200824718 九、發明說明： 【發明所屬之技術領域】 本發明爲有關美白劑。 【先前技術】 忽布（Humulus lupulus)爲製造啤酒不可欠缺之原料，予 ' . 以啤酒苦味或香味、改善發泡·久泡性，並提高清澄性， ' 也抑制雜菌之繁殖。又因忽布有健胃、促進消化等藥理作 用，以往也作爲藥用植物利用。 • 近年，著眼於如此忽布之有用性，試用其萃取物於啤酒 以外之用途。提案例如以忽布萃取物爲有效成分之耐熱性 嗜酸性菌增殖抑制劑（專利文獻1 )，以忽布萃取物爲有效 成分之米加工食品用香味改良劑（專利文獻2)，含有忽布 萃取物之入浴劑（專利文獻3)，具有活性酸素消去作用之 萃取物（專利文獻4)等用途。 對於忽布萃取物，也檢討用於皮膚外用劑，例如於含有 由半日花科（Cistaceae)之植物體萃取之成分之皮膚外用劑 ® ，與此成分倂用之植物萃取物之一例可舉出忽布萃取物（專 利文獻5)。 【專利文獻1】特開2005- 1 3724 1 【專利文獻2】特開2005-269988 【專利文獻3】特開平9-67245 【專利文獻4】特開平4-202138 【專利文獻5】特開2003-300859 【發明內容】 200824718 發明欲解決少課顆 但於忽布因多含萃取可能之成分，·故若萃取媒體或萃取 溫度等萃取條件相異，則得組成相異之萃取物，萃取物所 奏效也成異質者。故以忽布萃取物爲皮膚外用劑之一成分 來適用時’也由萃取條件而美白效果等外用劑之效果不充 • 分之場合也多。 ^ 於是本發明之目的爲提供含有忽布萃取物，而美白效果 特優之美白劑。 • 解決課穎之羊段 爲達成上述目的，本發明爲提供以忽布組織之冷水萃取 物爲有效成分之美白劑。 於此依冷水萃取之忽布萃取物以由羊齒精、羊齒精丙二 醯基葡萄糖苷、異櫟素、異櫟素丙二醯基葡萄糖苷、櫟精 丙二醯基葡萄糖苷、萘三羥黃酮醇芸香糖苷、萘三羥黃酮 醇丙二醯基葡萄糖苷、芸香素及根皮醯苯配糖體而成之群 選擇之類黃酮配糖體含有至少丨種較佳，根皮醯苯衍生物 ® 以由根皮異丁醯苯-2-0 _/3-D -葡萄卩比喃糖苷、根皮-2 -甲基 丁醯苯-2-01 -D-葡萄吡喃糖苷及根皮異戊醯苯-2-0- /3 -D-葡萄吡喃糖苷而成之群選擇之至少1種較佳。 因上述成分以高含有率萃取，故忽布組織以忽布之莖、 球花或葉較佳，乾燥之忽布花苞之粉碎物特佳。由同樣之 觀點，忽布組織以由乾燥忽布球花之粉碎物、忽布花粉 (lupulin)之大小以下之粉碎物之至少一部分去除者較佳， 乾燥之忽布球花之粉碎物可用乾燥之忽布球花之凍結物之 200824718 l 粉碎物。忽布組織可用由乾燥之忽布球花令以有機溶劑萃 取或超臨界流體萃取來萃取之物質之至少一部分，由該忽 布球花去除所得之忽布殘澄。 如此美白劑當然可以皮膚外用劑或其一成分使用，唯也 可以經口攝取型美白劑來機能。 - 發明之效果 • 提供含有忽布萃取物而美白效果特優之美白劑。 【實施方式】 • 實施發明之最佳形態 以下說明本發明美白劑之較佳實施形態。 本發明之美白劑之有效成分爲忽布組織之冷水萃取物， 任何品種之忽布皆可爲冷水萃取之對象。但依所得萃取成 分之美白效果高，以捷克產札字種、德國產哈拉島得拉得 雄種、日本產富良野18號、其中日本產等啤酒釀造用忽 布品種較佳、尤以捷克產札字種特佳。 本發明中忽布組織乃指忽布之任何組織或其一部分。冷 ® 水萃取所用忽布組織也可爲葉、莖及球花之任一，以球花 較佳，尤以忽布花苞更佳。忽布花苞乃指構成球花之花苞 葉，可由球花去除忽布花粉部分（黄色之顆粒）之至少一部 分而得。故本發明之冷水萃取所用忽布組織可爲啤酒等發 泡性酒精飮料之釀造所用忽布柱粒加工之際不粉碎成規定 大小而捨棄之忽布花苞，也可爲後述令忽布球花以超臨界 流體或有機溶劑萃取後殘留之忽布殘渣。 忽布組織之冷水萃取物爲令忽布組織以具備冷水萃取工 200824718 程之製法而得。於此「冷水」乃指室溫以下之水，通常指 超過0°C而5(TC以下之水。冷水之溫度以超過〇。(：而40°C 以下較佳，5°C以上30°C以下更佳，尤以10°C以上30°C以 下更較佳，以20±5°C (更以2(U3°C )特佳。使用如此溫度之 冷水，則萃取有效率，萃取物產量增多。使用〇°C以下之 冷水，則冷却成本增大，使用超過40°C之水，則有連誘發 炎症作用之成分也溶出之傾向。又爲縮短萃取時間，可於 水添加少量之醇，較佳爲乙醇，10質量％以下。 Φ 由忽布組織得萃取物之方法，可廣泛採用由植物以水萃 取天然物之方法，例如令忽布組織和一定量之冷水投入容 器，適宜攪拌並靜置所定時間，過濾萃取液而去除殘渣之 方法。欲完全去除混入之殘渣或不純物等，可更離心過濾 之萃取液，令其上清（以下稱離心上清）作爲冷水萃取物使 用。又所得之冷水萃取物也可濃縮、乾燥而使用。 忽布組織之冷水萃取物也可通經充塡合成吸著劑之柱而 精製來使用。合成吸著劑可爲例如Amberlite XAD-4、7及 ® 16(Organo公司）、活性碳、聚乙烯吡咯啶酮（PVPP;多酚吸 著劑），其中以Ambedite XAD-4較佳。具體而言，可令忽 布組織之冷水萃取物通經充塡合成吸著劑之柱，令其吸著 成分以例如水及甲醇之混合溶劑溶出，而使用溶出之劃份 本發明之美白劑以乾燥之忽布花苞之粉碎物之冷水萃取 物爲有效成分較佳，又以由乾燥之忽布球花之粉碎物去除 忽布花粉之大小以下之粉碎物之至少一部分者之冷水萃取 200824718 物爲有效成分較佳。冷水萃取所用乾燥忽布球花之粉碎物 可由具備例如令忽布球花乾燥而得乾燥忽布球花之乾燥工 程，令乾燥忽布球花粉碎而得粉碎物之粉碎工程’由此粉 碎物去除忽布花粉大小以下之粉碎物之選別工程之製法而 得。 於乾燥工程，可令忽布球花於100°c以下之溫度乾燥， 使忽布球花水分去除至保存可能之程度，唯以5 5 °C以下之 溫度乾燥至水分含量7〜9%較佳。於粉碎工程，可令忽布 球花有效率粉碎成微粉狀即可，可用例如針磨、錘磨、球 磨等粉碎機。於選別工程，可令粉碎物通過篩，例如令長 徑0.1mm以上之粉碎物作爲「超過忽布花粉之大小」者選 別。於此場合中以不通過篩之大小爲長徑0.3mm以上較佳 ，長徑0.5mm以上更佳。欲由乾燥忽布球花之粉碎物去除 忽布花粉之大小以下之粉碎物，則例如以篩孔0.1、0.3或 0·5mm之篩篩選乾燥忽布球花之粉碎物，回收不通過篩之 粉碎物即可。又由乾燥忽布球花之粉碎物去除忽布花粉之 大小以下之粉碎物之至少一部分者之冷水萃取物，可令如 此選別之乾燥忽布球花之粉碎物，依上述以冷水萃取之工 程記載之方法萃取即可。 更於本發明中使用之乾燥忽布球花之粉碎物以乾燥忽布 球花之凍結物之粉碎物較佳。令乾燥忽布球花凍結之方法 無特限，凍結溫度以-10°C以下較佳，-35t以下更佳。 又冷水萃取物可爲令由乾燥忽布球花依有機溶劑萃取或 超臨界流體萃取而萃取之物質之至少一部分，由該忽布球 200824718 花去除所得之忽布殘渣之冷水萃取物。有機溶劑萃取所用 有機溶劑可爲例如醇或己烷’以碳數1〜4之低醇較佳， 乙醇更佳。超臨界流體萃取所用超臨界流體可爲例如二氧 化碳、水、甲烷、乙烷、乙烯、丙烷、戊烷、甲醇、乙醇 ，以二氧化碳較佳。 冷水萃取物典型爲含有至少1種由羊齒精、羊齒精丙二 醯基葡萄糖苷、異櫟素、異櫟素丙二醯基葡萄糖苷、櫟精 丙二醯基葡萄糖苷、萘三羥黃酮醇芸香糖苷、萘三羥黃酮 醇丙二醯基葡萄糖苷、芸香素及根皮醯苯配糖體而成之群 選擇之類黃酮配糖體。 於此根皮醯苯配糖體可以如下式（1)表示。 R1 ΟΗ200824718 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a whitening agent. [Prior Art] Humulus lupulus is a raw material that is indispensable for the production of beer. It has a bitter taste or aroma, improves foaming and long-term foaming, and improves clarity. It also inhibits the growth of bacteria. In addition, it has been used as a medicinal plant in the past because it has a pharmacological effect such as strengthening the stomach and promoting digestion. • In recent years, with a view to the usefulness of such a flicker, try to use its extracts for purposes other than beer. For example, a heat-resistant eosinophilic growth inhibitor (Patent Document 1) in which the extract is used as an active ingredient, and a flavor-improving agent for rice processed foods containing the extract as an active ingredient (Patent Document 2) The bathing agent of the extract (Patent Document 3) has an active extract elimination effect (Patent Document 4) and the like. For the cloth extract, the skin external preparation is also reviewed, for example, a skin external preparation containing a component extracted from a plant of the family Cistaceae, and an example of the plant extract used for the ingredient is exemplified. The cloth extract is obtained (Patent Document 5). [Patent Document 1] Japanese Laid-Open Patent Publication No. 2005-269988 [Patent Document 3] Japanese Laid-Open Patent Publication No. Hei No. Hei No. Hei. -300859 [Summary of the Invention] 200824718 The invention is intended to solve the problem of less class, but it may contain components of extraction, so if the extraction conditions such as extraction medium or extraction temperature are different, the extracts of different compositions may be obtained. The effect is also heterogeneous. Therefore, when the extract is used as a component of the external preparation for skin, the effect of the external preparation such as the whitening effect by the extraction conditions is not sufficient. ^ Thus, the object of the present invention is to provide a whitening agent which contains a cloth extract and which has an excellent whitening effect. • Solving the sheep segment of the class In order to achieve the above object, the present invention provides a whitening agent which is provided with a cold water extract of the tissue as an active ingredient. According to the cold water extraction, the extract is made of fern, fern, propyl glucoside, isoquercein, isoindolin, glucosinolate, glucosinolate, naphthalene The flavonoid glycoside selected from the group consisting of trihydroxyflavonol rutinoside, naphthalene trihydroxyflavonol propylene diglucosylglucoside, ruthenium and phloridin glucoside contains at least one species, root bark Benzene derivatives® from root bark Isobutyl Benzene-2-0 _/3-D - glucoside glucoside, root bark-2 - methyl butyl benzophenone-2-01 -D-glucopyranoside and At least one selected from the group consisting of radicle isopenoquinone Benzene-2-0- /3 -D-glucopyranoside is preferred. Since the above components are extracted at a high content rate, it is preferable that the cloth is stalked, the spheroidal flower or the leaf is better, and the pulverized material of the dried cloth is particularly good. From the same point of view, it is better to remove at least a part of the pulverized material of the size of the smashed stalk and the sulphide of the dry cloth, and the pulverized material of the dry bud flower can be dried. The sudden freezing of the ball flower 200824718 l pulverized material. The cloth may be at least a part of the substance extracted by the organic solvent extraction or supercritical fluid extraction by drying the ball, and the resulting material is removed by the removal of the ball. Such a whitening agent can of course be used as a skin external preparation or a component thereof, and can also be used as an oral ingesting whitening agent. - Effect of the invention • Provides a whitening agent with a whitening effect and a whitening effect. [Embodiment] BEST MODE FOR CARRYING OUT THE INVENTION A preferred embodiment of the whitening agent of the present invention will be described below. The active ingredient of the whitening agent of the present invention is a cold water extract of the cloth, and any variety of cloth can be the object of cold water extraction. However, according to the high whitening effect of the extracted components, the Czech-made characters, the German-made Hala Island, the Japanese-made Furano No. 18, and the Japanese-made beer brewing varieties are better, especially in the Czech Republic. The production of the characters is particularly good. The organization in the present invention refers to any organization or a part thereof. The tissue used for cold ® water extraction can also be any of leaves, stems and globose flowers, preferably with globules, especially with buds. The bud flower bud is the flower bud leaf that constitutes the globose flower, which can be obtained by removing at least a portion of the pollen portion (yellow granule) from the globose flower. Therefore, the material used for the cold water extraction of the present invention can be used for the brewing of foaming alcoholic beverages such as beer, and the cloth is not pulverized into a predetermined size when the processing is performed, and the cloth can be discarded as described later. The residual residue remaining after extraction with a supercritical fluid or an organic solvent. The cold water extract of the organization is obtained by the method of making the cold water extractor. "Cold water" means water below room temperature, usually means more than 0 ° C and 5 (water below TC. The temperature of cold water exceeds 〇. (: preferably 40 ° C or less, 5 ° C or more 30 °) C or less is more preferable, particularly preferably 10 ° C or more and 30 ° C or less, more preferably 20 ± 5 ° C (more preferably 2 (U 3 ° C). Using cold water of such temperature, extraction efficiency, extract Increased production. When using cold water below 〇 °C, the cooling cost increases. When water exceeding 40 ° C is used, there is a tendency for the components that induce inflammation to be dissolved. In order to shorten the extraction time, a small amount of water can be added. The alcohol, preferably ethanol, is 10% by mass or less. Φ The method of extracting the extract from the cloth can be widely used as a method of extracting the natural product by water from the plant, for example, making the cloth and a certain amount of cold water into the container, suitable Stirring and standing for a predetermined period of time, filtering the extract to remove the residue. To completely remove the residue or impurities, the supernatant can be centrifuged to make the supernatant (hereinafter referred to as the supernatant) used as a cold water extract. The resulting cold water extract can also be concentrated and dried. The cold water extract of the tissue can also be refined by purifying the column of the synthetic sorbent. The synthetic sorbent can be, for example, Amberlite XAD-4, 7 and® 16 (Organo), activated