TW201212948A - Extracts from Peony's root cortex for application of antioxidation and method of preparing the same - Google Patents

Abstract

A method of preparing an extract from Peony's root cortex is disclosed, which is performed by at least one extraction step and a gel chromatography step, so as to obtaining the extract from Peony's root cortex. In addition, the extract from Peony's root cortex obtained by the aforementioned method is disclosed, in which the extract from Peony's root cortex includes the relatively high amount of total phenolic compounds and monoterpene glycosides, for example, tetragalloyl glucose, pentagalloyl glucose, galloylpaeoniflorin, benzyoyl-paeoniflorin and mudanpioside B, so as to effectively provide an antioxidative activity for preventing or delaying skin aging, thereby being applied to the cosmetic composition and food additives with antioxidative activity.

Description

201212948 六、發明說明： . 【發明所屬之技術領域】 本發明是有關於一種牡丹皮萃取物及其製造方法，特 別是有關於一種具有抗氧化活性之牡丹皮萃取物及其製造 方法。 【先前技術】 近年來’由於國民生活水準的提升，使得現代人對於 自己的儀谷越來越重視。惟臭氧層日漸破壞，使得人們對 於防曬、抗氧化及抗皮膚老化相關產品的需求量大增，因 而化粧保養品的市場規模逐年擴大。此外，現代人對於健 康觀念萌起’為避免因化學成分之化粧保養品傷害皮膚， 因此中草藥天然物在保健食品與美容上的應用以普遍受到 重視’尤其是將中草藥萃取物應用於化粧品中已成為全球 趨勢，亦是目前國内化妝品及生技產業積極開發的重點。 皮膚老化是必經的過程，老化的皮膚會產生各種組織 形態與生理功能的變化，例如皮膚萎縮、鬆弛、失去彈性 和光澤、產生皺紋、色素變化、皮膚傷口癒合能力及免疫 力降低等。除了因增齡而造成的内因性老化之外，皮膚尚 受到環境諸多不利的影響，而加速或提前老化。這些外因 性老化的原因’例如長期暴露於陽光或高能量放射線下、 飲食不乾淨、組織受損、微生物感染、生活壓力過大、疲 勞過度、或過多的油脂攝取等，也會增加自由基及黑色素 的產生或是代謝因子喪失。 自由基是指分子或原子其價電子具有不成對的電子， U] 4 201212948 例如.〇:〇 ·未成對電子，這種分子或原子呈現極不穩定 的狀態，會攻擊其他分子或原子，藉由搶奪另一個電子以 尋找本身的安定，但通常又造就另一個不安定的自由基生 成。生物體正常有氧代謝過程中會自然形成許多活性氧物 質(reactive 0Xygen species ; R〇s) ’活性氧物質包括超氧陰 離子(superoxide anion radical, 02 ·―)、過氧化氫(hydr〇gen peroxide ’ H202)、氫氧自由基(hydroxyl radical，OH ·)、 早重態氧(single oxygen ; 〇2)等，它代表一群性質活潑，會 攻擊皮膚組織或其它，造成老化與疾病的主要物質之一。 超氧陰離子(〇2·)，為氧分子捕獲一個e-，形成超氧自由 基。如下反應式：〇2 + e- —〇2 ·-。氫自由基·)，為 最強的氧化劑，由過氧化氫(H2〇2)獲得一個e-並失去一個 〇H ;或過氧化氫與超氧自由基反應。如下反應式：201212948 VI. Description of the Invention: [Technical Field] The present invention relates to a peony bark extract and a method for producing the same, and in particular to a peony bark extract having antioxidant activity and a method for producing the same. [Prior Art] In recent years, due to the improvement of the standard of living of the people, modern people have paid more and more attention to their own Yigu. However, the depletion of the ozone layer has led to an increase in demand for products related to sun protection, anti-oxidation and anti-aging skin, and the market size of cosmetic products has been expanding year by year. In addition, modern people have raised the concept of health. In order to avoid damage to the skin caused by chemical makeup products, the application of Chinese herbal medicines in health foods and beauty has been generally valued, especially in the application of Chinese herbal extracts to cosmetics. To become a global trend is also the focus of the active development of the domestic cosmetics and biotechnology industry. Skin aging is an inevitable process. Aging skin can cause changes in various tissue morphological and physiological functions, such as skin atrophy, relaxation, loss of elasticity and luster, wrinkles, pigment changes, skin wound healing, and decreased immunity. In addition to the intrinsic aging caused by ageing, the skin is subject to many adverse effects from the environment, accelerating or premature aging. These causes of exogenous aging, such as long-term exposure to sunlight or high-energy radiation, poor diet, tissue damage, microbial infections, excessive stress, excessive fatigue, or excessive oil intake, also increase free radicals and melanin. Production or loss of metabolic factors. Free radicals are molecules or atoms whose valence electrons have unpaired electrons, U] 4 201212948 For example, 〇:〇·unpaired electrons, such molecules or atoms exhibit a very unstable state, attacking other molecules or atoms, By robbing another electron to find its own stability, it usually creates another unstable free radical. Many active oxygen species (reactive 0Xygen species; R〇s) are naturally formed during normal aerobic metabolism in organisms. 'Reactive oxygen species include superoxide anion radical (02 · ―), hydrogen peroxide (hydr〇gen peroxide) ' H202), hydroxyl radical (OH ·), early oxygen ( 〇 2 ), etc., it represents a group of active substances, will attack skin tissue or other, one of the main substances causing aging and disease . Superoxide anion (〇2·), which captures an e- for oxygen molecules, forms a superoxide free radical. The following reaction formula: 〇 2 + e- - 〇 2 · -. Hydrogen radicals ·), the strongest oxidant, obtain an e- from hydrogen peroxide (H2〇2) and lose a 〇H; or hydrogen peroxide reacts with superoxide radicals. The following reaction formula:

H2〇2 + e— — HCT H2〇2 + 〇2 · ' -> HO · + OH" + 02 活性氧來源可分為内源和外源兩方面，内在來源包括 粒線體電子傳遞鍵（mitochondrial electron transport system) ’ 氧化反應（oxidation)、嗤菌體細胞（phagocytes)、 營養失調(nutritional imbalance)等；另外生物體亦可經由傳 染、離子幅射、空氣污染、抽煙與毒物之入侵而產生活性 氧物質；目前為止，包含内在及外在產生活性氧物質之路 徑，有部分之機轉仍未完全明暸。 這些具未成對電子的自由基化性相當活潑，可和體内 許多重要分子如核酸、蛋白質或生物膜上之多元性不飽和 脂肪酸反應，導致生物體氧化性傷害，主要反應為自由基 201212948 除易引發細胞膜上的不飽和脂肪酸進行脂質過氧化反應外 並會與膜上酵素或接受體行共價結合，導致細胞膜完整性 破壞’改變其結構的功能及通透性；另外自由基亦可和細 胞内之蛋白質進行交錯連結反應致使蛋白質變性或結構改 變’導致生物體内催化生化代謝反應進行所需之酵素活性 喪失’進而使細胞内之正常功能無法進行；而自由基亦會 f擊DMA分子’破壞其鹼基結構使其功能改變，造成基因 突變及毒性之產生；除此之外，脂質過氧化產物再經分子H2〇2 + e— HCT H2〇2 + 〇2 · ' -> HO · + OH" + 02 The source of active oxygen can be divided into endogenous and exogenous sources, and the intrinsic sources include the mitochondrial electron transfer bond ( Mitochondrial electron transport system) 'oxidation, phagocytes, nutritional imbalance, etc.; other organisms can also be produced through infection, ion radiation, air pollution, smoking and poison intrusion Reactive oxygen species; so far, the path of intrinsic and extrinsic production of reactive oxygen species has not been fully understood. These radicals with unpaired electrons are quite active and can react with many important molecules in the body such as nucleic acids, proteins or polyunsaturated fatty acids on biofilms, causing oxidative damage to organisms. The main reaction is free radicals 201212948. It is easy to trigger the lipid peroxidation on the cell membrane and covalently bind to the enzyme or receptor on the membrane, resulting in the destruction of cell membrane integrity. 'Change the function and permeability of the structure; The interstitial reaction of proteins in cells causes protein denaturation or structural changes to cause loss of enzyme activity required for catalytic biochemical metabolic reactions in the organism, which prevents the normal function of the cells from proceeding; and free radicals can also hit DMA molecules. 'Destroy its base structure to change its function, resulting in gene mutation and toxicity; in addition, lipid peroxidation products and molecules

内的環化、裂解專步驟所產生的丙二 (mal〇ndiaidehyde ; MDA)’亦具相當活性會和體内之脂質、蛋白質、核酸等分 子進行交錯連結反應。 有許多證據顯示自由基之堆積與破壞是造成老化或與 老化相關的退行性疾病如癌症、心血管疾病、關節炎及巴 金森氏症等疾病發生的重要因素，因此為了有效的防制疾 病的發生與發展’生物體必須抑制或清除活性氧物質，於 正吊It况了 ±物體中具有抗氧化之防禦系統，這個系統 由兩大部分組成’即抗氧化料及非酵素性之抗氧化物質 以交互協同之仙來移除活性氧與自由基，共同保護生物 體免受到活性氧物質之氧化性傷害。 近年來研九千者在針對疾病和老化此類研究中，有 了較新的發現，就是自由基與抗氧化物質的理論，也因此 於⑽的抗氧化物質產生極大興趣和期盼，積極 的尋找這類型的物質，例如維生素gw)、維 生素E (—__) 1胡蘿蔔鱗咖㈣，超氧歧化 帅up継ide dis_ase，s〇D)，植物萃取物成份等，以達 201212948 - 成延緩老化並對抗疾病的目標。 - ，纟不上所述，雖然皮膚内因性老化難以避免，但外因性 老化可以預防、避免、甚至治療的。有鑑於此，亟需提出 種月b具有抗氧化活性的植物性物質，以期能延緩甚立避 免皮膚組織受到破壞，減少皮膚的皺紋、缺乏彈性、乾燥 等老化現象。 【發明内容】 φ 因此，本發明之一態樣是在提供一種牡丹皮萃取物之 製造方法，其係利用至少一萃取步驟以及凝膠層析步驟， 藉由至少一有機溶液以從牡丹皮獲得具有抗氧化活性的牡 丹皮萃取物，且所得之牡丹皮萃取物並可進一步應用於化 妝品組合物及/或食品添加物。 其次，本發明之另一態樣是在提供一種牡丹皮萃取 物，其係利用上述萃取步驟以及凝膠層析步驟製得之牡丹 皮萃取物。由於所製得之牡丹皮萃取物具有抗氧化活性， • 因此可應用於製備化妝品組合物及/或食品添加物。 再者，本發明之另-態樣是在提供一種牡丹皮萃取物 於抗氧化之應用，其係利用上述萃取步驟以及凝膠層析步 驟，製得具有高效抗氧化活性之牡丹皮萃取物可應用於 延緩老化並對抗疾病。 根據本發明之上述態樣，提出一種牡丹皮萃取物之製 造方法。在一實施例中’首先進行第〜萃取步驟，其係利 用第一有機溶液萃取牡丹皮材料，以獲得第一萃取物。接 著’進行第二萃取步驟’其係利用第二有機溶液萃取前述 201212948 第一萃取物’以劃分出第一有機相以及水相，其中第一有 機相具有第二萃取物，而水相具有第三萃取物。然後’進 行第二卒取步驟，其係利用第三有機溶液萃取前述第_一举 取物，以劃分出第二有機相以及第三有機相，其中第二有 機相具有第四萃取物，而第三有機相具有第五萃取物。之 後’進行凝膠層析步驟，以由第五萃取物獲得具有抗氧化 活性之牡丹皮萃取物，其中此牡丹皮萃取物係以體積比8 : 2之曱醇與水再加10滴醋酸為展開液進行逆相薄層色層層 析片分析，依式（I)獲得例如0.436及0.564之展開阻滯 (retention factor ; Rf)値：The internal cyclization and cleavage-specific step of malondidiaidehyde (MDA) is also quite active and will interlace with lipids, proteins, nucleic acids and other molecules in the body. There is a lot of evidence that the accumulation and destruction of free radicals is an important factor in the development of diseases such as cancer, cardiovascular disease, arthritis and Parkinson's disease, which are caused by aging or aging, so in order to effectively prevent diseases. Occurrence and development 'The organism must inhibit or scavenge reactive oxygen species, and it has an anti-oxidant defense system in the object. This system consists of two major components, namely antioxidants and non-enzymatic antioxidants. The interaction synergy removes reactive oxygen species and free radicals to protect the organism from oxidative damage from reactive oxygen species. In recent years, nine thousand people have made new discoveries in such research on diseases and aging, that is, the theory of free radicals and antioxidants, and therefore the great interest and expectation of antioxidants in (10), positive Look for this type of substance, such as vitamin gw), vitamin E (-__) 1 carrot scale coffee (four), super oxygen disproportionate up継ide dis_ase, s〇D), plant extract ingredients, etc., to reach 201212948 - delaying aging And fight the disease's goals. - , not to mention, although skin aging is difficult to avoid, but external aging can prevent, avoid, or even treat. In view of this, it is urgent to propose a plant material having an antioxidant activity for the month b, in order to delay the damage of the skin tissue and reduce the aging of the skin, such as wrinkles, lack of elasticity, and drying. SUMMARY OF THE INVENTION Accordingly, one aspect of the present invention provides a method for producing a peony bark extract obtained by at least one extraction step and a gel chromatography step obtained from at least one organic solution from peony bark. The peony bark extract having antioxidant activity, and the obtained peony bark extract can be further applied to a cosmetic composition and/or a food additive. Further, another aspect of the present invention provides a peony bark extract which is obtained by the above extraction step and gel chromatography step. Since the prepared peony bark extract has antioxidant activity, it can be applied to the preparation of cosmetic compositions and/or food additives. Furthermore, another aspect of the present invention provides an anti-oxidation application of a peony bark extract, which utilizes the above extraction step and gel chromatography step to obtain a peony bark extract having high antioxidant activity. Used to delay aging and fight disease. According to the above aspect of the invention, a method for producing a peony bark extract is proposed. In one embodiment, the first extraction step is first performed by extracting the peony bark material with the first organic solution to obtain a first extract. Then 'performing a second extraction step', the second organic solution is used to extract the aforementioned 201212948 first extract' to separate the first organic phase and the aqueous phase, wherein the first organic phase has a second extract, and the aqueous phase has a Three extracts. Then performing a second stroke step of extracting the aforementioned first extract using a third organic solution to separate a second organic phase and a third organic phase, wherein the second organic phase has a fourth extract, and The triorganic phase has a fifth extract. Thereafter, a gel chromatography step is performed to obtain an anthocyanin extract having antioxidant activity from the fifth extract, wherein the peony bark extract is obtained by adding 10 drops of acetic acid to a volume ratio of 8:2 sterol and water. The developing solution is subjected to reverse phase thin layer chromatography analysis, and a retention factor (Rf) of, for example, 0.436 and 0.564 is obtained according to the formula (I):

