JP4804805B2 - Cell activator, UV damage relieving agent and melanin production inhibitor - Google Patents
Cell activator, UV damage relieving agent and melanin production inhibitor Download PDFInfo
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Description
本発明は、細胞賦活剤、紫外線障害緩和剤及びメラニン産生抑制剤に関する。さらに詳しくは、本発明は、天然物に由来して安全性が高く、皮膚の老化防止に有用な細胞賦活剤、紫外線障害緩和剤及びメラニン産生抑制剤に関する。 The present invention relates to a cell activator, a UV damage relieving agent, and a melanin production inhibitor. More specifically, the present invention relates to a cell activator, a UV damage relieving agent, and a melanin production inhibitor that are derived from natural products and have high safety and are useful for preventing skin aging.
皮膚真皮の線維芽細胞の機能低下、紫外線によるメラニンの産生などにより、皮膚の弾性の低下、しわ、しみの発生などの皮膚の老化症状が現れる。このような皮膚の老化症状を防止し、改善するために、ビタミン類、コラーゲン、合成高分子などを配合した化粧料が用いられているが、十分な効果は得られていない。このために、効果的に皮膚の老化を防止することができる成分の探索が行われ、提案されている。
例えば、真皮線維芽細胞の代謝活性を向上させ、紫外線などの外来ストレスによる皮膚の障害や老化を有効に防止あるいは改善する作用に優れる老化防止用皮膚外用剤として、ハナビラタケ子実体から極性溶媒により抽出された抽出物を含有する老化防止用皮膚外用剤が提案されている（特許文献１）。しかし、ハナビラタケは、針葉樹の切り株や樹皮の割れ目に生じるキノコであって、大量に採取することは容易ではない。また、皮膚の障壁機能を増強させる化粧品用組成物として、ココア豆の殻の水性−アルコール性抽出物からなるβ−エンドルフィン模倣性の植物抽出物の使用が提案され、ココア豆の殻の水／ブチレングリコール抽出物が例示されている（特許文献２）。しかし、カカオ殻の水性−アルコール性抽出物は、細胞賦活剤として十分な効果を有しない。
For example, as a skin external preparation for anti-aging that improves the metabolic activity of dermal fibroblasts and effectively prevents or ameliorates skin damage and aging caused by external stress such as ultraviolet rays, it is extracted from the fruit body of Hanabiratake with polar solvents An anti-aging skin external preparation containing an extracted extract has been proposed (Patent Document 1). However, Hanabiratake is a mushroom that arises in coniferous stumps and bark cracks, and it is not easy to collect large quantities. In addition, as a cosmetic composition for enhancing the skin barrier function, the use of a β-endorphin-mimetic plant extract composed of an aqueous-alcoholic extract of cocoa bean husk has been proposed, and cocoa bean husk water / A butylene glycol extract is exemplified (Patent Document 2). However, the aqueous-alcoholic extract of cocoa shell does not have a sufficient effect as a cell activator.
本発明は、天然物に由来して安全性が高く、皮膚の老化防止に有用な細胞賦活剤、紫外線障害緩和剤及びメラニン産生抑制剤を提供することを目的としてなされたものである。 The present invention has been made for the purpose of providing a cell activator, a UV damage relieving agent, and a melanin production inhibitor that are derived from natural products and are highly safe and useful for preventing skin aging.