carbon. , polyvinylpyrrolidone (PVPP; polyphenol sorbent), wherein Ambedite XAD-4 is preferred. Specifically, the cold water extract of the tissue can be passed through a column of a synthetic sorbent. The sorbing component is dissolved in a mixed solvent such as water and methanol, and the cold water extract of the pulverized material of the dried buds is preferably used as an active ingredient, and is dried by the use of the dissolved whitening agent of the present invention. The pulverized material of the cloth ball flower is used to remove at least a part of the pulverized material of the size of the pollen, and the cold water extraction is preferably the active ingredient. The pulverized material of the dry blister ball flower used for the cold water extraction may be provided with, for example, a blister ball flower. The drying process of drying and drying the ball flower is carried out, and the pulverization of the pulverized material is obtained by pulverizing the dried buds and smashing the pulverized material to remove the pulverized material of the size of the pulverized material. Drying process can make the cloth ball dry at a temperature below 100 °c, so that the water of the cloth ball can be removed to the extent that it can be preserved. It is only dried at a temperature below 5 5 °C until the moisture content is 7~9%. In the crushing project, the ball can be pulverized into a fine powder, which can be used, for example, by a pin mill, a hammer mill, a ball mill, etc. In the sorting process, the pulverized material can be passed through a sieve, for example, a long diameter of 0.1. The pulverized material of mm or more is selected as the "size larger than the size of the pollen." In this case, the size of the sieve is not longer than 0.3 mm, and the long diameter is preferably 0.5 mm or more. The pulverized material of the flower is used to remove the pulverized material of the size below the size of the pollen, for example, the pulverized material of the dried bud flower is screened by a sieve having a mesh size of 0.1, 0.3 or 0.5 mm, and the pulverized material which does not pass through the sieve is recovered. The cold water extract of at least a part of the pulverized material of the size of the fluffy pollen is removed from the pulverized material of the dry and smudged flower, so that the pulverized material of the dried and smashed flower can be selected according to the above-mentioned cold water extraction project. The method described can be extracted. More preferably, the pulverized material of the dried bud flower used in the present invention is preferably a pulverized product of the frozen material of the dried flower. The method for freezing the dry and dead bulbs is not limited, and the freezing temperature is preferably -10 ° C or less, preferably -35 t or less. The cold water extract may be at least a part of a substance obtained by extracting dry bulbs by organic solvent extraction or supercritical fluid extraction, and the cold water extract obtained by removing the resulting residue from the flower of the cloth. The organic solvent used for the organic solvent extraction may be, for example, an alcohol or hexane, preferably a lower alcohol having a carbon number of 1 to 4, more preferably ethanol. The supercritical fluid used for supercritical fluid extraction may be, for example, carbon dioxide, water, methane, ethane, ethylene, propane, pentane, methanol, ethanol, preferably carbon dioxide. The cold water extract typically contains at least one species derived from fern, fern propyl glucoside, isoquercein, isoindolin propyl diglucosylglucoside, quercetin glucosinolate, naphthalene trishydroxyl A flavonoid glycoside selected from the group consisting of flavonol rutinoside, naphthalene trihydroxyflavonol propylene diglucosylglucoside, ruthenium and phlorizin. The root bark benzene glycoside can be represented by the following formula (1). R1 ΟΗ
[式（1)中R1爲異丙基、異丁基或第二丁基]。 若式（1)中R1爲異丙基時，根皮醯苯配糖體爲如下式（2) 所示之根皮異丁釀苯- 2- O- ^-D -葡萄耻喃糖甘’右R爲異 丁基時，根皮醯苯配糖體爲如下式（3)所示之根皮-2-甲基 丁醯苯_2-0_/3 -D-葡萄吡喃糖苷’ R1爲第二丁基時，根皮 醯苯配糖體爲如下式（4)所示之根皮異戊醯苯-2-01 -D·葡 萄吡喃糖苷。 -10- (2) 200824718[R1 in the formula (1) is an isopropyl group, an isobutyl group or a second butyl group]. When R1 in the formula (1) is an isopropyl group, the phloridin benzene glycoside is a root-like butyl benzo-2-(O-^-D-glucopyranose) represented by the following formula (2) When the right R is an isobutyl group, the root bark phthalate glycoside is a root bark-2-methylbutazone 2-0_/3 -D-glucopyranoside R1 represented by the following formula (3). In the case of the second butyl group, the phlegm-endoquinone glycoside is a keto-p-isoamyl benzene-2-01-D·glucopyranoside represented by the following formula (4). -10- (2) 200824718