Rf=萃取物移動的距離 ^"液€動的距離 (； 依據本發明一實施例，上述之第一有機溶液例如可為 乙醇溶液，上述之第二有機溶液例如可為乙酸乙酯/水溶 液，且上述之第三有機溶液例如可為正己烷/甲醇溶液。 依據本發明一實施例，上述之第一有機相例如可為乙 酸乙酯相。 依據本發明一實施例，上述之第三有機相例如可為甲 醇相。 依據本發明一實施例，上述之牡丹皮萃取物可包括但 不限於總多酌·化合物(total phenolic compounds)以及單萜配 糖體類化合物（monoterpene glycosides)，例如四沒食子酿基 葡萄糖（tetragalloyl glucose)、三沒食子醯基葡萄糖 (pentagalloyl glucose)、 沒食子醯基芍藥苷 (galloylpaeoniflorin)、苯甲酿基芍藥 ^(benzyoylpaeoniflorin) 201212948 以及牡丹皮苷B(mudanpi〇side B)。 根據本發明之另一態樣，提出一種牡丹皮萃取物。在 一實施例中，此牡丹皮萃取物可利用上述萃取步驟以及凝 膠層析步驟而製得，且此牡丹皮萃取物係以體積比8 : 2之 甲醇與水再加10滴醋酸為展開液進行逆相薄層色層層析 片分析，依式（I)獲得例如〇 436及0.564之展開阻滯 (retention factor ; Rf)値。 根據本發明之其他態樣，提出一種化妝品組合物及/或 φ 食品添加物’其特徵在於此化妝品組合物及/或食品添加物 可包括利用上述萃取步驟以及凝膠層析步驟而製得具有抗 氧化活性之第一萃取物或牡丹皮萃取物，且此第一萃取物 或牡丹皮卒取物具有抗氧化活性。 應用本發明之牡丹皮萃取物及其製造方法’其係藉由 至少一萃取步驟以及凝膠層析步驟，從牡丹皮獲得具有提 高抗氧化活性之牡丹皮萃取物，並可進一步應用於化妝品 組合物及/或食品添加物。 【實施方式】 承前所述’本發明提供一種牡丹皮萃取物及其製造方 法，其係利用至少一萃取步驟以及凝膠層析步驟，以獲得 具有抗氧化活性的牡丹皮萃取物。 本文此處所稱之「牡丹皮」係指毛笑科(及⑽ 芍藥屬（尸β·α)牡丹（户似⑽/似心价妨.⑺扣）的根皮（root cortex)。牡丹皮是一種中草藥，在中醫上主要用來提供涼 血、清熱、活i及散瘀的功能。本發明使用之牡丹皮係經 201212948 林漢鈦博士確認後，即利用本發明以下揭露之方法，提供 具有抗氧化活性的牡丹皮萃取物。 本文此處所稱之「至少一萃取步驟」，實指利用不同極 f生的有機/谷劑糸統萃取牡丹皮。在一個例子中，前述之牡 丹皮在利用乙醇溶液萃取後，接著利用乙酸乙酯/水相、正 己烷/曱醇相等萃取出牡丹皮萃取物。在另一個例子中，前 述之牡丹皮在利用乙醇溶液萃取後，接著利用乙酸乙酯/水 相、正己烷/甲醇相等萃取出牡丹皮萃取物後，更進行凝膠 • 層析步驟，以獲得具有高效抗氧化活性之牡丹皮萃取物。 在又一例子中’前述所得之牡丹皮萃取物以體積比8: 2之 曱醇與水再加10滴醋酸為展開液進行逆相薄層色層層析 片分析’依式（I)獲得例如0.436及0.564之展開阻滯 (retention factor ; Rf)値： 萃取物移動的距離 展開液移動的距離 本文此處所稱之「抗氧化活性」係指前述所得之牡丹 皮萃取物可有效清除自由基、螯合金屬離子、提供還原力、 抑制脂質過氧化，進而達到避免或延緩皮膚老化之功效。 在本發明一個例子中，前述之抗氧化活性可藉由總多酚化 合物以及單莊配酿體類化合物提供，其中總多紛化合物例 如可為式(II)及/或式(III)所示之結構，而單萜配醣體類化合 物例如可為式(IV)之結構：Rf = distance of the movement of the extract ^ " the distance of the liquid movement (; according to an embodiment of the invention, the first organic solution may be, for example, an ethanol solution, and the second organic solution may be, for example, an ethyl acetate/water solution And the third organic solution may be, for example, a n-hexane/methanol solution. According to an embodiment of the invention, the first organic phase may be, for example, an ethyl acetate phase. According to an embodiment of the present invention, the third organic The phase may, for example, be a methanol phase. According to an embodiment of the invention, the above-mentioned peony bark extract may include, but is not limited to, total phenolic compounds and monoterpene glycosides, such as four. Tetragalloyl glucose, pentapalloyl glucose, galloylpaeoniflorin, benzyoylpaeoniflorin 201212948, and peony saponin B (benzinoylpaeoniflorin) Mudanpi〇side B). According to another aspect of the present invention, a peony bark extract is provided. In one embodiment, the peony bark extract The extraction step and the gel chromatography step are used, and the peony bark extract is subjected to reverse phase thin layer chromatography analysis with a volume ratio of 8:2 methanol and water plus 10 drops of acetic acid as a developing solution. According to formula (I), for example, re 436 and 0.564 of retention factor (Rf) 获得 are obtained. According to other aspects of the invention, a cosmetic composition and/or φ food additive is proposed, which is characterized by The composition and/or food additive may comprise a first extract or a peony bark extract having antioxidant activity by using the above extraction step and a gel chromatography step, and the first extract or the peony skin extract Having an antioxidant activity. The peony bark extract of the present invention and a method for producing the same are characterized in that the peony bark extract having an antioxidant activity is obtained from the peony bark by at least one extraction step and a gel chromatography step, and Further, it is applied to a cosmetic composition and/or a food additive. [Embodiment] The present invention provides a peony bark extract and a method for producing the same, which utilize at least one The extraction step and the gel chromatography step are carried out to obtain an extract of peony bark having antioxidant activity. The term "peony bark" as used herein refers to the genus of the genus Lama (and (10) peony (cadmium β·α) peony (household) (10)/like heart price. (7) buckle root root cortex. Peony skin is a kind of Chinese herbal medicine, which is mainly used to provide functions of cooling blood, clearing heat, living i and dilated phlegm in Chinese medicine. After confirmation by Dr. Lin Han Titan of 201212948, the method of the present invention disclosed below is used to provide an extract of peony bark having antioxidant activity. As used herein, "at least one extraction step" refers to the extraction of peony bark using different organic/grain extracts. In one example, the aforementioned sage is extracted with an ethanol solution, and then the peony bark extract is extracted by using an ethyl acetate/aqueous phase and n-hexane/nonanol. In another example, the aforementioned peony bark is extracted with an ethanol solution, and then extracted with an ethyl acetate/aqueous phase, n-hexane/methanol, and then subjected to a gel chromatography step. A peony bark extract with high antioxidant activity. In still another example, 'the aforementioned peony bark extract is obtained by inverse phase thin layer chromatography analysis with a volume ratio of 8:2 sterol and water plus 10 drops of acetic acid as a developing solution'. For example, 0.436 and 0.564 of the retention factor (Rf)値: the distance the extract moves away from the developing solution. The term “antioxidant activity” as used herein means that the aforementioned peony bark extract can effectively scavenge free radicals. Chelate metal ions, provide reducing power, inhibit lipid peroxidation, and thus achieve the effect of avoiding or delaying skin aging. In an example of the present invention, the aforementioned antioxidant activity can be provided by a total polyphenol compound and a single-flavored brewing compound, wherein the total compound can be, for example, represented by formula (II) and/or formula (III). The structure of the monoterpene glycoside compound can be, for example, the structure of the formula (IV):

