JP4025870B2 - Tyrosinase activity inhibitor - Google Patents
Tyrosinase activity inhibitor Download PDFInfo
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- JP4025870B2 JP4025870B2 JP2003033689A JP2003033689A JP4025870B2 JP 4025870 B2 JP4025870 B2 JP 4025870B2 JP 2003033689 A JP2003033689 A JP 2003033689A JP 2003033689 A JP2003033689 A JP 2003033689A JP 4025870 B2 JP4025870 B2 JP 4025870B2
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- flavonoid
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- catechin
- gallate
- tyrosinase activity
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Description
【0001】
【発明の属する技術分野】
本発明は、フラボノイドとアルデヒド類の重縮合体からなることを特徴とするチロシナーゼ活性阻害剤に関する。この阻害剤は、化粧品及び食品の分野で好適に利用される。
【0002】
【従来の技術】
【特許文献1】
特開平6−65085
【特許文献2】
特開平6−40883
従来より、チロシンやドーパなどの酸化を触媒するチロシナーゼの活性を阻害する化合物は、メラニン生成抑制作用を有することを利用して化粧品における美白剤として、また、食品の黒変防止剤として使用されている。このようなチロシナーゼ活性阻害剤として、アスコロビン酸類、グルタチオン、コウジ酸、アルブチン等が知られている。
【0003】
【発明が解決しようとする課題】
しかし、上記従来のチロシナーゼ活性阻害剤は、経時安定性が低い、細胞毒性が強い、水分存在下の酸化により阻害能力が低下する等の欠点があった。
そのため、本発明の課題は、チロシナーゼ活性阻害作用を有するうえに、従来のチロシナーゼ活性阻害剤の欠点を克服し、更にフラボノイドの持つ種々の機能をも有するチロシナーゼ活性阻害剤を提供することにある。
【0004】
【課題を解決するための手段】
その課題を解決するために、本発明のチロシナーゼ活性阻害剤は、
合成されたフラボノイド−アルデヒド重縮合体であって、フラボノイドがカテキン、エピカテキン、ガロカテキン、エピガロカテキン、カテキンガレート、エピカテキンガレート、ガロカテキンガレート及びエピガロカテキンガレートのうちから選ばれる1種以上であり、フラボノイドの6位と8位とで結合し、数平均分子量が500から100,000の範囲であるものを有効成分として含有することを特徴とする。
従来より、緑茶ポリフェノールの主成分であるカテキン、エピカテキン、エピガロカテキン、エピガロカテキンガレート等のフラボノイド類に抗酸化性や殺菌作用があることが知られている。さらに抗ガン作用、抗炎症作用、紫外線吸収作用、毛細血管の強化、血圧上昇抑制、血圧降下作用、記憶力向上、肝機能向上、脂肪吸収抑制、ストレス抑制、女性ホルモンバランス調整、抗腫瘍作用、突然変異抑制、血中コレステロール抑制、整腸作用等の効果も知られており、食品、化粧品素材や生医学分野への応用が期待されている。
【0005】
一方、カテキン類にはチロシナーゼ活性阻害はなく、むしろ、チロシンの酸化を促進する作用があり、チロシナーゼ活性阻害作用は見られない。
これに対して、フラボノイドとアルデヒドを重縮合させて高分子化したものは、一転してチロシナーゼ活性阻害作用を発揮する。特に前記フラボノイドがフェノール性水酸基を3つ以上含むものであるとき、その作用が顕著である。そして、重縮合反応によりアルデヒドのホルミル基は除かれるが、フラボノイドの骨格及び官能基は全て残っていることから、フラボノイドの上記各作用もそのまま維持される。
前記重縮合体は、典型的には下記一般式(1)で表される。
【0006】
【化2】
(式中、nは2以上の整数、Rはアルデヒドからホルミル基を除いた残基を示す。)
【0007】
【発明の実施の形態】
本発明のチロシナーゼ活性阻害剤を製造する際の重縮合反応で使用するフラボノイドの具体例としては、カテキン、エピカテキン、ガロカテキン、エピガロカテキン、カテキンガレート、エピカテキンガレート、ガロカテキンガレート、及びエピガロカテキンガレートが挙げられる。これらは単独あるいは混合物として使用される。