(4) 美白劑中，忽布組織之冷水萃取物之含有量爲以除去萃 取媒體之成分按美白劑全質量基準以0.0001〜100質量％較 佳，0.01〜100質量％更佳，1〜1〇〇質量％更較佳，尤以5 〜100質量％特佳。 美白劑除忽布組織之冷水萃取物之外，可含有浸潤劑、 油性成分、保濕劑、粉體、色素、乳化劑、分散助劑、可 溶化劑、洗淨劑、紫外線吸收劑、增粘劑、藥劑、香料、 樹脂、賦形劑、防菌防黴劑、消臭·脫臭劑、酵素、精製 -11- 200824718 水、醇。又可添加其他美白劑。 本發明之美白劑可以其單獨具有美白效果之皮膚外用劑 適用於皮膚等，也可於化粧料或藥劑添加而於這些附與美 白效果作爲美白效果附與劑來使用。更也可作爲服用而發 揮美白效果之經口攝取型美白劑利用。 【實施例】 以下基於實施例及比較例更具體說明本發明，但本發明 不受下列實施例限定。 # [製造例1] 冷水卒取: 令忽布（日本產富良野18號）之葉切碎，浸漬於10倍量 (w/v)之蒸餾水而於5°C靜置一晚。令此以9200G 15分之離 心後，回收上清，得忽布之葉之冷水萃取物。 冷水萃取物之鑑定： 所得之冷水萃取物移入分液漏斗，加己烷而捨棄己烷移 行成分。更於水層加乙酸乙酯，而捨棄乙酸乙酯移行成分 ® 。最後於水層加正甲醇，反復甲醇萃取操作3回所得甲醇 層予以合倂，減壓濃縮，得黃酮醇劃份（由忽布組織之冷水 萃取物分離之類黃酮配糖體）。 . 所得黃酮醇劃份先以高速液體層析（HPLC)分析。依 HPLC 之分析令 C18 柱（Waters Symmetry)於 40°C 使用，流 速作成0.2mL/分。移動相以0.05% TFA7水爲1液，以乙腈 爲2液，2液之比例作成至1 〇 %〜5 0 %以1 6分變化之線斜 率。檢出以350nm之UV檢出器施行。 -12- 200824718 更令上述黃酮醇劃份之各峰以分取用HPLC分離、鑑定 各峰之成分。依HPLC之分取用分離爲令C18柱（Waters SunFire)於40°C使用，流速作成 6mL/分。移動相爲令 10%MeCN保持10分，更以150分作成至60% MeCN變化 之線斜率。檢出以350nm之UV檢出器施行。HPLC之分析 結果如第1圖。 如第1圖所示，於忽布葉之冷水萃取物之黃酮醇劃份有 主峰3個存在，這些皆鑑定爲萘三羥黃酮醇配糖體。詳言 ϋ 之，第1圖之1所示峰爲萘三羥黃酮醇芸香糖苷、2所示 峰爲羊齒精、3所示峰爲萘三羥黃酮醇丙二醯基葡萄糖苷 [製造例2] 冷水萃取： 令忽布（捷克產札字種）之型90柱粒lkg投入蒸餾水10L ，於20°C適宜攪拌下使柱粒消失而靜置一晚。令此以 9 200G離心15分。離心機用日立公司製之CR21G。離心後 ，回收上清，其更予以濃縮，得1 50g之濃縮液（以下稱冷 水萃取物A)。 冷水萃取物之鑑定： 製造冷水萃取物A時所得上清移入分液漏斗，仿製造例 1之方法取得黃酮醇劃份’以HPLC分析而施行成分鑑定 。HPLC分析之結果如第2圖。如第2圖所示，於冷水萃 取物A之黃酮醇劃.份有主要峰3個存在，這些鑑定爲萘三 羥黃酮醇配糖體（羊齒精及萘三羥黃酮醇丙二醯基葡萄糖苷 -13- v 200824718 )和櫟精丙二醯基葡萄糖苷。詳言之，第2圖之1所示峰爲 萘三羥黃酮醇丙二醯基葡萄糖苷、2所示峰爲羊齒精、3 所示峰爲櫟精丙二醯基葡萄糖苷。又第2圖之4所示峰爲 芸香素、5所示峰爲異櫟素、6所示峰爲萘三羥黃酮醇芸 香糖苷。 [製造例3] 冷水卒取： 令乾燥忽布（中日本產）之球花予以超臨界C〇2萃取，其 Φ 殘渣予以柱粒化。此柱粒1000kg投入自來水20kL，於 20°C適宜攪拌下使柱粒消失而靜置12時間。令此以500G 離心後，回收上清，使其更濃縮，得960kg濃縮液（以下稱 冷水萃取物B)。冷水萃取物B中之固形分爲192kg。以下 實施例中，令此固形分調製成該濃度來使用。 冷水萃取物之鑑定： 令冷水萃取物B就製造時所得上清，仿製造例2之方法 施行HPLC分析，結果於冷水萃取物B之黃酮醇劃分有主 ® 要峰3個存在，這些鑑定爲萘三羥黃酮醇配糖體（羊齒精及 萘三羥黃酮醇丙二醯基葡萄糖苷）和櫟精丙二醯基葡萄糖苷 〇 [實施例1-1]酪胺酸酶活性抑制試驗1 於本試驗以冷水萃取物A(製造例2所得者）及冷水萃取 物B(製造例3所得者）爲試料使用。試驗依如下順序施行 〇 於容器加L—酪胺酸40mg和MilliQ水作成lOOmL。其 -14- 200824718 容器於有熱水之燒杯中加溫。更用超音波，使L-酪胺酸溶 解於MilliQ水，作成酪胺酸溶液。令酪胺酸溶液分注而冷 凍保存。 調製含有磷酸二氫鉀（KH2P〇4)4.5 3 6g之酸性MilliQ水溶 液500mL，作爲 A液。調製含有鱗酸氫二鈉（Na2HP〇4) 4.73g或磷酸氫二鈉 12水合物（NaaHPCU · 12水加成物 )1 1.9 3 8g之pH9程度之MilliQ水溶液500mL，作爲B液。 令A液與B液以1之比例混合，而調製PH6.8之1/15M • 磷酸緩衝液。 冷水萃取物A及冷水萃取物B各以MilliQ水稀釋，而 調製各濃度之試料溶液。每一濃度之試料溶液預備1 OmL 扭口試管4支。4支之試管中，2支作爲樣品（+ )用，殘留 之2支作爲樣品（-)用。又預備對照組用之扭口試管4支， 其中2支作爲對照組（+ )用，殘留之2支作爲對照組（-)用。 於此（+ )爲添加酪胺酸溶液，（-)爲不添加酪胺酸溶液。以 下調製各試管之反應溶液。表1乃示各試管之、反應溶液之 馨 組成。 -15- 200824718 【表1】(4) In the whitening agent, the content of the cold water extract in the tissue is such that the component for removing the extraction medium is preferably 0.0001 to 100% by mass based on the total mass of the whitening agent, more preferably 0.01 to 100% by mass, and 1 to 1 〇〇% by mass is more preferable, especially 5 to 100% by mass. The whitening agent may contain an sizing agent, an oily component, a moisturizing agent, a powder, a pigment, an emulsifier, a dispersing aid, a solubilizing agent, a detergent, a UV absorber, and a thickening agent in addition to the cold water extract of the cloth. Agents, pharmaceuticals, perfumes, resins, excipients, antibacterial and antifungal agents, deodorizing and deodorizing agents, enzymes, refining -11- 200824718 Water, alcohol. Other whitening agents can be added. The whitening agent of the present invention can be applied to the skin or the like by a skin external preparation having a whitening effect alone, or can be used as a whitening effect attachment agent by adding a cosmetic or a pharmaceutical agent. It can also be used as an oral ingesting whitening agent which is used for whitening. EXAMPLES Hereinafter, the present invention will be specifically described based on examples and comparative examples, but the present invention is not limited by the following examples. # [Production Example 1] Cold water stroke: The leaves of the cloth (Nurano No. 18, Japan) were chopped, immersed in 10 times (w/v) distilled water and allowed to stand at 5 ° C for one night. After the centrifugation at 9200G, the supernatant is recovered, and the cold water extract of the leaves of the cloth is obtained. Identification of cold water extract: The obtained cold water extract was transferred to a separatory funnel, and hexane was added to discard the hexane shift component. Add ethyl acetate to the aqueous layer and discard the ethyl acetate migration component ® . Finally, methanol was added to the aqueous layer, and the methanol layer obtained by repeated methanol extraction operations was combined and concentrated under reduced pressure to obtain a flavonol fraction (the flavonoid glycoside separated from the cold water extract of the tissue). The resulting flavonol fraction was first analyzed by high speed liquid chromatography (HPLC). The C18 column (Waters Symmetry) was used at 40 ° C according to HPLC analysis, and the flow rate was 0.2 mL/min. The mobile phase was prepared by using 0.05% TFA7 water as the first solution and acetonitrile as the 2 liquid, and the ratio of the 2 liquids to the ratio of 1 分 % to 50% to 16 °. The detection was performed with a 350 nm UV detector. -12- 200824718 Further, the peaks of the above flavonol fractions were separated and identified by HPLC to identify the components of each peak. Separation by HPLC was carried out using a C18 column (Waters SunFire) at 40 ° C, and a flow rate of 6 mL / min. The mobile phase is the slope of the line that keeps 10% MeCN for 10 minutes and 150 minutes to 60% MeCN. The detection was performed with a 350 nm UV detector. The results of HPLC analysis are shown in Figure 1. As shown in Fig. 1, the flavonol fraction