201212948201212948

在上述式(II)、式（III)及式(IV)所示之結構中，G代表 沒食子醯基(galloyl ; G)，a、c分別為1至2之正整數，b、 d分別為1至3之正整數，而R則可包括但不限於羥基 (〇H)、甲氧基(OCH3)或乙氧基(OC2H5)。在一例示中，上述 式(II)、式（III)或式（IV)所示之結構可包括但不限於四沒食 子醯基葡萄糖(tetragalloyl glucose、三沒食子醯基葡萄糖 (pentagalloyl glucose)、沒食子醯基苟藥苷 (galloylpaeoniflorin)、苯甲醯基芍藥苷 (benzyoylpaeoniflorin)、牡丹皮苷 B(mudanpioside B)或上 述之任意組合。 在本發明另一個例子中，前述之抗氧化活性可藉由例 如四沒食子醯基葡萄糖、三沒食子醯基葡萄糖、沒食子醯 基芍藥苷、苯甲醯基芍藥苷以及牡丹皮苷B提供。 201212948 在一實施例中，本發明之牡丹皮萃取物可利用以下方 法獲得。S青參閱帛1目’其係繪示根據本發明一實施例之 牡丹皮萃取物之製程流程圖。首先，進行第一萃取步驟， 其係利用粉碎機，在室溫或略低於室溫之溫度下，打碎約 18.0公斤之牡丹皮材料ιοί後’將打碎之牡丹皮材料1〇1 浸於95體積百分比之乙醇溶液中。接著，進行過濾步驟， 其係利用紗布、濾紙或其他過濾方式，過濾上述含有粉碎 牡丹皮材料101之95%乙醇溶液，重複四次後，以獲得濾 液。然後’利用減壓濃縮法去除乙醇溶液後，獲得第一萃 取物（以下或稱為Ps-l)103，其中第一萃取物1〇3為牡丹皮 之粗萃取物。 之後，利用乙酸乙酯/水溶液對上述第一萃取物1〇3進 行劃分(partition)步驟，藉以分層獲得第一有機相以及水 相，其中第一有機相為乙酸乙酯相且具有第二萃取物(以下 或稱為Ps-2) 105，而水相則具有第三萃取物（以下或稱為 Ps-3)107。 隨後，利用正己烷(n-hexane)/甲醇溶液對上述第二萃 取物105進行第二萃取步驟，以劃分出第二有機相以及第 二有機相，其中第二有機相（例如正己烧相）具有第四萃取 物（以下或稱為Ps-4)109，而第三有機相（例如甲醇相）具有 第五萃取物（以下或稱為Ps-5)111 ’且其中曱醇溶液可例如 90體積百分比。In the structures represented by the above formula (II), formula (III) and formula (IV), G represents a gallium group (G), a and c are positive integers of 1 to 2, respectively, b, d Each is a positive integer from 1 to 3, and R may include, but is not limited to, hydroxy (〇H), methoxy (OCH3) or ethoxy (OC2H5). In an example, the structure represented by the above formula (II), formula (III) or formula (IV) may include, but is not limited to, tetragalloyl glucose, pentagalloyl glucose. a gallium lp. galantopaeoniflorin, benzyoylpaeoniflorin, mudanpioside B or any combination thereof. In another example of the present invention, the aforementioned antioxidant The activity can be provided, for example, by tetragallium glucosylglucose, trigallocetylglucose, galloin-based paeoniflorin, benzamidine paeoniflorin, and pepiside B. 201212948 In an embodiment, The peony bark extract of the invention can be obtained by the following method. The process of the peony bark extract according to an embodiment of the present invention is shown in the following section. First, the first extraction step is performed, which utilizes The pulverizer pulverizes about 18.0 kg of the peony bark material ιοί at room temperature or slightly below room temperature, and then immerses the smashed peony bark material 1〇1 in 95 volume percent ethanol solution. Enter a filtration step of filtering the 95% ethanol solution containing the pulverized peony material 101 by using gauze, filter paper or other filtering means, and repeating four times to obtain a filtrate. Then, after removing the ethanol solution by a vacuum concentration method, A first extract (hereinafter referred to as Ps-1) 103 is obtained, wherein the first extract 1〇3 is a crude extract of peony bark. Thereafter, the first extract 1〇3 is subjected to ethyl acetate/water solution. a partitioning step whereby the first organic phase and the aqueous phase are obtained by layering, wherein the first organic phase is an ethyl acetate phase and has a second extract (hereinafter referred to as Ps-2) 105, and the aqueous phase has a third extract (hereinafter referred to as Ps-3) 107. Subsequently, the second extraction step 105 is subjected to a second extraction step using a n-hexane/methanol solution to separate the second organic phase and the first a second organic phase, wherein the second organic phase (eg, the normal burnt phase) has a fourth extract (hereinafter referred to as Ps-4) 109, and the third organic phase (eg, methanol phase) has a fifth extract (hereinafter referred to as Is Ps-5)111' and the sterol solution For example, 90 volume percent.

然後，進行凝膠層析步驟，其係將上述第五萃取物1 η 利用例如凝膠層析管柱，而得到牡丹皮萃取物。進一步而 言，凝膠層析步驟係將上述第五萃取物（曱醇相萃取物）1U 12 201212948 通過凝膠層析管柱’例如葡萄糖凝膠層析管柱（Sephadex LH-20 gel chromatography column)，利用！ 0〇 體積百分比之 甲醇沖提後’收集牡丹皮萃取物113。前述牡丹皮萃取物 113經薄層層析片分析後’依沖提收集的先後順序，分成 第一部分、第二部分以及第三部份，其中第三部分即為牡 丹皮萃取物（以下或稱為Ps-8)113。所得之牡丹皮萃取物ία 以體積比8 · 2之曱醇與水再加10滴醋酸為展開液進行逆 相薄層色層層析片分析’依式(I)獲得例如0.436及〇 564 之展開阻滯(retention factor ; Rf)値： 萃取物移動的距離 ΚΙ—----- 展開液移動的距離 值得一提的是，本發明之牡丹皮萃取物並非依溶劑極 性由尚至低的順序進行劃分步驟，而是依序利用乙酸乙酯 相以及曱醇相進行劃分步驟’以萃取出牡丹皮萃取物。由 於利用本發明之方法進行劃分步驟時，分層較為容易，且 不易起泡’因此可縮短製程時間。其次，本發明之方法可 找出牡丹皮中更好且更有效的成分。 本發明所得之牡丹皮萃取物經以下實施例證實，可藉 由萃取而得之特定組成的總多酚化合物以及單莊配醣體類 化合物，例如四沒食子醯基葡萄糖、三沒食子醯基葡萄糖、 沒食子醯基苟藥苷、苯曱醯基苟藥苷以及牡丹皮苷Β，提 供抗氧化活性，因此可應用於抗氧化之化妝品組合物及/或 食品添加物。 舉例而言，本發明所得之牡丹皮萃取物應用於化妝品 組合物時’其形式可包括但不限於水劑、乳劑、膏劑、粉 13 201212948 ' 剤防曬劑、抗氧化劑、抗老化劑或上述任意組合之化妝 品。此外，本發明所得之牡丹皮萃取物應用於食品添加物 時，其形式可包括但不限於抗氧化劑或抗老化劑之營養品 或保健食品。 σ 以下利用數個實施例以說明本發明之應用，然其並非 用以限定本發明’本發明技術領域中具有通常知識者，在 不脫離本發明之精神和範圍内，當可作各種之更動與潤飾。 • 實施例一：牡丹皮萃取物之製備 此實施例係利用第1圖之方法製造牡丹皮萃取物。首 先，進行第一萃取步驟，其係利用粉碎機，在室溫或略低 於室溫之溫度下，打碎約18.0公斤之牡丹皮材料後，將打 碎之牡丹皮材料浸於95體積百分比之乙醇溶液中。接著， 進行過濾步驟，其係利用紗布、濾紙或其他過濾方式，過 濾上述含有粉碎牡丹皮材料之95%乙醇溶液，重複四次 後，以獲得濾液。然後，利用減壓濃縮法去除乙醇溶液後， φ 獲得第一萃取物(Ps·1)10]，其中第一萃取物103為牡丹皮 之粗萃取物。 之後，利用乙酸乙酯/水溶液對上述第一萃取物1〇3進 行第二萃取步驟，以獲得第二萃取物斤3_2)丨〇5以及第三萃 取物(Ps-3) 107，而第二萃取物1〇5為乙酸乙酯萃取物。 隨後，利用正己燒(n-hexane)/甲醇溶液對上述第二萃 取物105進行第二萃取步驟，以劃分出第二有機相與第三 有機相’其中第二有機相（例如正己燒相）具有第四萃取物 (Ps-4) 109，而第二有機相（例如甲醇相）具有第五萃取物 201212948 (Ps-5)1U ’且甲醇溶液可例如9〇體積百分比。 然後’進行凝膠層析步驟，其係將上述第五萃取物 (Ps-5)111利用例如凝膠層析管柱，而得到牡丹皮萃取物， 其中凝膠層析步驟可利用例如葡萄糖凝膠層析管柱 (Sephadex LH-20 gel chromatography column ； Pharmacia Fine Chemicals AB Uppsala，Sweden)進行，並以 loo 體積 百分比之甲醇沖提後，收集各部份的牡丹皮萃取物113。 ，牡丹皮萃取物113經逆相薄層色層層析片分析後，依沖 • 提收集的，序’分成第-部分、第二部分以及第三部份， ，中第二部分(Ps-8)即為牡丹皮萃取物113。所得之牡丹皮 二取，113體積比8: 2之曱醇與水再加10滴醋酸為展開 及，『逆相薄層色層詹析片分析依式⑴獲得例如O we 564 之展開阻滯(retenti〇n factor ; Rf)値：Then, a gel chromatography step is carried out by using the above fifth extract 1 η using, for example, a gel chromatography column to obtain a peony bark extract. Further, the gel chromatography step is to pass the above fifth extract (sterol phase extract) 1U 12 201212948 through a gel chromatography column, such as a glucose gel chromatography column (Sephadex LH-20 gel chromatography column). ),use! 0〇 Volume percent of methanol was extracted and 'peony extract 113 was collected. The aforementioned peony bark extract 113 is analyzed by thin layer chromatography, and is divided into the first part, the second part and the third part according to the order of collection, wherein the third part is the peony bark extract (hereinafter referred to as For Ps-8) 113. The obtained peony bark extract ία is subjected to reverse phase thin-layer chromatography analysis with a volume ratio of 8.2 sterol and water plus 10 drops of acetic acid as a developing solution to obtain, for example, 0.436 and 〇564 according to the formula (I). Retention factor (Rf)値: The distance the extract moves ΚΙ----- The distance of the developing liquid is worth mentioning that the corundum extract of the present invention is not dependent on the polarity of the solvent. The dividing step is sequentially performed, and the ethyl acetate phase and the decyl alcohol phase are sequentially subjected to a dividing step to extract the peony bark extract. When the dividing step is carried out by the method of the present invention, delamination is easy and foaming is not easy' thus shortening the processing time. Second, the method of the present invention can find better and more effective ingredients in the peony bark. The peony bark extract obtained by the present invention is exemplified by the following examples, and the total polyphenol compound and the singly glycoside compound of a specific composition obtained by extraction, for example, tetragallium glucosylglucose, three gallans Mercaptoglucose, galloin-based paeoniflorin, phenylhydrazinine, and quercetin provide antioxidant activity and are therefore useful in antioxidant cosmetic compositions and/or food additives. For example, when the peony bark extract obtained by the present invention is applied to a cosmetic composition, its form may include, but is not limited to, a liquid, an emulsion, an ointment, a powder 13 201212948 '剤 a sunscreen agent, an antioxidant, an anti-aging agent or any of the above. Combination of cosmetics. Further, when the peony bark extract obtained by the present invention is applied to a food additive, the form thereof may include, but is not limited to, an antioxidant or an anti-aging agent nutrient or a health food. The following examples are used to illustrate the application of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes without departing from the spirit and scope of the invention. With retouching. • Example 1: Preparation of Cortex Mouth Extract This example uses the method of Figure 1 to produce a peony bark extract. First, a first extraction step is carried out by using a pulverizer to pulverize about 18.0 kg of peony bark material at room temperature or slightly below room temperature, and then immersing the smashed peony bark material at 95% by volume. In the ethanol solution. Next, a filtration step of filtering the above-mentioned 95% ethanol solution containing the pulverized peony material by gauze, filter paper or other filtration means, and repeating four times, to obtain a filtrate. Then, after removing the ethanol solution by a reduced pressure concentration method, φ obtains a first extract (Ps·1) 10], wherein the first extract 103 is a crude extract of peony bark. Thereafter, the first extract 1 〇 3 is subjected to a second extraction step using an ethyl acetate/water solution to obtain a second extract 3 2) 丨〇 5 and a third extract (Ps-3) 107, and a second The extract 1〇5 was an ethyl acetate extract. Subsequently, the second extraction step 105 is subjected to a second extraction step using a n-hexane/methanol solution to separate the second organic phase from the third organic phase, wherein the second organic phase (eg, the hexaphase phase) There is a fourth extract (Ps-4) 109, and a second organic phase (such as a methanol phase) has a fifth extract 201212948 (Ps-5) 1U ' and the methanol solution can be, for example, 9 volume percent. Then, a gel chromatography step is performed in which the above fifth extract (Ps-5) 111 is obtained by, for example, a gel chromatography column to obtain a peony bark extract, wherein the gel chromatography step can utilize, for example, glucose condensation. After the gel chromatography column (Sephadex LH-20 gel chromatography column; Pharmacia Fine Chemicals AB Uppsala, Sweden) was carried out and washed with loo volume percentage of methanol, each part of the peony bark extract 113 was collected. After the analysis of the peony bark extract 113 by the reverse phase thin layer chromatography, the sample is divided into the first part, the second part and the third part, and the second part (Ps- 8) It is the peony bark extract 113. The obtained peony bark is taken as a development ratio of 113:2 by volume of 8:2 sterol and water plus 10 drops of acetic acid, and the analysis of the reverse phase thin layer color layer is obtained according to the formula (1), for example, the expansion block of O we 564 is obtained. (retenti〇n factor ; Rf)値:

Rf=萃取物移動的距離 展開液移動的距離 （9Rf = distance the extract moves The distance the developer moves (9

實施例二：評估牡丹皮萃取物清除自由基之活性Example 2: Assessing the activity of extracting free radicals from peony bark extract

物匕實施例係利用實施例一不同萃取階段之牡丹皮萃取 ，评估其清除 U-二笨基-2·苦酿腓A ； DPPH * 及r〇l〇x當量的抗氧化能力。 1·Ι)ΡΡΗ·自由基清除能力測定 最士 fPPH•自由基是一種穩定的自由基，在波長51<7nm有 值變吸光值。藉由加入樣品乙醇溶液後測量其517 nm吸光 化及可換算出該濃度下之自由基抑制率。顏色由藍 15 201212948 紫轉為淡黃色，吸光値也隨之降低，因此可藉由517肺下 吸光値的變化，來評估抗氧化物清除自由基的能力，吸光 値越低時表不樣品抗氧化能力越佳。 此例示係以抗壞血酸(asc〇rbic acid，AA ;維生素C ·The material examples were evaluated by the extraction of the peony bark of the different extraction stages of Example 1, and the anti-oxidation ability of the U-di-phenyl-2, abalone, A, DPPH* and r〇l〇x equivalents was evaluated. 1·Ι)ΡΡΗ·Resistance of free radical scavenging ability FPPH•Free radical is a stable free radical with a value of absorbance at a wavelength of 51 < 7 nm. The absorbance at 517 nm was measured by adding a sample ethanol solution, and the radical inhibition rate at that concentration was converted. The color changes from blue 15 201212948 purple to pale yellow, and the absorption enthalpy is also reduced. Therefore, the ability of the antioxidant to scavenge free radicals can be evaluated by the change of 517 subcutaneous absorption ,, and the lower the absorbance, the sample resistance. The better the oxidation capacity. This example is ascorbic acid (asc〇rbic acid, AA; vitamin C ·

Sigma Chemical Co, St. Louis，U.S.A.)為標準品，將實施例Sigma Chemical Co, St. Louis, U.S.A.) is a standard, and the examples will be

-所得之^間萃取物（第-萃取物叫）、第二萃取物 (Ps-2)、第二萃取物(ps_3)、第四萃取物(ps_4)、第五萃取物 (Ps-5))、牡丹皮萃取物（第一部分，ps_6)、牡丹皮萃取物（第 二部分，Ps-7)以及牡丹皮萃取物（第三部分，ps_8)，分別配 製成不同的》辰度（0、1、5、10、50、1〇〇 pg/mL)之乙醇溶液 後，各取10 μι之樣品乙醇溶液，加入新鮮配製9⑼瓜的 100 μΜ DPPH乙醇溶液以及90 gL的DMSO，避光並均勻 混合後’以分光光度計檢測混合液於517 nm之吸光值，並 依下式(V)計算各萃取物的DPPH·自由基之清除效應百分 比（scavenging effects; %)，而所得之結果如第1表之所示， 其中第1表係列出DPPH·自由基之清除效應百分比(％)與 半數效應濃度（medium effective concentration ; EC50) ’ 而每 筆數值至少由大於或等於3個樣本數所得出： DPPH·自由基之清除效應百分比（％)- the resulting extract (the first extract), the second extract (Ps-2), the second extract (ps_3), the fourth extract (ps_4), the fifth extract (Ps-5) ), peony bark extract (Part 1 , ps_6), peony bark extract (Part 2, Ps-7) and peony bark extract (Part 3, ps_8), respectively, are formulated into different "Chen" degrees (0) After the ethanol solution of 1,5,10,50,1〇〇pg/mL), take 10 μl of the sample ethanol solution, add freshly prepared 9 (9) melon 100 μΜ DPPH ethanol solution and 90 gL DMSO, avoiding light and After uniform mixing, the absorbance of the mixture at 517 nm was measured by a spectrophotometer, and the DPPH·free radical scavenging effects (%) of each extract were calculated according to the following formula (V), and the results obtained were as follows. As shown in Table 1, the first table series shows the DPPH·free radical scavenging effect percentage (%) and the half effect concentration (EC50)', and each value is at least 3 or more samples. Out: Percentage of DPPH·free radical scavenging effect (%)

A517nm A517nm ^Hll)xl00% blank (V) 第1表 試劑 DPPH·自由基之清除效應百分比(％) 濃度(pg/mL) 1 5 10 50 1〇〇 EC50 (μβ/niL) 201212948 ---- -----— 第一萃取物(Ps-1) 29.〇±〇.3 44.1 士 1.2 50.0±2.7 56.5 土 4.4 85·7±0.7 10.0 第二萃取物(Ps-2) 32.6±2.5 32.6±〇·6 50.6±4.2 57.3±4.1 86.8±0·7 9.8 第三萃取物(Ps-3) 23.7±1.2 33.2±1·2 38.9±2.7 40.1 士 76.7±1.4 63.6 第四萃取物(Ps-4) 22.3±〇.4 24.4 土 1.6 26.5±2.1 31·9±〇·1 40.5±2.1 >100.0 第五萃取物(Ps-5) 34.〇±&7 56.9士4.8 75.2±2.2 86.6±5.5 89.7±1·0 3.8 牡丹皮萃取物 (第一部分，Ps-6) 23.3±2.5 29.6土〇_6 29·8±4·1 44.1 土 4.4 52.5±1·3 85.1 牡丹皮萃取物 (第二部分，Ps-7) 21-8±〇.6 45.0±2.0 58.2 士 0.2 89.3±〇.4 90.7±1.2 6.9 牡丹皮萃取物 (第三部分，Ps-8) 51.9±〇.9 75.7 士 Ο.3 79.2±0.1 79_6±0.1 81.7±1.7 0.7 維他命C 25.〇±〇.9 61.3±3.5 78_6土3.5 92.4±〇·7 91.4±3·1 3.8 由第1表之結果得知，實施例一所得之不同萃取階段 之牡丹皮萃取物對於清除DPPH·自由基之濃度與清除效 應百分比皆成正相關，惟牡丹皮萃取物（第三部分，ps-8) 之清除DPPH ·自由基的活性，顯著高於維生素C以及其 他不同萃取階段之萃取物。 2.ABTS · +陽離子自由基清除活性（ABTS radical scavenging activity) 2,2 -次偶氮基-雙（3-乙基笨並°塞σ坐嘛-6-績酸） (2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid ； ABTS) 在過氧化酶(peroxidase)的催化下，會產生ABTS · +自由基， 而呈現穩定的藍綠色。因此藉由加入抗氧化物參與反應， ABTS ·+得以還原成ABTS而抑制藍綠色生成，並經由測 定734 nm吸光值的變化，可評估抗氧化物的抗氧化能力， 201212948 • 且當吸光値越低時，表示樣品的抗氧化能力越佳。由於此 測定方式係以水溶性維他命 E(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid，Ίϊοίοχ)為正標準品，製作標準曲線以作為對照並評 估其總抗氧化能力，因此又稱為Tr〇l〇x當量的抗氧化能力 (Trolox Equivalent Antioxidant Capacity ； TEAC) ° 此例示係以Trolox為標準品，將實施例一所得之中間 萃取物（第一萃取物(Ps-Ι)、第二萃取物(Ps_2)、第三萃取物 φ (Ps-3)、第四萃取物(ps_4)、第五萃取物(Ps_5))、牡丹皮萃 取物（第一部分，ps_6)、牡丹皮萃取物（第二部分，Ps_7)以 及牡丹皮萃取物（第三部分，Ps-8)，分別配製成不同的濃度 (0、1、5、1〇、50、1〇〇 pg/mL)之樣品溶液後，取不同濃度 點之樣品溶液1.5 μί ’加入3.5 kL ddH2〇(二次去離子水) 以及145 pL之7 mM ABTS · +溶液，避光混合均勻後，於 室溫靜置20分鐘。 之後’利用分光光度計測定trolox於734 nm之吸光 φ 值’作出trolOX標準曲線，以藉由標準曲線換算出各樣品 之TEAC值以及半數效應濃度（medium effective concentration ; EC5〇)，並依下式（VI)計算各萃取物的 ABTS · +自由基之清除效應百分比。所得之結果如第2表 之所示’其中第2表係列出ABTS · +自由基之清除效應百 分比(％)、半數效應濃度(EC5Q)與TEAC(mgtrolox/g) ’每筆 數值至少由大於或等於3個樣本數所得出： ABTS · +自由基之清除效應百分比(％) 201212948 A734nm „amnle =(1--^^)xl00 % (VI) A734nm blankA517nm A517nm ^Hll)xl00% blank (V) Table 1 reagent DPPH·free radical scavenging effect percentage (%) concentration (pg/mL) 1 5 10 50 1〇〇EC50 (μβ/niL) 201212948 ---- ------ First extract (Ps-1) 29.〇±〇.3 44.1 ± 1.2 50.0±2.7 56.5 Soil 4.4 85·7±0.7 10.0 Second extract (Ps-2) 32.6±2.5 32.6 ±〇·6 50.6±4.2 57.3±4.1 86.8±0·7 9.8 Third extract (Ps-3) 23.7±1.2 33.2±1·2 38.9±2.7 40.1 ±76.7±1.4 63.6 Fourth extract (Ps-4 22.3±〇.4 24.4 Soil 1.6 26.5±2.1 31·9±〇·1 40.5±2.1 >100.0 Fifth extract (Ps-5) 34.〇±&7 56.9士4.8 75.2±2.2 86.6±5.5 89.7±1·0 3.8 Cortex peel extract (Part I, Ps-6) 23.3±2.5 29.6 Bandit _6 29·8±4·1 44.1 Soil 4.4 52.5±1·3 85.1 Cortex peel extract (Part 2 , Ps-7) 21-8±〇.6 45.0±2.0 58.2 ± 0.2 89.3±〇.4 90.7±1.2 6.9 Cortex peel extract (Part III, Ps-8) 51.9±〇.9 75.7 士Ο.3 79.2±0.1 79_6±0.1 81.7±1.7 0.7 Vitamin C 25.〇±〇.9 61.3±3.5 78_6 soil 3.5 92.4±〇·7 91.4±3·1 3.8 From the results of the first table The peony bark extract obtained in different extraction stages obtained in Example 1 has a positive correlation with the concentration of DPPH·free radicals and the percentage of scavenging effect, but the removal of DPPH from the peony bark extract (Part 3, ps-8) The activity is significantly higher than that of vitamin C and other extracts at different extraction stages. 2.ABTS · + cationic radical scavenging activity (ABTS radical scavenging activity) 2,2 - azo-bis (3-ethyl stupid ° 坐 坐 -6-6-acid) (2,2'- Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid; ABTS), under the catalysis of peroxidase, produces ABTS · + radicals and exhibits a stable blue-green color. Therefore, participation by adding antioxidants The reaction, ABTS · + can be reduced to ABTS to inhibit the formation of blue-green, and the antioxidant capacity of the antioxidant can be evaluated by measuring the change in absorbance at 734 nm, 201212948 • and the lower the absorbance, the oxidation resistance of the sample The better the ability, because this method is based on water-soluble vitamin E (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Ίϊοίοχ) as a positive standard, making a standard curve as a control and evaluating it. Total antioxidant capacity, therefore also known as Tr〇l〇xx equivalent of antioxidant capacity (Trolox Equivalent Antioxidant Capacity; TEAC) ° This example uses Trolox as a standard, the intermediate extract obtained in Example 1 (first extraction) (Ps-Ι), second extraction (Ps_2), third extract φ (Ps-3), fourth extract (ps_4), fifth extract (Ps_5)), peony bark extract (part one, ps_6), peony bark extract (second Part, Ps_7) and the peony bark extract (Part 3, Ps-8) were prepared into sample solutions of different concentrations (0, 1, 5, 1 〇, 50, 1 〇〇pg/mL). Add 1.5 μί of sample solution at different concentration points to 3.5 kL ddH2 〇 (secondary deionized water) and 145 pL of 7 mM ABTS · + solution, mix well in the dark, and let stand at room temperature for 20 minutes. The spectrophotometer measures the absorbance φ value of trolox at 734 nm' to make a trolOX standard curve to convert the TEAC value and the medium effect concentration (EC5〇) of each sample by the standard curve, and according to the following formula (VI) Calculate the percentage of the ABTS · + radical scavenging effect of each extract. The results obtained are shown in Table 2, where the second table series shows the ABTS · + radical scavenging effect percentage (%) and the half effect concentration (EC5Q). ) with TEAC (mgtrolox/g) 'each value is at least 3 samples greater than or equal to Number of results: ABTS · + Free radical scavenging effect percentage (%) 201212948 A734nm „amnle =(1--^^)xl00 % (VI) A734nm blank