本発明で使用するアルデヒドは、フラボノイドと反応するものであれば、特に制限されるものではない。具体例としては、ホルマリン、アセトアルデヒド、プロピオンアルデヒド、ブチルアルデヒド、カプロンアルデヒド、ピルビンアルデヒド、グリオキシル酸、ベンズアルデヒド、2−メトキシベンズアルデヒド、サリチルアルデヒド、4−ヒドロキシベンズアルデヒド、3−シアノベンズアルデヒド、p−ジフェニルアルデヒド、クロトンアルデヒド等が挙げられる。これらは単独あるいは混合物として使用され、フラボノイド1gに対して、0.1〜1000グラムの範囲で用いることが好ましい。
【0008】
フラボノイドとアルデヒドの重縮合反応においては、反応を効率よく進めるために酸触媒を加えても良い。用いる酸触媒は特に制限はなく、公知のものが使用できる。具体例として酢酸、トリフルオロ酢酸、塩酸、硫酸、硝酸、トリフルオロボレート、パラトルエンスルホン酸、メタンスルホン酸等が挙げられ、フラボノイド1gに対して、0.01mg〜100gの範囲で用いることが好ましい。フラボノイドとアルデヒドの重縮合反応に用いる溶媒としては、フラボノイドとアルデヒドと触媒が共に溶解するものが好ましい。具体例としてメタノール、エタノール、N,N−ジメチルホルムアミド、N,N−ジメチルアセトアミド、ジメチルスルホキシド、N−メチルピロリドン、ニトロメタン、ニトロベンゼン、ピリジン、1,4−ジオキサン、アセトン、メチルエチルケトン、テトラヒドロフラン、ジエチルエーテル、トルエン、ベンゼン、蒸留水、脱イオン水、緩衝液等が挙げられる。緩衝液を用いる場合はpHが2〜12の範囲が望ましい。緩衝液の種類としては、酢酸緩衝液、リン酸緩衝液、炭酸緩衝液等が望ましいが、これらに限定されるものではない。これらは単独あるいは混合物として使用され、任意の量を用いることができる。
重合温度は、反応が効率良く進行する温度が望ましい。好ましくは−20〜100℃の範囲であり、より好ましくは0〜60℃の範囲である。
上記重合反応によって得られる重縮合体の数平均分子量は500から100,000の範囲である。
【0009】
【実施例】
以下に、本発明を試験例により詳細に説明するが、本発明はこれらに限定されるものではない。
−実施例1−
100ミリリットルナスフラスコに、0.37グラム(1.3mmol)のカテキンを取り、0.5ミリリットルの酢酸、3ミリリットルのエタノール及び22ミリリットルの蒸留水の混合液に溶解させた。カテキンの溶解後、2.04ミリリットルの90%アセトアルデヒド溶液(32.5mmol)を加え、室温で24時間攪拌した。反応終了後、溶媒を減圧下で留去し、残査を真空乾燥することにより、収率93%で重縮合体(以下、ポリマーA)を得た。ポリマーAの分子量は水酸基を常法によりアセチル化した後に、クロロホルムを溶離液に用いてGPCにより測定した。その結果、数平均分子量が3700、分子量分布が1.8であった。また、ポリマーの構造解析をNMRを用いて行ったところ、カテキンの6位と8位で結合したカテキン−アセトアルデヒド重縮合体であることを確認した。1H NMR (DMSO-d6, ppm) 1.1-1.9 (br, CH3), 2.3-3.2 (br, H-4), 2.8-3.2 (br, H-3), 4.0-4.6 (br, H-2, CH), 6.5-7.2 (mH-2', H-5', H-6')13C NMR (DMSO-d6, ppm) 18-22 (m, CH3), 26-31 (m, C-4), 41 (s, CH), 69-71 (m, C-3), 85-87 (m, C-2), 105-107 (m, C-4a), 111-113, 117-118 (m, C-6, C-8), 118-119 (m, C-2', C-5'), 121-123 (m, C-6'), 131-134 (m, C-1'), 147-148 (m, C-3', C-4'), 152-157 (m, C-5, C-7, C8a)
【0010】
−実施例2−
実施例1におけるアセトアルデヒド溶液に代えてグリオキシル酸(32.5mmol)を用いたことと、酢酸を用いなかったこと以外は、実施例1と同一条件で原料モノマーを配合し、反応させることにより重縮合体(以下、ポリマーB)を合成した。ポリマーBの収率、数平均分子量、分子量分布の結果を実施例1の結果と併せて表1に示す。
【0011】
−実施例3−
実施例1におけるアセトアルデヒド溶液に代えてピルビンアルデヒド(32.5mmol)を用いたことと、0.5ミリリットルの酢酸を加えることに代えて1Mの塩酸を用いて混合液をpH=2に調整したこと以外は、実施例1と同一条件で原料モノマーを配合し、反応させることにより重縮合体(以下、ポリマーC)を合成した。ポリマーCの収率、数平均分子量、分子量分布の結果を実施例1の結果と併せて表1に示す。