of the cold water extract of the leaf was found to have three main peaks, which were identified as naphthalene flavonol glycoside. In detail, the peak shown in Fig. 1 is naphthyltriflavonol rutinoside, the peak shown in 2 is fern, and the peak at 3 is naphthalene flavonol propylene glycol glucoside [Production Example] 2] Cold water extraction: Let the 90 column lkg of the type of the cloth (Czech style) be put into 10L of distilled water, and the column particles disappeared at 20 °C and allowed to stand overnight. Let this centrifuge at 15 200G for 15 minutes. The centrifuge uses CR21G manufactured by Hitachi. After centrifugation, the supernatant was recovered, which was further concentrated to obtain 150 g of a concentrate (hereinafter referred to as cold water extract A). Identification of the cold water extract: The supernatant obtained in the production of the cold water extract A was transferred to a separatory funnel, and the flavonol fraction was obtained by the method of Production Example 1 by HPLC analysis. The results of HPLC analysis are shown in Figure 2. As shown in Fig. 2, the flavonols in the cold water extract A are divided into three main peaks, which are identified as naphthalene triolflavone glycosides (fern and naphthalene tris). Glucoside-13-v 200824718) and quercetin glucosinolate. In detail, the peak shown in Fig. 1 is naphthyltriolol propionate, the peak shown by 2 is fern, and the peak of 3 is bismuth glucosinolate. Further, the peak shown in Fig. 2 and Fig. 4 is rutin, the peak shown by 5 is isoquercetin, and the peak represented by 6 is naphthalene seroflavonol glucoside. [Production Example 3] Cold water stroke: The bulb of the dry cloth (made in Japan) was subjected to supercritical C〇2 extraction, and the Φ residue was subjected to column granulation. 1000 kg of this column pellet was charged with 20 kL of tap water, and the pellet was allowed to disappear under a suitable stirring at 20 ° C and allowed to stand for 12 hours. After centrifuging at 500 G, the supernatant was recovered and concentrated to obtain 960 kg of a concentrate (hereinafter referred to as cold water extract B). The solid form in the cold water extract B was divided into 192 kg. In the following examples, the solid fraction was prepared to be used in this concentration. Identification of the cold water extract: The supernatant obtained by the cold water extract B was subjected to HPLC analysis by the method of Production Example 2. As a result, the flavonol of the cold water extract B was divided into three main peaks, and these were identified as Naphthalene-triflavonol glycoside (frangipani and naphthalene tri-glycolol propylene diglucosylglucoside) and quercetin glucosinolate [Example 1-1] Tyrosinase activity inhibition test 1 In the test, cold water extract A (obtained in Production Example 2) and cold water extract B (produced in Production Example 3) were used as samples. The test was carried out in the following order: 容器 Add L-tyrosine 40 mg and MilliQ water to a container to make 100 mL. Its -14- 200824718 container is heated in a beaker with hot water. Further, by using ultrasonic waves, L-tyrosine was dissolved in MilliQ water to prepare a tyrosine acid solution. The tyrosine solution is dispensed and stored frozen. 500 mL of an acidic MilliQ aqueous solution containing 4.53 6 g of potassium dihydrogen phosphate (KH2P〇4) was prepared as a liquid A. A solution containing 4.73 g of disodium hydrogen citrate (Na2HP〇4) or disodium hydrogen phosphate 12 hydrate (NaaHPCU·12 water adduct) of 1 1.9 3 8 g of a MilliQ aqueous solution of pH 9 was prepared as a liquid B. Mix the liquid A and the liquid B in a ratio of 1 to prepare a 1/15 M phosphate buffer of pH 6.8. The cold water extract A and the cold water extract B were each diluted with MilliQ water to prepare a sample solution of each concentration. Prepare 4 OmL twisted test tubes for each concentration of sample solution. Of the four tubes, two were used as the sample (+), and the remaining two were used as the sample (-). Further, four test tubes for the control group were prepared, two of which were used as the control group (+), and the remaining two were used as the control group (-). Here, (+) is a tyrosine solution, and (-) is a tyrosine solution. The reaction solution of each test tube was prepared as follows. Table 1 shows the composition of each reaction tube and the reaction solution. -15- 200824718 [Table 1]