本発明者は、カカオ脂が多量のオレイン酸を含みながらカカオ豆中に安定に存在することから、カカオ殻中には抗酸化作用を有する成分が含まれると推定し、上記の課題を解決すべく鋭意研究を重ねた結果、カカオ殻のアルコール抽出物はほとんど薬効を有しないが、カカオ殻の水抽出物は優れた細胞賦活作用、紫外線障害緩和作用、メラニン産生抑制作用を有することを見いだし、この知見に基づいて本発明を完成するに至った。
すなわち、本発明は、
（１）カカオ殻の水抽出物を含有することを特徴とする細胞賦活剤、
（２）カカオ殻の水抽出物を含有することを特徴とする紫外線障害緩和剤、及び、
（３）カカオ殻の水抽出物を含有することを特徴とするメラニン産生抑制剤、
を提供するものである。
The present inventor presumed that cacao butter contains a large amount of oleic acid and stably exists in cacao beans, so that cacao shell contains an antioxidant component and solves the above problem. As a result of intensive research, we found that the alcohol extract of cocoa shell has little medicinal effect, but the water extract of cocoa shell has an excellent cell activation effect, UV damage mitigation effect, melanin production suppression effect, The present invention has been completed based on this finding.
That is, the present invention
(1) A cell activator comprising an aqueous extract of cocoa shells,
(2) A UV damage mitigating agent characterized by containing an aqueous extract of cocoa shells, and
(3) A melanin production inhibitor characterized by containing an aqueous extract of cocoa shells,
Is to provide.
本発明の薬剤は、カカオ殻の水抽出物を含有し、優れた細胞賦活作用、紫外線障害緩和作用、メラニン産生抑制作用を有する。本発明の薬剤が有効成分として含有するカカオ殻の水抽出物は、天然物に由来するので、安全性が極めて高い。カカオ殻は、チョコレート菓子などの原料として用いられるカカオ豆の加工工程において、副生物として発生するので、容易に大量かつ経済的に入手することができる。 The chemical | medical agent of this invention contains the water extract of a cocoa shell, and has the outstanding cell activation effect | action, ultraviolet-ray damage mitigation effect | action, and melanin production suppression effect. The water extract of cocoa shells contained in the drug of the present invention as an active ingredient is derived from a natural product, and thus has extremely high safety. Since cocoa shells are generated as a by-product in the process of cocoa beans used as a raw material for chocolate confectionery and the like, they can be easily obtained in large quantities and economically.
本発明の細胞賦活剤、紫外線障害緩和剤及びメラニン産生抑制剤は、カカオ殻の水抽出物を含有する。本発明の薬剤は、優れた細胞賦活作用、優れた紫外線障害緩和作用及び良好なメラニン産生抑制効果を有するので、老化防止用皮膚外用剤、美白化粧料などとして好適に用いることができる。
本発明に用いるカカオ殻は、ココア、チョコレート菓子などの原料を採取するために栽培されるアオギリ科の植物であるテオブロマ・カカオ（Ｔｈｅｏｂｒｏｍａ ｃａｃａｏ）の種子の殻である。テオブロマ・カカオは、ガーナ、ブラジル、トリニダード、スリ・ランカなどで大量に栽培され、その果実は食品の原料として安全衛生的に厳重な管理の下に処理されるので、カカオ殻は薬剤の原料として安全な状態で大量かつ経済的に入手することができる。
The cell activator, ultraviolet damage mitigating agent, and melanin production inhibitor of the present invention contain an aqueous extract of cocoa shells. Since the agent of the present invention has an excellent cell activation effect, an excellent UV damage mitigating action and a good melanin production inhibitory effect, it can be suitably used as an anti-aging skin external preparation, a whitening cosmetic, and the like.
The cocoa shell used in the present invention is a seed shell of Theobroma cacao, which is a plant of the family Aogiriaceae cultivated for collecting raw materials such as cocoa and chocolate confectionery. Theobroma cacao is cultivated in large quantities in Ghana, Brazil, Trinidad, Sri Lanka, etc., and its fruits are processed under strict safety and hygiene as food ingredients. It can be obtained in large quantities and economically in a safe state.