【0012】
−実施例4−
実施例1におけるアセトアルデヒド溶液に代えてサリチルアルデヒド(32.5mmol)を用いたことと、0.5ミリリットルの酢酸を加えることに代えて1Mの塩酸を用いて混合液をpH=2に調整したこと以外は、実施例1と同一条件で原料モノマーを配合し、反応させることにより重縮合体(以下、ポリマーD)を合成した。ポリマーDの収率、数平均分子量、分子量分布の結果を実施例1の結果と併せて表1に示す。
【0013】
【表1】
【0014】
[試験例1](モノフェノラーゼ作用の阻害能力の評価)
チロシナーゼの触媒作用としてチロシンをDOPAへ酸化するモノフェノラーゼ作用とDOPAを酸化するジフェノラーゼ作用が知られている。本例ではモノフェノラーゼ作用の阻害能力を評価するため、チロシナーゼによるL-チロシンの酸化における475nmの吸光度の経時変化を測定した。測定は、L-チロシン(2mM)、チロシナーゼ(100ユニット、マッシュルーム由来、SIGMA製)、及び阻害剤(ポリマーA〜D)をpH=6.8の0.1Mリン酸緩衝液と蒸留水との混合液(5:4重量比)に溶解させ、25℃で行った。
阻害剤におけるポリマー濃度の阻害能力への影響を調べた結果を図1に示す。尚、ポリマー濃度はカテキンユニット当たりで示したものである。これより阻害剤を加えない対照系と比較して、チロシンの酸化を阻害していることがわかる。また、ポリマー濃度が高いほどチロシンの酸化が抑制された。図2にポリマーA〜D及びカテキンのモノフェノラーゼ阻害能力を示す(ポリマー濃度40μM)。これより、カテキンでは吸光度の増加が対照系より高く、チロシンの酸化を促進していることがわかる。一方、ポリマーA〜Dではモノフェノラーゼ阻害が見られ、用いるアルデヒド種により阻害能力が異なり、アセトアルデヒドを用いて合成したポリマーAが最も阻害能力に優れていることがわかった。
【0015】
[試験例2](ジフェノラーゼ作用の阻害能力の評価)
本例ではジフェノラーゼ作用の阻害能力を評価するため、チロシナーゼによるDOPAの酸化における475nmの吸光度変化を測定した。測定は、チロシナーゼ(100ユニット、マッシュルーム由来、SIGMA製)と阻害剤(ポリマーA〜D:100μM)をpH=6.8の0.1Mリン酸緩衝液と蒸留水との混合液(5:4重量比)に溶解させ、25℃で10分間インキュベートし、その後にL-DOPA(1mM)を加えて更に10分間インキュベートさせて行った。阻害率(%)は以下の式から計算した。尚、対照実験は、阻害剤を混合液に添加しない以外は上記と同一条件とした。また、比較のために上記阻害剤に代えてルチン(100μM)又はアルブチン(100μM)を上記混合液に溶解させた以外は上記と同一条件で測定した。
【0016】
【数1】
P:対照実験における吸光度
Q:熱処理により活性を無くしたチロシナーゼを用いた場合の吸光度
R:阻害剤を加えた場合の吸光度
【0017】
評価結果を図3に示す。これより、重縮合体ではジフェノラーゼ阻害能力が認められるとともに、用いるアルデヒド種により阻害能力が異なり、アセトアルデヒドを用いて合成したポリマーAが最も阻害能力に優れていることがわかった。一方、カテキンではDOPAの酸化を促進しており、比較のために評価したルチン、アルブチンといった低分子のフェノール類はジフェノラーゼ活性が見られたもののカテキン−アルデヒド重縮合体より阻害能力が低いことがわかった。
【0018】
[試験例3](経時安定性)
ポリマーAを空気下、室温で半年以上放置後にNMRを測定したところ、合成直後と全く同一であり、着色も見られなかった。試験例1と同様にモノフェノラーゼ活性に対する阻害を評価したところ、合成直後(図2における20分後のポリマーA)と比べて20分後の吸光度の増加率が3%以下であった。また、試験例2と同様にジフェノラーゼ活性に対する阻害を評価したところ、阻害率の低下は5%以下であった。
[試験例4](細胞毒性)
200μMのポリマーA溶液を牛大動脈由来の血管内皮細胞に播種し、37℃でインキュベートした。24時間後のサンプルの顕微鏡観察を行ったところ、ほぼ全ての細胞が生存していたことから、血管内皮細胞に対するポリマーAの低い細胞毒性が確認された。
[試験例5](水中での耐酸化性)
ポリマーAを室温、蒸留水中で攪拌し、ろ過により回収した。回収物のNMRを測定したところ、合成直後と全く同一であり、着色も見られなかった。試験例1と同様にモノフェノラーゼ活性に対する阻害を評価したところ、合成直後(図2における20分後のポリマーA)と比べて20分後の吸光度の増加率が1%以下であった。また、試験例2と同様にジフェノラーゼ活性に対する阻害を評価したところ、阻害率の低下は3%以下であった。