第2表 試劑 ABTS · +自由基之清除效應百分比(％) TEAC 濃度 (pg/mL) 1 5 10 50 100 ec5〇 (pg/mL) (mg trolox/g) 第一萃取 物（Ps-1) 8.9±2.6 19.9±1.4 31.6 士 0.9 88.7±2.5 99.1±1.0 22.9 239 第二萃取 物（Ps-2) 14.4±1.2 28.4士2.2 48.3±1.7 99.7±0.3 100.5±0.1 11.4 485 第三萃取 物（Ps-3) 9·2±0·5 12.9±0.5 20.0±0.9 47.2±1.0 92.8±0.5 53.1 103 第四萃取 物（Ps-4) 13.0±0·5 36.6±1.1 62.8±0.4 99·6±0·1 100.1±0.2 7.5 727 第五萃取 物（Ps-5) 7.5±0.4 7.7±1.6 9.1±2.1 9.7±0.1 20.2±0.3 >100.0 — 牡丹皮萃取 物(第一部分 5·4±1.4 9.4±0.4 10.2±3.3 28.1±0.3 51.1±2.2 97.6 56.1 ，Ps-6) 牡丹皮萃取 物(第二部分 5.7 士 0.6 19.9±2.0 39.1±0.2 99.5 士 0.4 100.0±0.1 43.7 293 ，Ps-7) 牡丹皮萃取 物(第三部分 16.3±0.9 58.2±0.5 99.5±0.1 100.2±0.1 100.0±0.1 4.2 1299 ，Ps-8) Trolox 5.8±0.6 45.5±1.2 92.4±4.7 100.0 士 0.1 100.0±1.7 5.4 1000 由第2表之結果得知，實施例一所得之不同萃取階段 i Si 19 201212948 之牡^皮萃取物對於清除ABTS · +自由基之丨農度與清除效 應百分比皆成正相關，惟牡丹皮萃取物（第三部分，ps_8) 之清除ABTS · +自由基的活性，顯著高於水溶性維生素e (Trolox)以及其他不同萃取階段之萃取物。 實施例二：評估牡丹皮萃取物之總多紛化合物及單祐 配糖禮類化合物的含量、螯合金屬離子能力以及還原能力 此實施例係利用實施例一不同萃取階段之牡丹皮萃取 物’评估其總多齡化合物(totai phen〇iic conip〇un(is)以及單 萜配醋體類化合物(monoterpene glycosides)的含量，並評估 其螯合金屬離子能力以及還原能力。 1·總多酚化合物之測定 此例示可利用習知方法，以沒食子酸(gallic acid; Sigma Chemical Co, St· Louis, U.S.A.)為標準品。接著，將實施例 一所得之中間萃取物（第一萃取物（Ps-1)、第二萃取物 (Ps-2)、第三萃取物(Ps-3)、第四萃取物(Ps-4)、第五萃取物 (Ps-5))、牡丹皮萃取物（第一部分，Ps-6)、牡丹皮萃取物（第 二部分，Ps-7)以及牡丹皮萃取物（第三部分，Ps-8)，配製為 不同濃度後’各取50 pL ’再加入250 μ!>的Folin-Ciocalteu 酚類試劑(磷鉬酸化複合物，10倍稀釋），震盪混合約1分 鐘後，靜置約5分鐘。之後’加入約200 pL之碳酸鈉溶液 (7.5 % w/v)以及約500 μ!^之二次去離子水，於室溫避光1 小時後，利用分光光度計（波長760 nm)(Hitachi U-2001， Japan)測定沒食子酸溶液之吸光值（以蒸餾水之吸光值為空 白值），作出標準曲線’然後藉由標準曲線換算出各樣品之 20 201212948 總多酚化合物之含量。所得之結果如第3表之所示，其中 第3表係列出總多酚化合物之含量，每筆數值至少由大於 或等於3個樣本數所得出。上述各萃取物之總多酚化合物 之含量係換算成每公克之沒食子酸當量（gallic acid equivalent ; GAE)的毫克量(mg 〇f GAE/g)。 2.單萜配聽體類化合物之測定 此例示可利用習知方法，以芸香苷（rutin ; SigmaTable 2 Reagents ABTS · + Free radical scavenging effect percentage (%) TEAC concentration (pg/mL) 1 5 10 50 100 ec5 〇 (pg/mL) (mg trolox/g) First extract (Ps-1) 8.9±2.6 19.9±1.4 31.6 ± 0.9 88.7±2.5 99.1±1.0 22.9 239 Second extract (Ps-2) 14.4±1.2 28.4 ± 2.2 48.3±1.7 99.7±0.3 100.5±0.1 11.4 485 Third extract (Ps- 3) 9·2±0·5 12.9±0.5 20.0±0.9 47.2±1.0 92.8±0.5 53.1 103 Fourth extract (Ps-4) 13.0±0·5 36.6±1.1 62.8±0.4 99·6±0·1 100.1±0.2 7.5 727 Fifth extract (Ps-5) 7.5±0.4 7.7±1.6 9.1±2.1 9.7±0.1 20.2±0.3 >100.0 — Cortex peel extract (Part 1 5. 4±1.4 9.4±0.4 10.2± 3.3 28.1±0.3 51.1±2.2 97.6 56.1 , Ps-6) Cortex extract (Part II 5.7 ± 0.6 19.9 ± 2.0 39.1 ± 0.2 99.5 ± 0.4 100.0 ± 0.1 43.7 293 , Ps-7) Cortex extract (No. Three parts 16.3 ± 0.9 58.2 ± 0.5 99.5 ± 0.1 100.2 ± 0.1 100.0 ± 0.1 4.2 1299 , Ps-8) Trolox 5.8 ± 0.6 45.5 ± 1.2 92.4 ± 4.7 100.0 ± 0.1 100.0 ± 1.7 5.4 1000 by the As shown in the results of Table 2, the extracts of the different extraction stages i Si 19 201212948 obtained in Example 1 were positively correlated with the percentage of the removal of ABTS·+ radicals and the percentage of scavenging effect, but the peony bark extract ( In the third part, ps_8) removes ABTS · + free radical activity significantly higher than water-soluble vitamin e (Trolox) and other extracts of different extraction stages. Example 2: Evaluating the total amount of the compound of the peony bark extract and the content of the sedative compound, the ability to chelate the metal ion and the reducing ability. This example uses the peony peel extract of the different extraction stages of the first embodiment. To evaluate the content of total multi-age compounds (totai phen〇iic conip〇un(is) and monoterpene glycosides, and to evaluate their ability to chelate metal ions and reduce their ability. 1. Total polyphenolic compounds This example can be determined by a conventional method using gallic acid (Sigma Chemical Co, St. Louis, USA) as a standard. Next, the intermediate extract obtained in Example 1 (first extract ( Ps-1), second extract (Ps-2), third extract (Ps-3), fourth extract (Ps-4), fifth extract (Ps-5)), peony bark extract (Part I, Ps-6), Cortex Moutan Extract (Part 2, Ps-7) and Mudan Peel Extract (Part 3, Ps-8), prepared at different concentrations, then take 50 pL each and then add 250 μ!> Folin-Ciocalteu phenolic reagent (phosphomolybdate complex, 10-fold dilution After shaking for about 1 minute, let stand for about 5 minutes. Then add about 200 pL of sodium carbonate solution (7.5 % w/v) and about 500 μ!^ of twice deionized water to avoid light at room temperature. After an hour, the absorbance of the gallic acid solution was measured by a spectrophotometer (wavelength 760 nm) (Hitachi U-2001, Japan) (the absorbance value of the distilled water was a blank value), and a standard curve was made, and then converted by a standard curve. The content of the total polyphenol compound in each sample is 20 201212948. The results obtained are shown in Table 3, wherein the third table series contains the total polyphenol compound content, and each value is at least 3 samples or more. The total polyphenolic compound content of each of the above extracts is converted into milligrams of gallic acid equivalent (GAE) per gram (mg 〇f GAE/g). Determination of a class of compounds This example can be determined using conventional methods, rutin (rutin; Sigma