本発明の薬剤に用いるカカオ殻の抽出物は、水を溶媒とする抽出物である。カカオ殻をエタノールなどのアルコールを溶媒として抽出しても、抽出物が得られる。しかし、カカオ殻の水抽出物が優れた細胞賦活作用と紫外線障害緩和作用を有するのに対して、カカオ殻のアルコール抽出物は、細胞賦活作用も紫外線障害緩和作用も全く有しない。さらに、カカオ殻の水抽出物がメラニン産生抑制作用を有するのに対して、カカオ殻のアルコール抽出物は逆にメラニン産生促進作用を有する。カカオ殻の水抽出物とアルコール抽出物は、カカオ殻からの抽出量が同程度であっても、全く異なる作用を有する成分が含有されると推定される。カカオ殻の水抽出物は、細胞賦活作用、紫外線障害緩和作用及びメラニン産生抑制作用以外の他の薬効をも有することが期待される。
本発明に用いるカカオ殻の水抽出物は、カカオ殻を水で処理して、カカオ殻中に含まれる水に可溶性の成分を、水に不溶性又は難溶性の成分と分離することにより得ることができる。カカオ殻の水抽出に用いる装置に特に制限はなく、例えば、ソックスレー抽出器、バッテリー抽出機、ボールマン抽出機、ロトセル抽出機、ルルギ抽出機、ケネディー抽出機などの浸透貫流型固液抽出装置、パチューカタンク、ボノトー抽出機、ヒルデブランド抽出機などの固体分散型固液抽出装置、千代田式Ｌ型抽出機、ミアグ抽出機などの貫流−浸漬併用型固液抽出装置などを挙げることができる。
The cocoa shell extract used in the drug of the present invention is an extract using water as a solvent. An extract can be obtained by extracting cocoa shells with alcohol such as ethanol as a solvent. However, the cocoa shell water extract has an excellent cell activation effect and UV damage mitigating action, whereas the cocoa shell alcohol extract has neither cell activation action nor UV damage mitigating action. Furthermore, the water extract of cocoa shell has a melanin production inhibitory action, whereas the alcohol extract of cocoa shell has a melanin production promoting action. It is estimated that the water extract of cocoa shell and the alcohol extract contain components having completely different actions even if the extraction amount from the cocoa shell is the same. The water extract of cocoa shell is expected to have a medicinal effect other than the cell activation effect, the UV damage mitigation effect, and the melanin production suppression effect.
The water extract of cocoa shells used in the present invention can be obtained by treating the cocoa shells with water and separating the water-soluble components contained in the cocoa shells from the water-insoluble or sparingly soluble components. it can. There is no particular limitation on the apparatus used for water extraction of cocoa shell, for example, a soxhlet extractor, battery extractor, ball man extractor, Rotocell extractor, Lurgi extractor, Kennedy extractor, etc. Examples thereof include solid-dispersed solid-liquid extraction devices such as Pachuca tanks, Bonoto extractors and Hildebrand extractors, and cross-flow / immersion combined solid-liquid extractors such as Chiyoda L-type extractors and Miagu extractors.
本発明において、カカオ殻の水抽出温度に特に制限はなく、例えば、５〜１００℃の任意の温度で抽出することができ、あるいは、加圧下に１００℃を超える温度で抽出することもできる。カカオ殻から取得する水抽出物の量は、カカオ殻の３〜１１重量％であることが好ましく、５〜９重量％であることがより好ましい。水抽出物の量がカカオ殻の３重量％未満であると、カカオ殻が含有する有効成分を十分に利用し得ないおそれがある。水抽出物の量がカカオ殻の１１重量％を超えると、抽出が困難になるのみならず、薬効の乏しい成分まで抽出されるおそれがある。カカオ殻から水抽出により得られた抽出液は、水分を蒸発させたのち凍結乾燥することにより、水抽出物を乾固物とすることができる。 In this invention, there is no restriction | limiting in particular in the water extraction temperature of a cocoa shell, For example, it can extract at arbitrary temperatures of 5-100 degreeC, or can also extract at the temperature exceeding 100 degreeC under pressure. The amount of the water extract obtained from the cocoa shell is preferably 3 to 11% by weight of the cocoa shell, and more preferably 5 to 9% by weight. If the amount of the water extract is less than 3% by weight of the cocoa shell, the active ingredient contained in the cocoa shell may not be sufficiently utilized. When the amount of the water extract exceeds 11% by weight of the cocoa shell, extraction may be difficult, and components having poor medicinal effects may be extracted. The extract obtained by water extraction from the cocoa shells can be converted into a solid product by freeze-drying after evaporating the water.