【0019】
【発明の効果】
本発明によると、フラボノイドをアルデヒド類との重縮合することにより、チロシナーゼ活性阻害作用を有するポリマーを提供することができる。これらのフラボノイド重合物はこの酵素阻害作用以外にもフラボノイドに起因する種々の機能を有しており、香粧品用及び食品用素材として有望である。
【図面の簡単な説明】
【図1】実施例1のポリマー濃度とモノフェノラーゼ活性阻害作用との関係を示すグラフである。
【図2】実施例1〜4のポリマーのモノフェノラーゼ活性阻害作用を示すグラフである。
【図3】実施例1〜4のポリマーのジフェノラーゼ活性阻害作用を示すグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a tyrosinase activity inhibitor comprising a polycondensate of a flavonoid and an aldehyde. This inhibitor is suitably used in the cosmetics and food fields.
[0002]
[Prior art]
[Patent Document 1]
JP-A-6-65085
[Patent Document 2]
JP-A-6-40883
Conventionally, compounds that inhibit the activity of tyrosinase that catalyzes the oxidation of tyrosine and dopa have been used as a whitening agent in cosmetics and as an anti-blackening agent in foods because of their ability to suppress melanin production. Yes. As such tyrosinase activity inhibitors, ascorbic acids, glutathione, kojic acid, arbutin and the like are known.
[0003]
[Problems to be solved by the invention]
However, the conventional tyrosinase activity inhibitors described above have drawbacks such as low stability over time, strong cytotoxicity, and reduced inhibition ability due to oxidation in the presence of moisture.
Therefore, an object of the present invention is to provide a tyrosinase activity inhibitor having a tyrosinase activity inhibitory action, overcoming the drawbacks of conventional tyrosinase activity inhibitors, and further having various functions of flavonoids.
[0004]
[Means for Solving the Problems]
In order to solve the problem, the tyrosinase activity inhibitor of the present invention comprises:
A flavonoid-aldehyde polycondensate synthesized, wherein the flavonoid is one or more selected from catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate and epigallocatechin gallate It is characterized by containing as an active ingredient a flavonoid bonded at the 6th and 8th positions and having a number average molecular weight in the range of 500 to 100,000 .
