract of quince and quince of the present invention has excellent tyrosinase inhibitor and α-amylase inhibitor activity and is useful as an additive for cosmetics or diet foods.

Claims (6)

An enzyme inhibitor having α-amylase inhibitory activity or tyrosinase inhibitory activity, which is obtained by extracting from the fruit of karin or quince. A diet food comprising the enzyme inhibitor having the α-amylase inhibitor activity according to claim 1. A cosmetic having tyrosinase inhibitor activity, which is obtained by extracting from the fruit of karin or quince. A solvent such as ethanol is added to the raw karin material obtained by removing the skin and core of the raw karin, and the mixture is pulverized well by a mixer or the like, then left at room temperature for about one week to perform extraction, and then filtered through gauze or the like. The filtrate was concentrated under reduced pressure and dried to dryness. A method for producing an enzyme inhibitor having α-amylase inhibitor activity or tyrosinase inhibitor activity, comprising adding lactose, mixing well, then drying and pulverizing with warm air. After peeling the quince and separating the fruit part from the fruit core and seed part, add a solution consisting of water such as methanol and water, pulverize with a mixer, etc. and leave it at room temperature for about 2 days and night to extract. Then, after filtration through a filter medium, the filtrate is further subjected to vacuum filtration, and the obtained filtrate is concentrated under reduced pressure to obtain an enzyme inhibitor having an α-amylase inhibitor activity or a tyrosinase inhibitor activity. Method. Karin extract extract contains stearyl alcohol, stearic acid, hydrogenated lanolin, octyldodecyl alcohol, polyoxyethylene (20 mol) monocetyl, alcohol ether, glycerin monostearate, provylene glycol, purified water, fragrance, and preservatives. A method for producing a whitening cream, comprising adding one or more components.