Chemical Co, St. Louis，U.S.A.)為標準品。接著，將實施例 • 一所得之中間萃取物（第一萃取物（Ps-1)、第二萃取物 (Ps-2)、第三萃取物(ps_3)、第四萃取物(ps 4)、第五萃取物 (Ps-5))、牡丹皮萃取物（第一部分，ps_6)、牡丹皮萃取物（第 二部分，Ps-7)以及牡丹皮萃取物（第三部分，ps_8)，配製為 不同濃度後，各取37 μί，再加入49〇 之二次去離子水 以及約3 pL之亞硝酸鈉溶液(5 % w/v)，反應約6分鐘。之 後，加入3〇01^之1〇%三氣化鋁(八1(：13)溶液並反應6分鐘 後’再加入400 pL之4°/。氫氧化鈉(Na〇H)溶液及4〇吣 籲之-次去離子水震盪混合均勻後，靜置ls分鐘。然後，利 用刀光光度计（Hitachi U-2001，japa_定芸香苦溶液於 51〇 nm之吸光值（以蒸餘水之吸光值為空白值），作出標準 曲線:然後藉由標準曲線換算出各樣品之總類黃銅化合物 之含:E。所得之結果如第3表之所示，其中第3表係列出 總類黃酮化合物之含量，每筆數值至少由大於或等於3個 樣本數所知出。上述各萃取物之總類黃綱化合物之含量係 換算成每公克之芸香普當量的亳克量㈣Gf加响）。 3·螯合金屬離子能力之測定 21 201212948 - 在多種金屬離子中，亞鐵離子(Fe2+)是最具影響力的促 氧化劑，會促進脂質氧化作用的進行。利用亞鐵離子與亞 鐵嗪(4,4’-[3·(2_吡啶基）-1，2,4-三嗪-5,6-二基]-二苯磺酸二 納鹽 ’ 4,4丨-(3 -(2-pyridinyl)-1，2,4-triazine-5,6-diyl) bisbenzenesulfonic acid，disodium salt ; ferrozine)的複合物 在波長562 nm之下會有呈色反應，玎以測得樣品對Fe2+ 的螯合此力。當樣品螯合Fe2+時，會造成562 nm之吸光值 的降低。因此吸光值越低表示樣品的螯合能力越強。是以， 此例示係藉由評估樣品螯合亞鐵離子的能力，從而測定其 抑制脂質過氧化的能力。 此例示可利用習知方法，以乙二胺四乙酸 ((ethylenedinitrilo)tetraacetic acid ； EDTA ； Sigma Chemical Co, St· Louis’U.S.A.)為標準品’將實施例一所得之中間萃 取物（第一萃取物(Psd)、第二萃取物(Ps_2)、第三萃取物 (Ps-3)、第四萃取物(ps_4)、第五萃取物(ps_5))、牡丹皮萃 取物（第一部分，PS_6)、牡丹皮萃取物（第二部分，ps_7)以 φ 及牡丹皮萃取物（第三部分，Ps·8)各取1 μί，依序加入93 pL 二次去離子水以及2 pL之2 mM之氣化亞鐵(FeCl2)溶液 靜置30秒後’再加入4pL之5mM的亞鐵嗪，反應1〇分 鐘後，以分光光度計檢測其於562 nm之吸光值，並依下式 (VII)計算EDTA溶液與各萃取物之螯合亞鐵能力 所得之結果如第3表之所示’其中第3表更列出$合亞鐵 能力百分比，每筆數值至少由大於或等於3個樣本數所得 出。上述各萃取物之螯合亞鐵能力百分比係換算成每公克 之 EDTA 當量（EDTA equivalent)的毫克量（mg 〇f ⑹。 Γ Γ· Λ ί Μ 22 201212948 螯合亞鐵能力百分比(％) =(1-4STsampie)x100 % (VII) A562nm bIank 4·還原能力之測定 此例示係利用普魯士藍[Fe4(Fe(CN)6)3]之生成量，作為 測定還原能力之指標。其原理則是將赤血鹽（potassium ferrocyanide ; K3Fe(CN)6)提供的三價鐵離子（Fe3+)，還原成 二價鐵離子(Fe2+)之黃血鹽(K4Fe(CN)6)，黃血鹽再與三氯化 鐵(FeCh)提供的Fe3+反應形成普魯士藍，藉由700 nm處吸 光值偵測普魯士藍的含量，以得知其還原力大小，因此吸 光值越高表示樣品的還原力越強。 此例示可利用習知方法，以維生素C(AA ;抗壞血酸) 為標準品’將實施例一所得之中間萃取物（第一萃取物 (Ps-Ι)、第二萃取物(Ps_2)、第三萃取物(Ps-3)、第四萃取物 (Ps-4)、第五萃取物（ps_5))、牡丹皮萃取物（第一部分， Ps·6)、牡丹皮萃取物（第二部分，Ps-7)以及牡丹皮萃取物（第 三部分’ Ps-8) ’配製成不同濃度後，各取20 μί之樣品溶 液，分別加入50 pL之0.2 Μ磷酸緩衝溶液(phosphate buffer ’ pH 6.6，含 1%赤血鹽[K3Fe(cN)6]，於 50°C 之水浴 中反應20分鐘後，再加入5〇 μΙ>之ι〇〇/0三氣醋酸 (trichloroacetic add ; TCA)溶液，經離心（轉速為 loooo rpm，10分鐘）後，取25吣之上清液加入65 9吣之二次 去離子水及9·1 gL之0.1%三氣化鐵(FeCl3)溶液反應後，以 分光光度計檢測其於700 nm之吸光值。 利用分光光度計（波長700 nm)測定維生素C之吸光 23 201212948 值，作出標準曲線’然後藉由標準曲線換算出各樣品之還 原力。所得之結果如第3表之所示，其中第3表每筆數值 至少由大於或等於3個樣本數所得出。上述各萃取物之還 原力係換算成每公克之維生素c當量(AA equivalent)的毫 克量(mg AA/g)。 第3表 萃取物 總多酚化合物 (mg of GAE/g) 總類黃酮 化合物 (mg of rutin/g) 螯合亞鐵能力 還原能力 (mgofEDTA/g) (mgofAA/g) 第一萃取物（Ps-1) 17.3±0.4 2.2±0.2 1.9±1.6 1.7±0.5 第二萃取物（Ps-2) 30·2±0.8 21.2±0.5 1.7±1.4 1.9±1.5 第三萃取物（Ps-3) 9·2±0.1 — 1.1±0.5 1.1±0.2 第四萃取物（Ps-4) 一 — 0.9±0.8 0.5±0.1 第五萃取物（Ps-5) 44_1±0.6 29.8±1.1 2.5±1.7 1 ·3 土 0*5 牡丹皮萃取物 (第一部分，Ps_6) 7·0±1.0 6.0 士 0.2 2.1±1.5 1.1±0_3 牡丹皮萃取物 (第二部分，Ps-7) 38.9±0·4 5.1 土 1.2 2.1±1.3 1.5±0.2 -------- 牡丹皮萃取物 80.8±0.3 l〇1.8±1.9 4.0±0.5 1.7士1.5 (第三部分，Ps-8)Chemical Co, St. Louis, U.S.A.) is a standard. Next, the obtained intermediate extract (first extract (Ps-1), second extract (Ps-2), third extract (ps_3), fourth extract (ps 4), Fifth extract (Ps-5)), peony bark extract (Part 1 , ps_6), peony bark extract (Part 2, Ps-7) and peony bark extract (Part 3, ps_8), formulated as After different concentrations, each was taken for 37 μί, then 49 〇 of twice deionized water and about 3 pL of sodium nitrite solution (5 % w/v) were added for about 6 minutes. Then, add 3〇01^ of 1〇% tri-aluminized aluminum (eight 1 (:13) solution and react for 6 minutes' then add 400 pL of 4°/. sodium hydroxide (Na〇H) solution and 4〇吣 之 - 次 - 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次 次The absorbance value is a blank value), and a standard curve is prepared: then the total brass compound content of each sample is converted by the standard curve: E. The results obtained are shown in Table 3, wherein the third table is a general class. The content of the flavonoid compound is known by at least three sample numbers per sample. The content of the total class of the yellow class compound of each of the above extracts is converted into the amount of gram per gram of eucalyptus equivalent (four) Gf plus) 3. Determination of the ability to chelate metal ions 21 201212948 - Among various metal ions, ferrous ions (Fe2+) are the most influential oxidants, which promote the oxidation of lipids. The use of ferrous ions and ferrous salts (4,4'-[3·(2_pyridyl)-1,2,4-triazin-5,6-diyl]-diphenylsulfonic acid The salt of '4,4丨-(3-pyridinyl)-1,2,4-triazine-5,6-diyl) bisbenzenesulfonic acid, disodium salt; ferrozine) will have a wavelength of 562 nm. In the color reaction, 玎 is used to measure the chelation of Fe2+ by the sample. When the sample chelate Fe2+, it will decrease the absorbance at 562 nm. Therefore, the lower the absorbance value, the stronger the chelation ability of the sample. This example demonstrates its ability to inhibit lipid peroxidation by assessing the ability of a sample to sequester ferrous ions. This example can be accomplished using conventional methods with ethylenediaminetetraacetic acid (EDA); EDTA; Sigma Chemical Co, St. Louis' USA) is the standard product of the intermediate extract obtained in Example 1 (first extract (Psd), second extract (Ps_2), third extract (Ps-3), Four extracts (ps_4), fifth extract (ps_5)), peony bark extract (Part I, PS_6), peony bark extract (Part II, ps_7) with φ and peony bark extract (Part III, Ps·8) Take 1 μί each, sequentially add 93 pL of secondary deionized water and 2 pL of 2 mM gasification After the solution of (FeCl2) was allowed to stand for 30 seconds, 4 pL of 5 mM ferrous iron was added, and after 1 minute of reaction, the absorbance at 562 nm was measured by spectrophotometer, and the EDTA solution was calculated according to the following formula (VII). The results of the chelating ferrous ability of each extract are shown in Table 3, where Table 3 further lists the percentage of ferrous iron capacity, and each value is obtained by at least 3 sample numbers. The percentage of chelating ferrous capacity of each of the above extracts is converted into milligrams per gram of EDTA equivalent (EDTA equivalent) (mg 〇f (6). Γ Γ · Λ ί Μ 22 201212948 Percentage of chelating ferrous capacity (%) = (1-4STsampie) x100 % (VII) A562nm bIank 4·Measurement of reducing ability This example uses the amount of Prussian blue [Fe4(Fe(CN)6)3] produced as an index for measuring the reducing power. The principle is The ferric salt (Fe3+) provided by potassium ferrocyanide (K3Fe(CN)6) is reduced to the divalent iron ion (Fe2+) yellow blood salt (K4Fe(CN)6), and the yellow blood salt is re- The Fe3+ reaction provided by ferric chloride (FeCh) forms Prussian blue, and the content of Prussian blue is detected by the absorbance at 700 nm to know the reducing power. Therefore, the higher the absorbance value, the stronger the reducing power of the sample. This example can be obtained by using a conventional method using vitamin C (AA; ascorbic acid) as a standard. The intermediate extract obtained in Example 1 (first extract (Ps-Ι), second extract (Ps_2), third Extract (Ps-3), fourth extract (Ps-4), fifth extract (ps_5)), peony bark extract ( The first part, Ps·6), the peony bark extract (Part 2, Ps-7) and the peony bark extract (Part 3 'Ps-8)' are formulated into different concentrations, each taking 20 μί sample solution Add 50 pL of 0.2 Μ phosphate buffer solution (phosphate buffer 'pH 6.6, containing 1% red blood salt [K3Fe(cN)6], react in a water bath at 50 °C for 20 minutes, then add 5 〇μΙ> The solution of trichloroacetic acid (TCA) was centrifuged (roto rpm, 10 minutes), and the supernatant was added to 25 吣 of the second deionized water and 9·9. After reacting 1 gL of 0.1% iron trioxide (FeCl3) solution, the absorbance at 700 nm was measured by spectrophotometer. The absorbance of vitamin C was measured by spectrophotometer (wavelength 700 nm), and the standard curve was made. 'The reducing power of each sample is then converted by the standard curve. The results obtained are shown in Table 3, wherein each value of the third table is obtained by at least 3 sample numbers. The reduction of each of the above extracts The amount of milligrams (mg AA) converted to vitamin C equivalent per gram (AA equivalent) /g). Table 3 extract total polyphenolic compound (mg of GAE/g) total flavonoid compound (mg of rutin/g) chelated ferrous ability reducing ability (mgofEDTA/g) (mgofAA/g) first Extract (Ps-1) 17.3±0.4 2.2±0.2 1.9±1.6 1.7±0.5 Second extract (Ps-2) 30·2±0.8 21.2±0.5 1.7±1.4 1.9±1.5 Third extract (Ps-3) 9·2±0.1 — 1.1±0.5 1.1±0.2 Fourth extract (Ps-4) I—0.9±0.8 0.5±0.1 Fifth extract (Ps-5) 44_1±0.6 29.8±1.1 2.5±1.7 1 · 3 0*5 Mudan peel extract (Part I, Ps_6) 7·0±1.0 6.0 ± 0.2 2.1±1.5 1.1±0_3 Cortex peel extract (Part 2, Ps-7) 38.9±0·4 5.1 Soil 1.2 2.1±1.3 1.5±0.2 -------- Cortex peel extract 80.8±0.3 l〇1.8±1.9 4.0±0.5 1.7 ± 1.5 (Part III, Ps-8)

由第3表之結果得知，實施例一所得之牡丹皮萃取物 (第三部分’ ps-8)之總多酚化合物及總類黃_化合物的含 量、螯合亞鐵能力以及還原能力，顯著高於其他不同萃取 階段之萃取物。 實施例四：評估牡丹皮萃取物之抑制脂質過氧化能力 24 201212948 此實施例係利用微脂粒脂質雙層結構類似細胞膜的特 性，來測試體外脂質過氧化實驗。由於脂質過氧化的產物 丙二齡(malondialdehyde ; MDA)在高溫下可與硫代巴比妥 酸(thiobarbituric acid ; TBA)形成紅色的破代巴比妥酸反應From the results of the third table, the total polyphenolic compound and the total yellow-like compound content of the peony bark extract (Part 3 'ps-8) obtained in Example 1, the chelating ability and the reducing ability, Significantly higher extracts than other different extraction stages. Example 4: Evaluation of the ability of the peony bark extract to inhibit lipid peroxidation 24 201212948 This example tests the in vitro lipid peroxidation experiment using the characteristics of the cell membrane of the liposome lipid bilayer similar to the cell membrane. Due to the lipid peroxidation product, malondialdehyde (MDA) can form a red deactivated barbituric acid reaction with thiobarbituric acid (TBA) at high temperature.