本発明の細胞賦活剤、紫外線障害緩和剤又はメラニン産生抑制剤において、カカオ殻の水抽出物の含有量に特に制限はないが、０.０００１〜５重量％であることが好ましい。カカオ殻の水抽出物の含有量が０.０００１重量％未満であると、十分な薬効が発現しないおそれがある。カカオ殻の水抽出物の含有量が５重量％を超えると、カカオ殻の水抽出物が有する薬効が十分に利用されないおそれがある。
本発明の細胞賦活剤、紫外線障害緩和剤又はメラニン産生抑制剤においては、必要に応じて、紫外線吸収剤、保湿剤、増粘剤、酸化防止剤、防腐剤、香料、着色料、皮膚栄養剤などを配合することができる。
In the cell activator, ultraviolet damage mitigating agent or melanin production inhibitor of the present invention, the content of the cocoa shell water extract is not particularly limited, but is preferably 0.0001 to 5% by weight. If the content of the cocoa shell water extract is less than 0.0001% by weight, sufficient medicinal effects may not be exhibited. If the content of the cocoa shell water extract exceeds 5% by weight, the medicinal effect of the cocoa shell water extract may not be sufficiently utilized.
In the cell activator, ultraviolet damage mitigating agent or melanin production inhibitor of the present invention, an ultraviolet absorber, a humectant, a thickener, an antioxidant, an antiseptic, a fragrance, a colorant, and a skin nutrient as needed. Etc. can be blended.
以下に、実施例を挙げて本発明をさらに詳細に説明するが、本発明はこれらの実施例によりなんら限定されるものではない。
実施例１
カカオ殻３０ｇを粉砕し、ソックスレー抽出器を用いて精製水３００ｍＬで７時間抽出を行った。抽出液から水分を留去したのち、残渣を凍結乾燥してカカオ殻の水抽出物２.０ｇを得た。
５重量％ウシ血清［ＦＢＳ，ＥＱＵＩＴＥＣＨ−ＢＩＯ ＩＮＣ.］を添加したダルベッコ改変イーグル培地［ＤＭＥＭ、(株)医学生物学研究所］を用いて維持した正常ヒト線維芽細胞［クラボウ(株)］を、細胞賦活作用の試験に供した。
臭化３−(４,５−ジメチル−２−チアゾリル)−２,５−ジフェニル−２Ｈ−テトラゾリウム（ＭＴＴ）は、ミトコンドリアにおいて細胞内のエネルギー代謝過程で生じるニコチンアミドアデニンジヌクレオチドホスフェート（ＮＡＤＰＨ）又はニコチンアミドアデニンジヌクレオチド（ＮＡＤＨ）により開裂し、ホルマザンを生成する。このために、ホルマザンの生成量を、細胞賦活作用の指標とした。
正常ヒト線維芽細胞を、１ウェルあたり２.０×１０4個の細胞密度で、９６穴マイクロプレートに播種した。１重量％ウシ血清（ＦＢＳ）を含有するダルベッコ改変イーグル培地（ＤＭＥＭ）にて２４時間培養したのち、所定の濃度のカカオ殻の水抽出物を含有した１重量％ウシ血清（ＦＢＳ）を含有するダルベッコ改変イーグル培地（ＤＭＥＭ）に交換した。さらに４８時間培養したのち、臭化３−(４,５−ジメチル−２−チアゾリル)−２,５−ジフェニル−２Ｈ−テトラゾリウム［ＭＴＴ、ナカライテスク(株)］０.４ｍｇ／ｍＬを含有する１重量％ウシ血清（ＦＢＳ）を含有するダルベッコ改変イーグル培地（ＤＭＥＭ）に交換して２時間培養した。細胞を洗浄したのち、２−プロパノールにて細胞内に生成したホルマザンを溶解し、マイクロプレートリーダーを用いて５６０ｎｍ及び６６０ｎｍの吸光度を測定し、両者の差をもって、細胞内に生成したホルマザン量として評価した。細胞賦活作用は、抽出物無添加培養細胞（コントロール）の吸光度（ＡＢＳ）を１００とした賦活指数（％）で表した。なお、試験ごとの陽性コントロール（ＰＣ）として、５重量％ウシ血清（ＦＢＳ）を含有するダルベッコ改変イーグル培地（ＤＭＥＭ）にて培養した細胞を用いた。
賦活指数（％）
＝{ＡＢＳ(抽出物添加培養細胞)／ＡＢＳ(抽出物無添加培養細胞)}×１００
比較例１
カカオ殻３０ｇを粉砕し、エタノール３００ｇに浸漬して撹拌しながら７日間抽出を行った。固形分をろ別したのち、エタノールを留去し、カカオ殻のエタノール抽出物２.３ｇを得た。
カカオ殻の水抽出物の代わりに、カカオ殻のエタノール抽出物を用いた以外は実施例１と同様にして、細胞賦活作用を評価した。
実施例１及び比較例１の結果を、第１表に示す。すべての結果は、平均値±標準偏差で表した。有意差検定にはＳｔｕｄｅｎｔのｔ−検定を用い、ｐ値が０.０５未満のとき有意であると判定した。
Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
Example 1