As Ab As， Ao 樣品⑴ 對照組㈩ 樣品ω 對照組Θ 酪胺酸溶液之有無 + + 輸 衡 試料溶液 2ml dnL ΙΙΠ~ J\\\ 2ml 無 1/15M磷酸緩衝液 2ml 2ml 2ml 2ml 酪胺酸溶液 0.5ml 0.5ml 無 ^ΊΓΓ 無 MilliQ水(液量調整） Μ 2ml 0.5ml 2.5 ml 酵素溶液 0.5ml 0.5ml 0.5ml 0.5ml 依表1，於樣品（+ )用試管及樣品（-)用試管，各添加試料 溶液2mL。於對照組（+ )用試管及對照組㈠用試管，試料溶 液代之以MilliQ水各添加2mL。次於所有之試管各添加 PH6.8之1/15M磷酸緩衝液2mL。 於樣品（+ )用試管及對照組（+ )用試管，各添加酪胺酸溶 液0.5mL。於樣品（-)用試管及對照組（_)用試管，酪胺酸溶 液代之以MilliQ水各添加0.5mL。 於Falcon管投入必要量程度之酪胺酸酶，量其重量。依 酪胺酸酶之量加MilliQ水，調製1〇〇〇單位/mL之酵素溶 液。酵素溶液乃於使用即前調製。所有試管皆添加酵素溶 液0 · 5 m L，試管加蓋，以渦流攪拌之作業每隔1 5秒施行。 所有試管設定於保持37°C之恒溫振盪器内，以70/分振 盪1小時。由恒溫振盪器取出所有試管，混合2、3次， 爲使酵素反應停止而冰冷5分。於充分冷却之96穴板， 令各試管之溶液注意不加溫並各添加300 // L，用板讀機測 定475nm之吸光度。歸零以MilliQ水施行。 -16 - 200824718 由樣品（+)、對照組（+)、樣品（-)、對照組㈠用試管之溶 液測定之吸光度之値各爲As、Ab、As>、Ao時，依下式求 出値作爲酪胺酸酶活性抑制率。第3圖爲展示冷水萃取物 A及冷水萃取物B之酪胺酸酶活性抑制率（％)之圖。作爲 正對照組使用麴酸之MilliQ水稀釋液。 [(Ab-(As-As^))/(Ab-Ao)]xl00(%) 如第3圖所示，因冷水萃取物A及冷水萃取物B之試料 溶液而酪胺酸酶活性抑制率上昇。也即得知於冷水萃取物 • A及冷水萃取物B具有酪胺酸酶活性抑制作用。尤其於添 加冷水萃取物A400ppm之樣品，與對照組相較具有顯著之 酪胺酸酶活性抑制作用。 [實施例1-2]酪胺酸酶活性抑制試驗2 於本試驗，以冷水萃取物C(製造例1所得者）爲試料使 用。試驗仿實施例1 -1之順序施行。 試料溶液乃將冷水萃取物C以MilliQ水稀釋，調製冷水 萃取物C之1倍稀釋溶液（原液）（χΐ)、2倍稀釋溶液（xl/2) ® 、及10倍稀釋溶液（Χίο)。酵素反應後之各試管之溶液則 於96穴板各添加200 μ L。其他則仿實施例1 -1之順序， 求出酪胺酸酶活性抑制率。第4圖展示冷水萃取物C之酪 胺酸酶活性抑制率（％)，以對照組（MilliQ水）之酪胺酸酶活 性抑制率爲0%時之相對値之圖。 如第4圖所示，因冷水萃取物c之試料溶液而濃度依存 地酪胺酸酶活性抑制率減少。也即得知與冷水萃取物C濃 度依存之酪胺酸酶活性抑制作用。 -17- 200824718 [實施例2]對B 16黑色素瘤培養細胞之黑色素生成抑制試 驗， 於本試驗，以冷水萃取物A、冷水萃取物B及冷水萃取 物C爲試料使用。試驗以如下順序施行。 於含有10%之FBS(牛胎兒血清）、青徽素及鏈徽素之 Yeagle MEM培養基，懸浮B16黑色素瘤細胞（人類科學資 源庫〗CRB0202)呈lxlO5細胞/mL，而於板播種。移入調整 爲5 %C〇2之保溫箱，於37 °C培養24小時。於板添加溶解 於DMSO之試料，於同條件培養3日。又試料代之以 DMSO添加時作爲對照組，添加熊果苷（albtin)時爲正對照 組。 培養後，由胰蛋白酶處理來回收細胞，以血球計算盤測 定細胞數。基此算出每穴之細胞數，求出細胞生存率。第 5圖、第7圖、第9圖各爲冷水萃取物A、冷水萃取物B 、冷水萃取物C添加時之黑色素瘤細胞之細胞生存率，以 對照組之細胞生存率爲1〇〇%時之相對値展示之圖。 又細胞數測定後，以磷酸緩衝液PBS(-)將細胞洗淨而回 收。回收之黑色素瘤細胞之白色化狀況以肉眼目視判定。 表2乃示目視判定之基準。第6圖、第8圖、第1〇圖各 爲添加冷水萃取物A、冷水萃取物B、冷水萃取物C時之 黑色素瘤細胞之白色化狀況和目視判定之結果之照片圖。 -18- 200824718 【表2】 判定 基準 + + 與對照組相較極白色 + 與對照組相較白色 土 與對照組相較稍白色 • 與對照組同黑色 由第5圖〜第〗0圖得知，冷水萃取物a、冷水萃取物b 及冷7K萃取物c於無毒性之範圍內依濃度而呈示黑色素生 成抑制作用。 [實施例3]對正常人皮膚3次元模式之黑色素生成抑制試 驗 於本試驗，以冷水萃取物A作爲試料使用。試驗仿如下 順序施行。 於人正常皮膚3次兀模式（MEL-300A(Asian donor)、Lot 、No.676 1、倉紡），UVB以31.5mJ/cm2照射。UV照射乃以 透射照明器DT-20MP(ATTO)施行。UV量之測定乃將UVX 數位輻射計（UVP Inc·)裝在 UVX-31檢出器，而測定 UV310nm之強度。 第11圖爲展示皮膚模式杯之構造之斜視圖（a)及斷面圖 (b)。皮膚模式杯1〇〇爲於組織培養穴2之内部具備培養内 管4。於培養内管之内側依水平方向伸張膜8，組織培養 穴2充塡培養液6至膜8之高度。於培養内管4中之膜8 上，設置由人正常表皮角化細胞或黑細胞而成之組織1 〇。 皮膚模式杯1 00中，由培養液6通過膜8向組織10供給營 -19- 200824718 養。 UV照射後，於皮膚模式杯之培養液添加試料溶液。試 料溶液代之以培養液添加時爲對照組。於調整爲5%C〇2之 保溫箱移入皮膚模式杯，於37。(：開始培養。培養使用EPI-100-LLMM長期維持培養基來施行。24小時之培養後，更 換培養基，再添加試料溶液。3日後，再更換培養基，添 加試料溶液。更2日後，施行培養基更換和試料添加，24 小時後施行細胞毒性試驗。 ® 細胞毒性試驗乃以、MTT分析施行。MTT分析乃依如下 順序施行。皮膚模式杯以PBS250 // L洗淨3回。於注入 MTT溶液300 # L之24穴板投入組織，於調整爲5%C〇2之 37°C保溫箱培養3小時。再皮膚模式杯以PBS洗淨。於含 有0.04N之鹽酸之異丙醇2mL之24穴板投入組織，萃取2 小時。令萃取液200 /z L移入96穴板，測定560nm之吸光 度。又參照物爲65 5nm。由吸光度之値，求出組織細胞之 生存率。第1 2圖爲令冷水萃取物A添加時之組織細胞之 ® 生存率，以對照組之生存率爲100%時之相對値展示之圖 〇 培養後，施行各組織之照片撮影。第1 3圖爲展示對照 組之黑色素生成狀況之照片，第14圖爲展示添加冷水萃 取物A之0.08質量％溶液時之黑色素生成狀況之照片，第 15圖爲展示添加冷水萃取物A之0.008質量％溶液時之黑 .色素生成狀況之照片。 由第12圖〜第15圖得知，添加冷水萃取物A時，與對 -20- 200824718 照組相較組織無黑化。尤其添加冷水萃取物A之0.08 %溶 液時，與對照組相較甚白。也即冷水萃取物A於無細胞毒 性之範圍，依濃度而展示黑色素生成抑制作用。 [產業上之利用可能性] 本發明提供含有忽布萃取物而美白效果特優之美白劑。 【圖式簡單說明】 第1圖由忽布（日本產富良野18號）之葉以水萃取之黃酮 醇劃份之HPLC圖。 ® 第2圖由忽布（捷克產札字種）之柱粒以水萃取之黃酮醇 劃份之HPLC圖。 第3圖展示冷水萃取物A及冷水萃取物B之酪胺酸酶活 性抑制率（％)之圖。 第4圖冷水萃取物C之酪胺酸酶活性抑制率（％)，以對 /照組之酪胺酸酶活性抑制率爲0%時之相對値展示之圖。 第5圖於實施例2添加冷水萃取物A時之黑色素瘤細胞 之細胞生存率（％)，以對照組之細胞生存率爲100%時之相 ^ 對値展示之圖。 第6圖於實施例2令添加冷水萃取物A時之黑色素瘤細 胞之白色化狀況和目視判定之結果之照片圖。 第7圖於實施例2添加冷水萃取物B時之黑色素瘤細胞 之細胞生存率（％)，以對照組之細胞生存率爲1〇〇%時之相 對値展示之圖。 第8圖於實施例2添加冷水萃取物B時之黑色素瘤細胞 之白色化狀況和目視判定之結果之照片圖。 -21- 200824718 第9圖於實施例2添加冷水萃取物C時之黑色素瘤細胞 之細胞生存率（％)，以對照組之細胞生存率爲1 〇〇 %時之相 對値展示之圖。 第1 0圖於實施例2添加冷水萃取物C時之黑色素瘤細 胞之白色化狀況和目視判定之結果之照片圖。 第11圖U)爲展示皮膚模式杯之構造之斜視圖、（b)展示 皮膚模式杯之構造之斷面圖。 第1 2圖於實施例3添加冷水萃取物A時之組織細胞之 Φ 生存率，以對照組之生存率爲1 00%時之相對値展示之圖 〇 第1 3圖展示實施例3中對照組之黑色素生成狀況之照 片。 第14圖展示實施例3中添加冷水萃取物人之0.08質量 %溶液時之黑色素生成狀況之照片。 第15圖展示實施例3中添加冷水萃取物A之0.008質量 %溶液時之黑色素生成狀況之照片。 ® 【主要元件符號說明】 2 組織培養穴 4 培養内管 6 培養液 8 膜 10 組織 100 皮膚模式杯 -22-As Ab As, Ao sample (1) control group (10) sample ω control group 有 tyrosine solution presence + + balance sample solution 2ml dnL ΙΙΠ~ J\\\ 2ml no 1/15M phosphate buffer 2ml 2ml 2ml 2ml tyrosine Solution 0.5ml 0.5ml No^ΊΓΓ No MilliQ water (liquid volume adjustment) Μ 2ml 0.5ml 2.5 ml Enzyme solution 0.5ml 0.5ml 0.5ml 0.5ml According to Table 1, test tube and sample (-) test tube in sample (+) Each sample solution was added in an amount of 2 mL. In the control group (+), a test tube and a control group (1) were used, and the test solution was replaced with 2 mL of MilliQ water. Next to all tubes, 2 mL of 1/15 M phosphate buffer of pH 6.8 was added. To the sample (+), a test tube and a control group (+) were used, and 0.5 mL of a tyrosine solution was added to each. In the sample (-), a test tube and a control group (_) were used, and a tyrosine solution was added instead of 0.5 mL each of MilliQ water. The necessary amount of tyrosinase was added to the Falcon tube and the weight was measured. An amount of 1 〇〇〇 unit/mL of the enzyme solution was prepared by adding MilliQ water to the amount of tyrosinase. The enzyme solution is prepared immediately before use. All test tubes were supplemented with an enzyme solution of 0 · 5 m L, and the test tubes were capped. The operation of vortex stirring was performed every 15 seconds. All tubes were set in a constant temperature shaker maintained at 37 ° C and shaken for 1 hour at 70 / min. All tubes were taken out by a constant temperature oscillator and mixed 2 or 3 times, and chilled for 5 minutes to stop the enzyme reaction. In a well-cooled 96-well plate, the solution of each test tube was taken out without heating and each was added at 300 // L, and the absorbance at 475 nm was measured with a plate reader. Return to zero with MilliQ water. -16 - 200824718 From the sample (+), the control group (+), the sample (-), and the control group (1), the absorbance measured by the test tube solution is As, Ab, As > and Ao, and the following formula is obtained.