清浪測責 性物質（TBA-reactive substances ; TBARS)，可藉由測定於 532 nm之吸光值，而偵測MDA含量，從而判定脂質過氧 化程度。吸光值越低，表示樣品抑制脂質過氧化能驗（egg 上述微脂粒的主要脂質成分可例如卵構^ EI>C phosphatidylcholine ; EPC)，其係利用體積比1 · 7 (即陶水 與去礦物質水於水浴環境中間接進行超音波震225 進行超音波震盪）’形成微脂粒，其平均粒輕树妒.容浪（包 nm至250 nm。在此實施例中，取3.2 μΐ^的橄腊m]V〇 括10mM之卵磷脂膽鹼）、ii pL的硫酸亞鐵’外扣“ 溶液、11 pL的維生素c(10mM)溶液、151.5 之严不等 !'出-11(：1緩衝溶液(1)117.4)、2.5 01^的實施例/之/^^之 的各萃取物’於37°C下反應1小時。接著，加入 μίν之 TCA(10%)試劑（内含 ι〇/0 ΤΒΑ 溶液）以及 1〇·6 12〇〇〇 EDTA(0.1 M)，加熱至1〇〇。〇放置20分鐘。經泠卻炎以 rpm之轉速離心1〇分鐘沉澱後，各取1〇〇 μΐ之上 其於532 nm之吸光值，以獲得MDA的含耋。TBA-reactive substances (TBARS) can detect the degree of lipid peroxidation by measuring the absorbance at 532 nm and detecting the MDA content. The lower the absorbance value, the inhibition of lipid peroxidation by the sample (eg, the main lipid component of the above-mentioned vesicles can be, for example, egg structure EI> C phosphatidylcholine; EPC), which utilizes a volume ratio of 1.7 (ie, pottery water and Mineral water indirect in the water bath environment ultrasonic wave 225 for ultrasonic vibration) 'forms a liposome, the average grain light tree 妒. Rong wave (package nm to 250 nm. In this example, take 3.2 μΐ ^ Olive m m] V includes 10 mM lecithin choline), ii pL ferrous sulfate 'external buckle' solution, 11 pL of vitamin c (10 mM) solution, 151.5 rigorous! 'Out-11 (: 1 buffer solution (1) 117.4), 2.5 01^ of each of the extracts of / ^ ^ ^ reaction at 37 ° C for 1 hour. Next, add μίν of TCA (10%) reagent (with ι 〇/0 ΤΒΑ solution) and 1〇·6 12〇〇〇EDTA (0.1 M), heated to 1 〇〇. Place for 20 minutes. After centrifugation at rpm for 1 minute, precipitate, take 1 The absorbance at 532 nm above 〇〇μΐ to obtain the enthalpy of MDA.

25 201212948 48.3±3.4 48·9±3.4 66·7±2.225 201212948 48.3±3.4 48·9±3.4 66·7±2.2

第一萃取物（Ps-l) 26·0±2·1 55.1±1.9 58.2±3.4 77.2±2·0 第二萃取物（Ps-2) 41·5±1·6 59.6士2.7 66.8±1.5 83.8±2,1 第三萃取物（Ps_3) 14.2±〇.7 36.2±1.0 43.2土2.1 70.8±2.7 第四萃取物（Ps-4) 5.0±0.9 24.0±0.5 33·7±2.8 49.9±3·3 第五萃取物（Ps-5) 牡丹皮萃取物 23·8±5.1 42.2±3·8 56·4±0·2 89.2±0.3 94 )+·? (第一部分，Ps_6) 3· 牡丹皮萃取物 36.1±〇.4 56.9±2.8 62.2±2.5 70.〇±〇.2 (第二部分，Ps-7) 牡丹皮萃取物 40.6±0.3 67.9±2.1 70.4±1.9 72.3±1.8 (第三部分> Ps-8) 7S.〇±〇.2 3.7First extract (Ps-l) 26·0±2·1 55.1±1.9 58.2±3.4 77.2±2·0 Second extract (Ps-2) 41·5±1·6 59.6 2.7 66.8±1.5 83.8 ±2,1 third extract (Ps_3) 14.2±〇.7 36.2±1.0 43.2 soil 2.1 70.8±2.7 fourth extract (Ps-4) 5.0±0.9 24.0±0.5 33·7±2.8 49.9±3·3 Fifth extract (Ps-5) Cortex peel extract 23·8±5.1 42.2±3·8 56·4±0·2 89.2±0.3 94 )+·? (Part 1, Ps_6) 3. Mutan peel extract 36.1±〇.4 56.9±2.8 62.2±2.5 70.〇±〇.2 (Part 2, Ps-7) Cortex peel extract 40.6±0.3 67.9±2.1 70.4±1.9 72.3±1.8 (Part 3) Ps -8) 7S.〇±〇.2 3.7

維他命CVitamin C

Trolox 76·2±1.8 73·6±8.0 80.7士6.7 91·6±0.9 74·8±1.6 12·2±0·5 14.1±5.5 34·1±〇.4Trolox 76·2±1.8 73·6±8.0 80.7±6.7 91·6±0.9 74·8±1.6 12·2±0·5 14.1±5.5 34·1±〇.4

由第4表之結果得知，實施例一之牡丹皮萃取物（第= 部分)之抑制微脂粒過氧化的比率，顯著高於其他不同萃^ 階段之萃取物以及水溶性維他命E(Trolox)，因此證實牡丹 皮萃取物（第三部分，Ps-8)可作為食品添加物，以延長食品 的保存期。 實施例五：分析牡丹皮萃取物之組成份 此實施例係進一步分析實施例一獲得的牡丹皮萃取 物（Ps-8)之高效能液相層析（high performance liquid chromat〇graphy ; HPLC)指紋圖譜（finger profile)。在此實 施例中’所使用的HPLC系統可為高效液相層析儀/二極體 陣列檢剛器（diode array detector)系統，例如 Agilent 1100 26 201212948 series HPLC-DAD 系統（Agilent Technologies，Inc. CA， U.S.A.)。層析條件例示如下：層析管柱例如可為AgilentFrom the results of Table 4, the ratio of inhibition of liposome peroxidation in the peony bark extract (part = part) of Example 1 was significantly higher than that of other extracts and water-soluble vitamin E (Trolox). ), thus confirming that the peony bark extract (Part III, Ps-8) can be used as a food additive to extend the shelf life of the food. Example 5: Analysis of the composition of the peony bark extract This example is a further analysis of the high performance liquid chromatography (HPLC) fingerprint of the peony bark extract (Ps-8) obtained in Example 1. Finger profile. In this embodiment, the HPLC system used may be a high performance liquid chromatography/diode array detector system such as the Agilent 1100 26 201212948 series HPLC-DAD system (Agilent Technologies, Inc.). CA, USA). The chromatographic conditions are exemplified as follows: the chromatography column can be, for example, Agilent

Eclipse XDB-C18 管柱（5 μπι，2.1 mm X 150 mm)或其他性 質相當者。移動相由甲醇（溶劑A)以及含0.1%甲酸之試 劑水（溶劑B)所組成《溶劑梯度程式設定：〇分鐘至35 分鐘係注入10 %至80 %之甲醇，35分鐘至42分鐘係注 入80 %至100 %之曱醇’而42分鐘至65分鐘則注入1〇〇 %之甲醇。流速例如可控制在0.59 mL/min。層析溫度可 維持在25 °C。牡丹皮萃取物的樣品可先溶解在曱醇中並 鲁 利用〇.45μιη之濾膜過濾後’利用超音波振盪器（例如 Branson 5210Ε-ΜΤΗ 超音波振盪器，Branson Ultrasonics Corporation，CT，U.S.A.)振盪萃取後，進行HPLC指紋圖 譜之分析，其中注入HPLC-DAD系統之樣本體積例如可 為10 μ!^。偵測之UV光譜的波長例如可為200 nm至400 nm。另外，利用液相層析串聯質譜（liquid chromatography-tandem mass spectrometry ； LC-MS-MS) 的技術，例如具有電喷麗離子源（electrospray ionization _ source)之 Esquire HCT LC-MS-MS 系統（Bruker Daltinics， MA，U.S.A.)，進行牡丹皮萃取物之質譜分析《所得之結果 如第2圖所示。 請參閱第2圖，其係顯示根據本發明一實施例之牡丹 皮萃取物的HPLC指紋圖譜，其中第2圖之縱軸表示吸光 度單位(absorbance unit; a.u.)，橫軸表示滯留時間（retention time ;單位：分鐘）。由第2圖之結果可知，實施例一所得 之牡丹皮萃取物(Ps_8)的HPLC指紋圖譜具有14個峰值， 27 201212948 其中第3個峰值為内標準品(internal standard)，例如笨甲酿 基芍藥苷，以用於進行定量分析。經分析後，在—個例示 中’牡丹皮萃取物至少包含10重量百分比至15重量百分 比之四沒食子醯基葡萄糖、25重量百分比至3〇重量百= 比之三沒食子醯基葡萄糖、5重量百分比至1〇重量百八比 之沒食子醯基芍藥苷、1〇重量百分比至15重量百分2之 苯甲醯基芍藥苷以及10重量百分比至15重量百分=== 丹皮苷B。在另一個例示中，牡丹皮萃取物至少包含u iThe Eclipse XDB-C18 column (5 μm, 2.1 mm X 150 mm) or other equivalent. The mobile phase consists of methanol (solvent A) and reagent water (solvent B) containing 0.1% formic acid. Solvent gradient program setting: 10% to 80% methanol is injected from 〇 minute to 35 minutes, and injected from 35 minutes to 42 minutes. 80% to 100% sterol' and 42% to 65 minutes are injected with 1% methanol. The flow rate can be controlled, for example, at 0.59 mL/min. The chromatographic temperature can be maintained at 25 °C. Samples of the peony bark extract can be first dissolved in sterol and filtered through a filter of 45.45μιη. 'Using an ultrasonic oscillator (eg Branson 5210Ε-ΜΤΗ ultrasonic oscillator, Branson Ultrasonics Corporation, CT, USA) After the extraction, an HPLC fingerprint analysis is performed, wherein the sample volume injected into the HPLC-DAD system can be, for example, 10 μ! The wavelength of the detected UV spectrum can be, for example, 200 nm to 400 nm. In addition, a technique using liquid chromatography-tandem mass spectrometry (LC-MS-MS), such as an Esquire HCT LC-MS-MS system with an electrospray ionization source (Bruker) Daltinics, MA, USA), Mass spectrometric analysis of the peony bark extract. The results obtained are shown in Figure 2. Referring to FIG. 2, there is shown an HPLC fingerprint of a peony bark extract according to an embodiment of the present invention, wherein the vertical axis of FIG. 2 represents an absorbance unit (au) and the horizontal axis represents a retention time (retention time). ;Unit: minutes). As can be seen from the results of Fig. 2, the HPLC fingerprint of the peony bark extract (Ps_8) obtained in Example 1 has 14 peaks, 27 201212948, wherein the third peak is an internal standard, such as a stupid base. Paeoniflorin for quantitative analysis. After analysis, in an illustration, the 'peony bark extract contains at least 10 to 15% by weight of four galloglucosylglucose, 25 to 30% by weight = three ratios of gallic glucosylglucose. 5 parts by weight to 1 〇 weight 8% of the galloin-based paeoniflorin, 1% by weight to 15% by weight of the benzylidene-based paeoniflorin and 10% by weight to 15% by weight === Dan Derinoside B. In another illustration, the peony bark extract contains at least u i