30 g of cocoa shells were pulverized and extracted with 300 mL of purified water for 7 hours using a Soxhlet extractor. After water was distilled off from the extract, the residue was freeze-dried to obtain 2.0 g of a cacao shell water extract.
Normal human fibroblast cells [Kurabo Co., Ltd.] maintained using Dulbecco's modified Eagle medium [DMEM, Institute of Medical Biology] supplemented with 5% by weight bovine serum [FBS, EQUITECH-BIO INC.] Then, the cells were subjected to a cell activation test.
3- (4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) is a nicotinamide adenine dinucleotide phosphate (NADPH) produced during intracellular energy metabolism in mitochondria or Cleavage with nicotinamide adenine dinucleotide (NADH) to produce formazan. For this reason, the amount of formazan produced was used as an index for cell activation.
Normal human fibroblasts were seeded in 96-well microplates at a density of 2.0 × 10 4 cells per well. After 24 hours of culture in Dulbecco's modified Eagle's medium (DMEM) containing 1% by weight bovine serum (FBS), it contains 1% by weight bovine serum (FBS) containing an aqueous extract of cocoa shells at a predetermined concentration. It was replaced with Dulbecco's modified Eagle medium (DMEM). After further incubation for 48 hours, 1 containing 0.4 mg / mL of 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide [MTT, Nacalai Tesque, Inc.] The culture was replaced with Dulbecco's modified Eagle medium (DMEM) containing wt% bovine serum (FBS) and cultured for 2 hours. After washing the cells, the formazan produced in the cells with 2-propanol was dissolved, the absorbance at 560 nm and 660 nm was measured using a microplate reader, and the difference between the two was evaluated as the amount of formazan produced in the cells. did. The cell activation action was expressed as an activation index (%) with the absorbance (ABS) of the culture cells without addition of the extract (control) as 100. As a positive control (PC) for each test, cells cultured in Dulbecco's modified Eagle medium (DMEM) containing 5% by weight bovine serum (FBS) were used.
Activation index (%)
= {ABS (cultured cells with extract added) / ABS (cultured cells without extract)} × 100
Comparative Example 1
30 g of cocoa shells were pulverized, immersed in 300 g of ethanol and extracted for 7 days with stirring. After filtering off the solid content, ethanol was distilled off to obtain 2.3 g of an ethanol extract of cocoa shells.