Conventionally, flavonoids such as catechin, epicatechin, epigallocatechin, and epigallocatechin gallate, which are the main components of green tea polyphenols, are known to have antioxidant and bactericidal effects. Furthermore, anti-cancer action, anti-inflammatory action, ultraviolet absorption action, strengthening of capillaries, suppression of blood pressure increase, blood pressure reduction action, improvement of memory ability, liver function improvement, fat absorption suppression, stress suppression, female hormone balance adjustment, anti-tumor action, suddenly Effects such as mutation suppression, blood cholesterol suppression and intestinal regulation are also known, and application to food, cosmetic materials and biomedical fields is expected.
[0005]
On the other hand, catechins have no tyrosinase activity inhibition, but rather have an effect of promoting tyrosine oxidation, and no tyrosinase activity inhibition effect is observed.
In contrast, a polymer obtained by polycondensation of a flavonoid and an aldehyde turns around and exhibits a tyrosinase activity inhibitory action. In particular, when the flavonoid contains three or more phenolic hydroxyl groups, the action is remarkable. Then, although the formyl group of the aldehyde is removed by the polycondensation reaction, since all the flavonoid skeleton and functional group remain, the above-mentioned actions of the flavonoid are maintained as they are.
The polycondensate is typically represented by the following general formula (1).
[0006]
[Chemical 2]
(In the formula, n represents an integer of 2 or more, and R represents a residue obtained by removing a formyl group from an aldehyde.)
[0007]
DETAILED DESCRIPTION OF THE INVENTION
Specific examples of flavonoids used in the polycondensation reaction in producing the tyrosinase activity inhibitor of the present invention include catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, and epigallo Catechin gallate is mentioned . These are used alone or as a mixture.
The aldehyde used in the present invention is not particularly limited as long as it reacts with flavonoids. Specific examples include formalin, acetaldehyde, propionaldehyde, butyraldehyde, capronaldehyde, pyruvinaldehyde, glyoxylic acid, benzaldehyde, 2-methoxybenzaldehyde, salicylaldehyde, 4-hydroxybenzaldehyde, 3-cyanobenzaldehyde, p-diphenylaldehyde, croton. Examples include aldehydes. These are used alone or as a mixture, and are preferably used in the range of 0.1 to 1000 grams per 1 g of flavonoid.
[0008]
In the polycondensation reaction between the flavonoid and the aldehyde, an acid catalyst may be added in order to advance the reaction efficiently. There is no restriction | limiting in particular in the acid catalyst to be used, A well-known thing can be used. Specific examples include acetic acid, trifluoroacetic acid, hydrochloric acid, sulfuric acid, nitric acid, trifluoroborate, p-toluenesulfonic acid, methanesulfonic acid, and the like. It is preferably used in a range of 0.01 mg to 100 g with respect to 1 g of flavonoid. . As the solvent used for the polycondensation reaction between the flavonoid and the aldehyde, a solvent in which both the flavonoid, the aldehyde and the catalyst are dissolved is preferable. Specific examples include methanol, ethanol, N, N-dimethylformamide, N, N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone, nitromethane, nitrobenzene, pyridine, 1,4-dioxane, acetone, methyl ethyl ketone, tetrahydrofuran, diethyl ether, Examples include toluene, benzene, distilled water, deionized water, and buffer solution. When a buffer solution is used, the pH is preferably in the range of 2-12. As a kind of buffer solution, an acetate buffer solution, a phosphate buffer solution, a carbonate buffer solution and the like are desirable, but not limited thereto. These are used alone or as a mixture, and any amount can be used.
The polymerization temperature is preferably a temperature at which the reaction proceeds efficiently. Preferably it is the range of -20-100 degreeC, More preferably, it is the range of 0-60 degreeC.
The number average molecular weight of the polycondensate obtained by the polymerization reaction is in the range of 500 to 100,000.
[0009]
【Example】
Hereinafter, the present invention will be described in detail by test examples, but the present invention is not limited thereto.