値 as the inhibition rate of tyrosinase activity. Fig. 3 is a graph showing the inhibition rate (%) of tyrosinase activity of cold water extract A and cold water extract B. As a positive control group, MilliQ water dilution of citric acid was used. [(Ab-(As-As^))/(Ab-Ao)]xl00(%) As shown in Fig. 3, the inhibition rate of tyrosinase activity due to the sample solution of cold water extract A and cold water extract B rise. That is, it was found that the cold water extract • A and the cold water extract B have an inhibitory effect on tyrosinase activity. In particular, a sample of 400 ml of cold water extract was added, which showed significant inhibition of tyrosinase activity compared with the control group. [Example 1-2] Tyrosinase activity inhibition test 2 In this test, cold water extract C (obtained in Production Example 1) was used as a sample. The test was carried out in the same manner as in Example 1-1. The sample solution was diluted with cold water extract C with MilliQ water, and the diluted water extract C was diluted 1 times (stock solution) (χΐ), 2 times diluted solution (xl/2) ® , and 10 times diluted solution (Χίο). The solution of each tube after the enzyme reaction was added to each of the 96-well plates to add 200 μL. Otherwise, the inhibition rate of tyrosinase activity was determined in the same manner as in Example 1-1. Fig. 4 is a graph showing the relative inhibition of the tyrosinase activity inhibition rate (%) of the cold water extract C in the control group (MilliQ water) with a tyrosinase activity inhibition rate of 0%. As shown in Fig. 4, the concentration of the tyrosinase activity was decreased depending on the sample solution of the cold water extract c. That is, the inhibition of tyrosinase activity depending on the concentration of the cold water extract C was known. -17- 200824718 [Example 2] A melanin production inhibition test was performed on B 16 melanoma cultured cells, and in this test, cold water extract A, cold water extract B, and cold water extract C were used as samples. The tests were carried out in the following order. In a Yeagle MEM medium containing 10% FBS (bovine fetal serum), chlorin and aglycone, suspended B16 melanoma cells (Human Science Resource Bank, CRB0202) showed lxlO5 cells/mL, and were seeded on the plate. Transfer to an incubator adjusted to 5% C〇2 and incubate at 37 °C for 24 hours. A sample dissolved in DMSO was added to the plate, and cultured for 3 days under the same conditions. The sample was replaced with DMSO as a control group, and arbutin (albtin) was added as a positive control group. After the culture, the cells were recovered by trypsin treatment, and the number of cells was measured by a blood cell calculation plate. Based on this, the number of cells per hole was calculated, and the cell survival rate was determined. Fig. 5, Fig. 7, and Fig. 9 are cell survival rates of melanoma cells when cold water extract A, cold water extract B, and cold water extract C were added, and the cell survival rate of the control group was 1%. The picture of the time relative to the display. After the number of cells was measured, the cells were washed with phosphate buffer PBS (-) and recovered. The whitening condition of the recovered melanoma cells was visually judged by the naked eye. Table 2 shows the basis for visual judgment. Fig. 6, Fig. 8, and Fig. 1 are each a photograph showing the whitening condition and the result of visual judgment of melanoma cells when cold water extract A, cold water extract B, and cold water extract C were added. -18- 200824718 [Table 2] Judging Criteria + + Compared with the control group, it was extremely white + Compared with the control group, the white soil was slightly whiter than the control group. • The same black color as the control group was obtained from the 5th to the 0th It is known that the cold water extract a, the cold water extract b, and the cold 7K extract c exhibit melanin production inhibition depending on the concentration in a non-toxic range. [Example 3] Test for melanin production inhibition in normal human skin 3-dimensional mode In this test, cold water extract A was used as a sample. The test was carried out in the following order. In normal human skin 3 times sputum mode (MEL-300A (Asian donor), Lot, No. 676 1, spur spinning), UVB was irradiated at 31.5 mJ/cm 2 . UV irradiation was performed with a transmission illuminator DT-20MP (ATTO). The amount of UV was measured by mounting a UVX digital radiometer (UVP Inc.) on a UVX-31 detector to measure the intensity of UV 310 nm. Figure 11 is a perspective view (a) and a sectional view (b) showing the construction of the skin mode cup. The skin mode cup 1 is provided with a culture inner tube 4 inside the tissue culture hole 2. The film 8 is stretched in the horizontal direction on the inner side of the culture inner tube, and the tissue culture hole 2 is filled with the height of the culture solution 6 to the film 8. On the membrane 8 in the culture inner tube 4, a tissue made of human normal epidermal