The cell activation effect was evaluated in the same manner as in Example 1 except that the cocoa shell ethanol extract was used instead of the cocoa shell water extract.
The results of Example 1 and Comparative Example 1 are shown in Table 1. All results were expressed as mean ± standard deviation. Student's t-test was used for the significant difference test, and it was determined to be significant when the p-value was less than 0.05.
第１表に見られるように、実施例１のカカオ殻の水抽出物は、濃度１２５.０〜１,０００.０μｇ／ｍＬにおいて細胞賦活作用が認められるが、比較例１のカカオ殻のエタノール抽出物には細胞賦活作用が認められない。
実施例２
実施例１で調製したカカオ殻の水抽出物で正常ヒト線維芽細胞を処理したのち紫外線を照射し、細胞生存率をニュートラルレッド法で測定することにより、紫外線障害緩和作用を評価した。
正常ヒト線維芽細胞を、１ウェルあたり２.０×１０4個の細胞密度で、９６穴マイクロプレートに播種した。５重量％ウシ血清（ＦＢＳ）を含有するダルベッコ改変イーグル培地（ＤＭＥＭ）にて２４時間培養したのち、所定の濃度のカカオ殻の水抽出物を含有した５重量％ウシ血清（ＦＢＳ）を含有するダルベッコ改変イーグル培地（ＤＭＥＭ）に交換した。さらに２４時間培養したのち細胞を洗浄し、所定の濃度のカカオ殻の水抽出物を含有したＨａｎｋ'ｓ緩衝液（カルシウム、マグネシウム含有）に交換し、紫外線７５ｍＪ／ｃｍ2［(株)東芝、ＦＬ２０ＳＥランプ］を照射した。照射後、緩衝液を所定の濃度のカカオ殻の水抽物を含有した５重量％ウシ血清（ＦＢＳ）を含有するダルベッコ改変イーグル培地（ＤＭＥＭ）に交換し、さらに２４時間培養したのち、細胞を３０μｇ／ｍＬのニュートラルレッド及び５重量％ウシ血清（ＦＢＳ）を含有するダルベッコ改変イーグル培地（ＤＭＥＭ）に交換し２時間培養した。細胞を洗浄したのち、細胞に取り込まれたニュートラルレッドをニュートラルレッド用細胞溶解液（３０重量％のエタノールを含有する０.１モル／Ｌ塩酸）を用いて溶解し、マイクロプレートリーダーを用いて５６０ｎｍ及び６６０ｎｍの吸光度を測定し、両者の差をもってニュートラルレッド量として評価した。細胞生存率は、紫外線未照射細胞（コントロール）の吸光度（ＡＢＳ）を１００として、細胞生存率（％）で表した。
細胞生存率（％）
＝（紫外線照射細胞のＡＢＳ／紫外線未照射細胞のＡＢＳ）×１００
比較例２
カカオ殻の水抽出物の代わりに、比較例１で調製したカカオ殻のエタノール抽出物を用いた以外は実施例２と同様にして、紫外線障害緩和作用を評価した。
実施例２及び比較例２の結果を、第２表に示す。すべての結果は、平均値±標準偏差で表した。有意差検定にはＳｔｕｄｅｎｔのｔ−検定を用い、ｐ値が０.０５未満のとき有意であると判定した。
As can be seen from Table 1, the cocoa shell water extract of Example 1 has a cell activation effect at a concentration of 125.0 to 15,000 μg / mL. There is no cell activation effect in the extract.
Example 2
Normal human fibroblasts were treated with the cacao shell aqueous extract prepared in Example 1 and then irradiated with ultraviolet rays, and the cell viability was measured by the neutral red method to evaluate the ultraviolet damage mitigating action.