Example 1
In a 100 ml eggplant flask, 0.37 g (1.3 mmol) of catechin was taken and dissolved in a mixture of 0.5 ml acetic acid, 3 ml ethanol and 22 ml distilled water. After dissolution of catechin, 2.04 ml of 90% acetaldehyde solution (32.5 mmol) was added and stirred at room temperature for 24 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, and the residue was vacuum-dried to obtain a polycondensate (hereinafter, polymer A) with a yield of 93%. The molecular weight of the polymer A was measured by GPC using chloroform as an eluent after acetylating the hydroxyl group by a conventional method. As a result, the number average molecular weight was 3700, and the molecular weight distribution was 1.8. Moreover, when the structural analysis of the polymer was performed using NMR, it confirmed that it was a catechin-acetaldehyde polycondensate couple | bonded by 6th-position and 8th-position of catechin. 1 H NMR (DMSO-d 6 , ppm) 1.1-1.9 (br, CH 3 ), 2.3-3.2 (br, H-4), 2.8-3.2 (br, H-3), 4.0-4.6 (br, H -2, CH), 6.5-7.2 (mH-2 ', H-5', H-6 ') 13 C NMR (DMSO-d 6 , ppm) 18-22 (m, CH 3 ), 26-31 ( m, C-4), 41 (s, CH), 69-71 (m, C-3), 85-87 (m, C-2), 105-107 (m, C-4a), 111-113 , 117-118 (m, C-6, C-8), 118-119 (m, C-2 ', C-5'), 121-123 (m, C-6 '), 131-134 (m , C-1 '), 147-148 (m, C-3', C-4 '), 152-157 (m, C-5, C-7, C8a)
[0010]
-Example 2-
Polycondensation by mixing and reacting raw material monomers under the same conditions as in Example 1 except that glyoxylic acid (32.5 mmol) was used in place of the acetaldehyde solution in Example 1 and acetic acid was not used. The body (hereinafter referred to as polymer B) was synthesized. The results of the yield, number average molecular weight, and molecular weight distribution of polymer B are shown in Table 1 together with the results of Example 1.
[0011]
-Example 3-
Using pyruvaldehyde (32.5 mmol) instead of the acetaldehyde solution in Example 1 and adjusting the mixture to pH = 2 using 1 M hydrochloric acid instead of adding 0.5 ml acetic acid. Except for the above, a polycondensate (hereinafter referred to as polymer C) was synthesized by blending and reacting raw material monomers under the same conditions as in Example 1. The results of the yield of polymer C, number average molecular weight, and molecular weight distribution are shown in Table 1 together with the results of Example 1.
[0012]
Example 4
The salicylaldehyde (32.5 mmol) was used in place of the acetaldehyde solution in Example 1, and the mixture was adjusted to pH = 2 using 1M hydrochloric acid instead of adding 0.5 ml of acetic acid. Except for the above, a raw material monomer was blended and reacted under the same conditions as in Example 1 to synthesize a polycondensate (hereinafter, polymer D). The results of the yield, number average molecular weight, and molecular weight distribution of polymer D are shown in Table 1 together with the results of Example 1.
[0013]
[Table 1]
[0014]
[Test Example 1] (Evaluation of ability to inhibit monophenolase action)
As a catalytic action of tyrosinase, a monophenolase action for oxidizing tyrosine to DOPA and a diphenolase action for oxidizing DOPA are known. In this example, in order to evaluate the ability to inhibit the monophenolase action, the change with time in the absorbance at 475 nm in the oxidation of L-tyrosine by tyrosinase was measured. The measurement was performed using L-tyrosine (2 mM), tyrosinase (100 units, derived from mushrooms, manufactured by SIGMA), and an inhibitor (polymers A to D) of 0.1 M phosphate buffer having pH = 6.8 and distilled water. It was dissolved in a mixed solution (5: 4 weight ratio) and carried out at 25 ° C.
The results of examining the influence of the polymer concentration on the inhibitory ability of the inhibitor are shown in FIG. The polymer concentration is indicated per catechin unit. This shows that tyrosine oxidation is inhibited as compared with the control system in which no inhibitor was added. Moreover, the higher the polymer concentration, the more tyrosine oxidation was suppressed. FIG. 2 shows the monophenolase inhibitory ability of polymers A to D and catechin (polymer concentration 40 μM). This shows that the increase in absorbance of catechin is higher than that of the control system and promotes the oxidation of tyrosine. On the other hand, in the polymers A to D, monophenolase inhibition was observed, and the inhibition ability was different depending on the aldehyde species used, and it was found that the polymer A synthesized using acetaldehyde was most excellent in inhibition ability.