keratinocytes or black cells is placed. In the skin mode cup 100, the culture solution 6 is supplied to the tissue 10 through the membrane 8 to supply the -19-200824718. After the UV irradiation, the sample solution was added to the culture solution of the skin mode cup. The test solution was replaced with a control solution when the culture solution was added. Move the incubator adjusted to 5% C〇2 into the skin mode cup at 37. (: Start culture. The culture was carried out using EPI-100-LLMM long-term maintenance medium. After 24 hours of culture, the medium was changed, and the sample solution was added. After 3 days, the medium was replaced and the sample solution was added. After 2 days, the medium was replaced. The sample was added and the cytotoxicity test was performed 24 hours later. ® The cytotoxicity test was performed by MTT assay. The MTT assay was performed in the following order. The skin mode cup was washed 3 times with PBS250 // L. The injection of MTT solution 300 # L The 24 hole plate was put into the tissue and cultured in a 37 ° C incubator adjusted to 5% C 2 for 3 hours. The skin model cup was washed with PBS. The plate was placed in a 24-well plate containing 2 mL of isopropanol containing 0.04 N hydrochloric acid. The tissue was extracted for 2 hours. The extract 200 / z L was transferred to a 96-well plate, and the absorbance at 560 nm was measured. The reference material was 65 5 nm. The survival rate of the tissue cells was determined from the absorbance. Figure 12 is for cold water. The survival rate of tissue cells when extract A was added was compared with the map of the control group when the survival rate of the control group was 100%. After the culture, photographs of each tissue were performed. Figure 13 shows the melanin of the control group. Photograph of the condition, Fig. 14 is a photograph showing the state of melanin production when a 0.08 mass% solution of cold water extract A was added, and Fig. 15 is a photograph showing the state of black pigmentation when a solution of cold water extract A was added in a 0.008 mass% solution. Photographs from Fig. 12 to Fig. 15 show that when adding cold water extract A, there is no blackening compared with the group of -20-200824718. Especially when adding 0.08% solution of cold water extract A, The group is relatively white. That is, the cold water extract A exhibits melanin production inhibition depending on the concentration in the range of no cytotoxicity. [Industrial Applicability] The present invention provides a whitening effect containing a cloth extract and an excellent whitening effect. Whitening agent. [Simple description of the figure] Figure 1 is an HPLC chart of the flavonol fraction extracted from the leaves of the cloth (Nippon Furano No. 18). ® Fig. 2 is a piece of cloth (Czech type) HPLC chart of flavonol fraction extracted from water by column. Fig. 3 is a graph showing inhibition rate (%) of tyrosinase activity of cold water extract A and cold water extract B. Fig. 4 Cold water extract C Tyrosinase activity inhibition rate (%), The relative 値 display of the tyrosinase activity inhibition rate of the control group is 0%. Figure 5 shows the cell survival rate (%) of the melanoma cells when the cold water extract A is added in Example 2, Fig. 6 is a photograph showing the whitening condition and visual judgment result of melanoma cells when cold water extract A was added in Example 2, in the case where the cell survival rate of the group was 100%. 7 is a graph showing the cell survival rate (%) of melanoma cells when cold water extract B was added in Example 2, and the relative 値 display of the control group when the cell survival rate was 1%. Fig. 8 is a photographic diagram showing the whitening condition and visual judgment result of melanoma cells in the case of adding cold water extract B in Example 2. -21- 200824718 Fig. 9 shows the cell survival rate (%) of melanoma cells in the case of adding cold water extract C in Example 2, and the relative cell display in the control group with a cell survival rate of 1%. Fig. 10 is a photographic diagram showing the whitening condition of the melanoma cells and the results of visual observation when the cold water extract C was added in Example 2. Fig. 11 is a perspective view showing the structure of the skin pattern cup, and (b) a sectional view showing the structure of the skin pattern cup. Fig. 1 is a graph showing the Φ survival rate of tissue cells when cold water extract A was added in Example 3, and the relative 値 display of the survival rate of the control group was 100%. Fig. 13 shows the control in Example 3. A photograph of the melanin production status of the group. Fig. 14 is a photograph showing the state of melanin production in the case of adding 0.08 mass% of a solution of cold water extract to human in Example 3. Fig. 15 is a photograph showing the state of melanin production in the case of adding a 0.008 mass% solution of cold water extract A in Example 3. ® [Main component symbol description] 2 Tissue culture hole 4 Culture inner tube 6 Culture medium 8 Membrane 10 Tissue 100 Skin mode cup -22-