Normal human fibroblasts were seeded in 96-well microplates at a density of 2.0 × 10 4 cells per well. After culturing in Dulbecco's Modified Eagle Medium (DMEM) containing 5% by weight bovine serum (FBS) for 24 hours, it contains 5% by weight bovine serum (FBS) containing an aqueous extract of cocoa shells at a predetermined concentration. It was replaced with Dulbecco's modified Eagle medium (DMEM). After further culturing for 24 hours, the cells were washed and replaced with Hank's buffer (containing calcium and magnesium) containing a cacao shell aqueous extract with a predetermined concentration, and ultraviolet rays of 75 mJ / cm 2 [Toshiba Corporation, FL20SE lamp]. After the irradiation, the buffer solution was replaced with Dulbecco's modified Eagle medium (DMEM) containing 5% by weight bovine serum (FBS) containing a water extract of cocoa shells of a predetermined concentration, and further cultured for 24 hours. The culture medium was replaced with Dulbecco's modified Eagle medium (DMEM) containing 30 μg / mL neutral red and 5% by weight bovine serum (FBS) and cultured for 2 hours. After washing the cells, the neutral red incorporated into the cells was lysed using a cell lysate for neutral red (0.1 mol / L hydrochloric acid containing 30% by weight of ethanol), and 560 nm using a microplate reader. The absorbance at 660 nm was measured, and the difference between the two was evaluated as the neutral red amount. The cell viability was expressed in terms of cell viability (%), where the absorbance (ABS) of unirradiated cells (control) was 100.
Cell viability (%)
= (ABS of UV-irradiated cells / ABS of non-UV-irradiated cells) × 100
Comparative Example 2
The UV damage mitigating action was evaluated in the same manner as in Example 2, except that the cocoa shell ethanol extract prepared in Comparative Example 1 was used instead of the cocoa shell water extract.
The results of Example 2 and Comparative Example 2 are shown in Table 2. All results were expressed as mean ± standard deviation. Student's t-test was used for the significant difference test, and it was determined to be significant when the p-value was less than 0.05.
第２表に見られるように、実施例２のカカオ殻の水抽出物は、濃度６２.５〜１,０００.０μｇ／ｍＬにおいて紫外線障害緩和作用が認められるが、比較例２のカカオ殻のエタノール抽出物には紫外線障害緩和作用が認められない。
実施例３
実施例１で調製したカカオ殻の水抽出物について、Ｂ１６メラノーマ細胞のトータルメラニン産生に対する作用を評価した。
５重量％ウシ血清（ＦＢＳ）を含有するダルベッコ改変イーグル培地（ＤＭＥＭ）を用いて維持したＢ１６マウスメラノーマＦ０株［Ｂ１６Ｆ０、大日本製薬(株)］を、１ウェルあたり１.８×１０4個の細胞密度で、６穴プレートに播種した。５重量％ウシ血清（ＦＢＳ）を含有するダルベッコ改変イーグル培地（ＤＭＥＭ）にて２４時間培養したのち、所定の濃度のカカオ殻の水抽出物を含有する５重量％ウシ血清（ＦＢＳ）を含有するダルベッコ改変イーグル培地（ＤＭＥＭ）に交換した。さらに５日間培養したのち、細胞をトリプシン処理にて剥離し、タンパク量を測定するとともに、細胞ペレットを作製し、目視判定により細胞ペレットの色調を、１白−５黒の５段階スコアで評価した。なお、陽性コントロール（ＰＣ）として、５０ミリモル／Ｌ乳酸ナトリウムと５重量％ウシ血清（ＦＢＳ）を含有するダルベッコ改変イーグル培地（ＤＭＥＭ）にて培養した細胞を用いた。
比較例３
カカオ殻の水抽出物の代わりに、比較例１で調製したカカオ殻のエタノール抽出物を用いた以外は実施例３と同様にして、Ｂ１６メラノーマ細胞のトータルメラニン産生に対する作用を評価した。
実施例３及び比較例３の結果を、第３表に示す。すべての結果は、平均値±標準偏差で表した。有意差検定にはＳｔｕｄｅｎｔのｔ−検定を用い、ｐ値が０.０５未満のとき有意であると判定した。
As can be seen in Table 2, the aqueous extract of cocoa shells of Example 2 has a UV damage mitigating action at a concentration of 62.5 to 1,000. 0 μg / mL. Ethanol extract does not show any UV damage mitigating action.