[0015]
[Test Example 2] (Evaluation of ability to inhibit diphenolase action)
In this example, in order to evaluate the inhibitory ability of the diphenolase action, the change in absorbance at 475 nm in the oxidation of DOPA by tyrosinase was measured. The measurement was performed by mixing a tyrosinase (100 units, derived from mushrooms, manufactured by SIGMA) and an inhibitor (polymers A to D: 100 μM) of a 0.1M phosphate buffer solution having pH = 6.8 and distilled water (5: 4). (Weight ratio) and incubated at 25 ° C. for 10 minutes, followed by addition of L-DOPA (1 mM) and further incubation for 10 minutes. The inhibition rate (%) was calculated from the following formula. The control experiment was performed under the same conditions as above except that the inhibitor was not added to the mixed solution. For comparison, measurement was performed under the same conditions as above except that rutin (100 μM) or arbutin (100 μM) was dissolved in the mixed solution instead of the inhibitor.
[0016]
[Expression 1]
P: Absorbance in the control experiment Q: Absorbance when using tyrosinase whose activity has been eliminated by heat treatment R: Absorbance when adding an inhibitor
The evaluation results are shown in FIG. From this, it was found that the polycondensate has the ability to inhibit diphenolase, and the inhibitory ability varies depending on the aldehyde species used, and the polymer A synthesized using acetaldehyde has the highest inhibitory ability. On the other hand, catechin promotes the oxidation of DOPA, and low molecular weight phenols such as rutin and arbutin evaluated for comparison are found to have lower inhibitory ability than catechin-aldehyde polycondensates although diphenolase activity was observed. It was.
[0018]
[Test Example 3] (Stability with time)
When NMR was measured after polymer A was allowed to stand at room temperature for half a year or more in air, it was exactly the same as that immediately after synthesis, and no coloring was observed. When the inhibition of monophenolase activity was evaluated in the same manner as in Test Example 1, the rate of increase in absorbance after 20 minutes was 3% or less compared to immediately after synthesis (polymer A after 20 minutes in FIG. 2). Moreover, when the inhibition with respect to diphenolase activity was evaluated similarly to Test Example 2, the fall of the inhibition rate was 5% or less.
[Test Example 4] (Cytotoxicity)
A 200 μM polymer A solution was seeded on bovine aorta-derived vascular endothelial cells and incubated at 37 ° C. Microscopic observation of the sample after 24 hours revealed that almost all cells were alive, so that low cytotoxicity of polymer A against vascular endothelial cells was confirmed.
[Test Example 5] (Oxidation resistance in water)
Polymer A was stirred at room temperature in distilled water and collected by filtration. When NMR of the recovered product was measured, it was exactly the same as that immediately after the synthesis, and no coloring was observed. When the inhibition to monophenolase activity was evaluated in the same manner as in Test Example 1, the rate of increase in absorbance after 20 minutes was 1% or less compared to immediately after synthesis (polymer A after 20 minutes in FIG. 2). Moreover, when the inhibition with respect to the diphenolase activity was evaluated similarly to Test Example 2, the reduction in the inhibition rate was 3% or less.
[0019]
【The invention's effect】
According to the present invention, a polymer having an inhibitory action on tyrosinase activity can be provided by polycondensation of flavonoids with aldehydes. These flavonoid polymers have various functions attributable to flavonoids in addition to the enzyme inhibitory action, and are promising as materials for cosmetics and foods.
[Brief description of the drawings]
FIG. 1 is a graph showing the relationship between the polymer concentration of Example 1 and the monophenolase activity inhibitory action.
FIG. 2 is a graph showing the monophenolase activity inhibitory action of the polymers of Examples 1 to 4.
FIG. 3 is a graph showing the diphenolase activity inhibitory action of the polymers of Examples 1 to 4.
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