重量百分比之四沒食子醢基葡萄糖、29·4重量百分=之二 沒食子醯基葡萄糖、8.7重量百分比之沒食子醯基芍 10.2重畺百分比之苯甲醯基芍藥苷以及重量百 牡丹皮苷Β。 日刀比之 ，·…〜个我％躍从符疋的製程條件、特定八械 ::或特定儀器為例示’說明本發明之牡丹皮 ： 製造方法之應用，惟本發明所屬技術領域中任何且 =知太本發明並不限於此，在不脫離本發明= ^ ㈣之牡丹皮萃取物可使用其他製程條件、 其他分析方法或儀器進行。 汆仟 並製施例可知’本發明之牡丹皮萃取物及 明之:法進= 應用於化妝品組:=二=得之牡丹皮萃取物可 雖然本發明已以數個實施例揭露如上，然其並非用以 28 201212948 限定本發明，在本發明所屬技術領域中任何具有通常知識 者，在不脫離本發明之精神和範圍内，當可作各種之更動 與潤飾，因此本發明之保護範圍當視後附之申請專利範圍 所界定者為準。 【圖式簡單說明】 為讓本發明之上述和其他目的、特徵、優點與實施例 能更明顯易懂，所附圖式之詳細說明如下·· 第1圖係繪示根據本發明一實施例之牡丹皮萃取物之 製程流程圖。 第2圖係顯示根據本發明一實施例之牡丹皮萃取物的 HPLC指紋圖譜。 【主要元件符號說明】 101 牡丹皮材料 109 :第四萃取物 103 第一萃取物 111 :第五萃取物 105 第二萃取物 113 :牡丹皮萃取物 107 第三萃取物 29Weight percentage of four gallic acid glucosinolate, 29.4 weight percent = two gallic ruthenium ruthenium, 8.7 weight percent gallic acid based on 10.2 weight percent of benzyl hydrazide paeoniflorin and weight Hundreds of peony saponins. Japanese knives, .... I am a member of the process conditions, specific eight:: or a specific instrument as an example 'describe the corundum of the present invention: the application of the manufacturing method, but any of the technical fields of the present invention And the invention is not limited thereto, and the peony bark extract can be used using other process conditions, other analytical methods or instruments without departing from the invention.汆仟 制 施 施 ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' 牡丹 牡丹 牡丹 牡丹 牡丹 牡丹 牡丹 牡丹 牡丹 牡丹 牡丹</ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; The scope defined in the patent application is subject to change. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; Process flow chart of the peony bark extract. Fig. 2 is a view showing an HPLC fingerprint of a peony bark extract according to an embodiment of the present invention. [Main component symbol description] 101 Coraba skin material 109: Fourth extract 103 First extract 111: Fifth extract 105 Second extract 113: Cortex peel extract 107 Third extract 29

Claims (1)

201212948 七、申請專利範圍： 1. 一種牡丹皮萃取物之製造方法，至少包含： 進行一第一萃取步驟，其係利用一第一有機溶液萃取 一牡丹皮材料，以獲得一第一萃取物； 進行一第二萃取步驟，其係利用一第二有機溶液萃取 該第一萃取物’以劃分出一第一有機相以及一水相’其中 該第一有機相具有一第二萃取物，且該水相具有一第三萃 取物； _ 進行一第三萃取步驟，其係利用一第三有機溶液萃取 該第二萃取物，以劃分出一第二有機相以及一第三有機 相’其中該第二有機相具有一第四萃取物，且該第三有機 相具有一第五萃取物；以及 進行一凝膠層析步驟，以由該第五萃取物獲得具有抗 氧化活性之一牡丹皮萃取物，其中該牡丹皮萃取物係以體 積比8 : 2之曱醇與水再加10滴醋酸為一展開液進行一逆 相薄層色層層析片分析，而依式⑴獲得0.436及0.564之一 # 展開阻滞(retention factor ; Rf)値： Rf=萃取物移動的距離 _展開液移動的距離_ () 2·根據申請專利範圍第1項所述之牡丹皮萃取物之 =造方法，其中該第一有機溶液為乙醇溶液，該第二有機 4液為乙酸乙酯/水溶液’且該第三有機溶液為正己烷/甲醇 溶液。 30 201212948 3. 根據申請專利範圍第1項所述之牡丹皮萃取物之 製造方法，其中該第一有機溶液為95體積百分比之乙醇溶 液，該第二有機溶液為體積比3 : 1之乙酸乙酯/水溶液’ 且該第三有機溶液為體積比1 : 1之正己烷/90體積百分比 之甲醇溶液。 4. 根據申請專利範圍第1項所述之牡丹皮萃取物之 製造方法，其中該第一有機相為乙酸乙酯相。 5. 根據申請專利範圍第1項所述之牡丹皮萃取物之 製造方法’其中該第三有機相為曱醇相。 6. 根據申請專利範圍第1項所述之牡丹皮萃取物之 製造方法，其中該凝膠層析步驟係利用葡萄糖凝膠層析管 柱(Sephadex LH-20 gel chromatography c〇lumn)進行0 • 7.根據申請專利範圍第1項所述之牡丹皮萃取物之 製造方法，其中該牡丹皮萃取物至少包含10重量百分比至 15重量百分比之四沒食子醯基葡萄糖（tetragalloyl glucose)、25重量百分比至30重量百分比之三沒食子醯基 葡萄糖(pentagalloyl glucose)、5重量百分比至1〇重量百分 比之沒食子醯基芍藥苷（galloylpaeoniflorin)、10重量百分 比至15重量百分比之苯甲醯基芍藥苷 (benzyoylpaeoniflorin)以及10重量百分比至15重量百分比 之牡丹皮苷 B(mudanpioside B)。 r 31 201212948 8. 種牡丹皮萃取物，其特徵在於一牡丹皮材料係 根據申請專利範圍第〗項至第7項之任—方法而製得具有 抗氧化活性之該牡丹皮萃取物，且該牡丹皮萃取物係以體 積比8 : 2之甲醇與水再加1〇滴醋酸為一展開液進行一逆 相薄層色層層析片分析，依式(I)獲得0.436及0.564之展開 阻滯(Rf)値： Rf=萃取物移動的距離 ⑴ 展開液移動的距離 9·根據申請專利範圍第7項所述之牡丹皮萃取物， 其中該牡丹皮萃取物至少包含10重量百分比至15重量百 分比之四沒食子醯基葡萄糖、25重量百分比至3〇重量百 分比之三沒食子醯基葡萄糖、5重量百分比至10重量百分 比之沒食子醯基苟藥苷、1〇重量百分比至15重量百分比 之笨甲醯基芍藥苷以及10重量百分比至15重量百分比之 牡丹皮苷B。 10. —種化妝品組合物，其特徵在於該化妝品組合物 至少包含根據申請專利範圍第丨項至第7項之任一方法而 製得之一牡丹皮萃取物，且該杜丹皮萃取物具有抗氧化活 性，其中該化妝品組合物為水劑、乳劑、膏劑、粉劑、防 曬劑、抗氧化劑及抗老化劑之化妝品。 11. 一種食品添加物，其特徵在於該食品添加物至少 32 201212948201212948 VII. Patent application scope: 1. A method for manufacturing a peony bark extract, comprising at least: performing a first extraction step of extracting a peony bark material by using a first organic solution to obtain a first extract; Performing a second extraction step of extracting the first extract by using a second organic solution to divide a first organic phase and an aqueous phase, wherein the first organic phase has a second extract, and the The aqueous phase has a third extract; _ performing a third extraction step of extracting the second extract with a third organic solution to define a second organic phase and a third organic phase The second organic phase has a fourth extract, and the third organic phase has a fifth extract; and a gel chromatography step is performed to obtain a cortex peel extract having antioxidant activity from the fifth extract , wherein the peony bark extract is subjected to a reverse phase thin layer chromatography analysis with a volume ratio of 8:2 sterol and water plus 10 drops of acetic acid as a developing solution, and 0.436 and 0.564 are obtained according to formula (1). A #retention factor (Rf)値: Rf=the distance the extract moves _ the distance the developing liquid moves _ () 2· The method of making the peony bark extract according to item 1 of the patent application scope, Wherein the first organic solution is an ethanol solution, the second organic 4 liquid is an ethyl acetate/aqueous solution', and the third organic solution is a n-hexane/methanol solution. The method of manufacturing the peony bark extract according to claim 1, wherein the first organic solution is a 95 volume percent ethanol solution, and the second organic solution is a volume ratio of 3:1 acetic acid. The ester/aqueous solution' and the third organic solution is a methanol solution having a volume ratio of 1:1 n-hexane/90 volume percent. 4. The method for producing a peony bark extract according to claim 1, wherein the first organic phase is an ethyl acetate phase. 5. The method for producing a peony bark extract according to claim 1, wherein the third organic phase is a sterol phase. 6. The method for producing a peony bark extract according to claim 1, wherein the gel chromatography step is performed by using a glucose gel chromatography column (Sephadex LH-20 gel chromatography c〇lumn). 7. The method for producing a peony bark extract according to claim 1, wherein the peony bark extract contains at least 10% by weight to 15% by weight of tetragalloyl glucose, 25 parts by weight. Percentage to 30% by weight of pentatalloyl glucose, 5 to 1% by weight of galloidlpaeoniflorin, 10% to 15% by weight of benzamidine Paeonioylpaeoniflorin and 10% to 15% by weight of mudanpioside B. r 31 201212948 8. A peony bark extract, characterized in that a peony bark material is obtained according to the method of claim 7-1 to item 7 to obtain an antioxidant activity of the peony bark extract, and The peony bark extract was analyzed by a reverse phase thin-layer chromatography layer with a volume ratio of 8:2 methanol and water plus 1 liter of acetic acid as a developing solution, and the expansion resistance of 0.436 and 0.564 was obtained according to the formula (I).滞(Rf)値: Rf=distance of the movement of the extract (1) The distance from which the liquid is moved. The peony bark extract according to claim 7, wherein the peony bark extract contains at least 10% by weight to 15% by weight. a percentage of four gallic acid glucosinolates, from 25 weight percent to 3 weight percent of three galloglucosylglucose, from 5 weight percent to 10 weight percent gallic acid glucosides, from 1 weight percent to 15 weight percent Percent by weight of the carbaryl glucoside and 10 to 15 weight percent of peony saponin B. 10. A cosmetic composition, characterized in that the cosmetic composition comprises at least one of the peony peel extracts according to any one of the methods of the present invention, wherein the Dudan peel extract has Antioxidant activity, wherein the cosmetic composition is a cosmetic of a liquid, an emulsion, an ointment, a powder, a sunscreen, an antioxidant, and an anti-aging agent. 11. A food additive characterized in that the food additive is at least 32 201212948 包含根據申請專利範圍第1項至第7項之任一方法而製得 之一牡丹皮萃取物，且該牡丹皮萃取物具有抗氧化活性， 其中該食品添加物為抗氧化劑或抗老化劑之營養品或保健 食品。 33A peony bark extract obtained by the method according to any one of claims 1 to 7, wherein the peony bark extract has antioxidant activity, wherein the food additive is an antioxidant or an anti-aging agent Nutrition or health food. 33