Example 3
About the water extract of the cocoa shell prepared in Example 1, the effect | action with respect to the total melanin production of a B16 melanoma cell was evaluated.
1.8 × 10 4 B16 mouse melanoma strains F0 [B16F0, Dainippon Pharmaceutical Co., Ltd.] maintained using Dulbecco's modified Eagle's medium (DMEM) containing 5% by weight bovine serum (FBS) per well At a cell density of 6 wells. After culturing in Dulbecco's modified Eagle medium (DMEM) containing 5% by weight bovine serum (FBS) for 24 hours, it contains 5% by weight bovine serum (FBS) containing an aqueous extract of cocoa shells at a predetermined concentration. It was replaced with Dulbecco's modified Eagle medium (DMEM). After further culturing for 5 days, the cells were detached by trypsin treatment, the amount of protein was measured, a cell pellet was produced, and the color tone of the cell pellet was evaluated by visual judgment with a 5-step score of 1 white-5 black. . As a positive control (PC), cells cultured in Dulbecco's modified Eagle medium (DMEM) containing 50 mmol / L sodium lactate and 5% by weight bovine serum (FBS) were used.
Comparative Example 3
The effect of B16 melanoma cells on total melanin production was evaluated in the same manner as in Example 3 except that the cocoa shell ethanol extract prepared in Comparative Example 1 was used instead of the cocoa shell water extract.
The results of Example 3 and Comparative Example 3 are shown in Table 3. All results were expressed as mean ± standard deviation. Student's t-test was used for the significant difference test, and it was determined to be significant when the p-value was less than 0.05.
第３表に見られるように、実施例３のカカオ殻の水抽出物は、濃度６２.５〜１２５.０μｇ／ｍＬにおいて、タンパク量がコントロールより少なく、色調スコアがコントロールより低く、Ｂ１６メラノーマ細胞のトータルメラニン産生抑制作用が認められる。これに対して、比較例３のカカオ殻のエタノール抽出物は、タンパク量がコントロールより多く、色調スコアがコントロールより高く、Ｂ１６メラノーマ細胞のトータルメラニン産生促進作用が認められる。 As can be seen in Table 3, the cacao shell water extract of Example 3 has a lower protein content and lower color score than the control at a concentration of 62.5-125.0 μg / mL, and B16 melanoma cells. Can suppress the total melanin production. In contrast, the ethanol extract of the cocoa shell of Comparative Example 3 has a higher protein content than the control and a higher color tone score than the control, and the action of promoting the total melanin production of B16 melanoma cells is observed.
本発明の細胞賦活剤、紫外線障害緩和剤及びメラニン産生抑制剤は、カカオ殻の水抽出物を有効成分とするので、ココアやチョコレート菓子などの製造に際して大量に発生するカカオ殻を活用することができる。カカオの種子は、天然物であり、食品の原料として衛生的に管理されているので、副生物としてのカカオ殻も、薬剤の原料として安全に使用することができる。カカオ殻の水抽出は、有機溶媒を使用することなく、安全かつ経済的に行うことができる。 Since the cell activator, UV damage mitigating agent and melanin production inhibitor of the present invention contain a cacao shell aqueous extract as an active ingredient, it is possible to utilize the cacao shell generated in large quantities in the production of cocoa, chocolate confectionery, etc. it can. Since cocoa seeds are natural products and are hygienically managed as a raw material for food, cocoa shells as a by-product can also be used safely as a raw material for drugs. The water extraction of cocoa shells can be performed safely and economically without using an organic solvent.
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