TWI223982B

TWI223982B - Edible composition isolated from cherries and useful for inhibiting oxidation in a living mammal

Info

Publication number: TWI223982B
Application number: TW088121631A
Authority: TW
Inventors: Muraleedharan G Nair; Haibo Wang; James I Gray; Alden M Booren; Gale M Strasburg
Original assignee: Univ Michigan
Priority date: 1998-12-11
Filing date: 1999-12-10
Publication date: 2004-11-21
Also published as: CN1334740A; WO2000033667A3; WO2000033667A2; AU2175400A; CN100394916C; JP2002531473A

Abstract

An edible composition useful for inhibiting oxidation in a living mammal, particularly a human, and isolated from cherries. Said edible composition comprises anthocyanins, bioflavonoids, phenolics and food grade carriers.

Description

1223982 A7 B7 五、發明說明（01) 使用樓桃革離士日/it 4、片 H;— 摘要：描述一種使用樓桃衍生物於哺供營養療效或植物療效之、V 疋人類體内提質中抑制氧化的方法。生命* 苷、生物類黃酮、酚類或其混合物。 *桃〈化色素詳細說明：本發明提出一種使用具有單離物以提供益處之方法。 ‘、’或植物療效之櫻桃本發明亦有關—種使用得自櫻桃命生物物質中抑制氧化之方法。本發明卢於需要之生飲食補充劑或食品添加劑之化合物或組成匕物。！，：種作為花色::由::類黃酮及如合二 5牛夕由植物衍生之化合物皆可藥或，，營養療效/植物療效II特性，因心=…醫反應性氧中間體而作為抗氧劑、藉著抑 1低濃度（作為抗發炎劑、或作為參與細胞增殖抑：素合成而活性對於舒緩慢性疾病包括癌症、 2制刮。此等而言’極為重要⑻nseua等人，Food;ech=血管疾病 3))。因此’使用天然產物，飲食補充，食品 89(199 療/植物治療公司有機會採用不僅可如同業，營養治效地促進食品安定性，亦可對消費者提供；鞋乳剩般有的化合物。 ’、月^ <健康益本紙張尺涵財關家鮮（CNS)A4規格（21〇]1223982 A7 B7 V. Description of the invention (01) Use of Lou Tao Leather Lishi Day / it 4, Tablet H; — Abstract: Describes the use of Lou Tao Derivatives for nutritional or botanical effects in humans. Inhibition of oxidation in quality. Life * glycosides, bioflavonoids, phenols or mixtures thereof. * Peach (chemical pigment) Detailed description: The present invention proposes a method of using a single ion to provide benefits. This invention is also related to a method for inhibiting oxidation using a biological substance derived from cherry. The present invention is a compound or composition for a dietary supplement or food additive. !! :: as a flower color ::::::: flavonoids and Ruhe 5 New Year's Eve are plant-derived compounds can be medicine or, nutritional efficacy / plant efficacy II characteristics, because the heart = ... medical reactive oxygen intermediate Antioxidants, by inhibiting low concentrations (as anti-inflammatory agents or as inhibitors involved in cell proliferation: hormone synthesis and activity for soothing chronic diseases including cancer, curative system. These are 'extremely important' nseua et al., Food ; ech = vascular disease 3)). Therefore, the use of natural products, dietary supplements, food 89 (199) / phytotherapy companies have the opportunity to adopt compounds that not only promote nutritional effectiveness and promote food stability, but also provide consumers with the same compounds as shoe milk. ', Month ^ < Health Benefit Paper Ruler Han Cai Guan Jiaxian (CNS) A4 Specification (21〇)

It----------! (請先閱讀背面之注意事項再填寫本頁) -4- 經濟部智慧財產局員工消費合作社印製 1223982 A7 __ B7 五、發明說明（02) 櫻桃通常被推測具有健康益處。其中一種較佳櫻桃係為歐洲酸櫻桃（薔薇科），常見品種M0NTM0RENCY係為美國所大量種植之主要苦櫻桃（tart cherry)，為了挑戰 MONTMORENCY單一品種，新植株BALAT〇N(苦櫻桃（ tart cherry)Ujferbertoi £11以〇3)於 1 9 84 年被引進美國，已於密西根州、猶他州及威斯康辛州進行試驗。 BALATON所結果貫顏色較MONTMORENCY果實暗。最近I結果顯tf花色素苷諸如花青素_ 3 _糖苷具有強抗氧劑活性（TSUda，T·等人，j.Agric.Fo〇d Chem· 42:24 0 7 - 2 4 1 0 ( 1 9 9 4 ))。添加抗氧化劑係為一般增加食品使用期限之晋遍方法，推測與脂質過氧化有關。天然抗氧化劑於致癌過程之預防上扮演重要角色。食品抗氧化劑可於生命系統中對抗過氧化損害（H a 1 i w e 11，B ·及J . M . C . G u 11 e-i*idge，生物及醫藥中之自由基，〇xf〇rci Unix；ersity Press ’New York 416-494(1989);Osawa，T·等人，食品抗氧化劑於防止氧化扣、害、抗突變及抗癌形成機制中所扮演之角色 ;Kuroda,Y.;Shankel5D.M.,Waters, Μ . D . E d s .; P 1 e n u mIt ----------! (Please read the notes on the back before filling out this page) -4- Printed by the Consumer Consumption Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1223982 A7 __ B7 V. Invention Description (02) Cherry Health benefits are usually speculated. One of the better cherries is the sour cherry (Rosaceae). The common variety M0NTM0RENCY is the main bitter cherry grown in the United States. In order to challenge the MONTMORENCY single variety, a new plant BALATON (tart cherry Ujferbertoi (£ 11 to 〇3) was introduced to the United States in 1984 and has been tested in Michigan, Utah, and Wisconsin. The results of BALATON are darker than those of MONTMORENCY. Recent I results show that tf anthocyanins such as anthocyanins_3_ glycosides have strong antioxidant activity (TSUda, T. et al., J. Agric. Food Chem. 42:24 0 7-2 4 1 0 ( 1 9 9 4)). The addition of antioxidants is a general method to increase the shelf life of foods, and it is presumed to be related to lipid peroxidation. Natural antioxidants play an important role in the prevention of carcinogenic processes. Food antioxidants can fight peroxidative damage in living systems (H a 1 iwe 11, B · and J. M. C. G u 11 ei * idge, free radicals in biology and medicine, 0xf〇rci Unix; ersity Press' New York 416-494 (1989); Osawa, T., et al. The role of food antioxidants in preventing oxidative knock, damage, anti-mutation and anti-cancer formation mechanisms; Kuroda, Y .; Shankel 5D.M. , Waters, Μ. D. E ds.; P 1 enum

Publishinh.New York 1 3 9 - 1 5 3 ( 1 9 9 0 )) 〇早期研究顯示MONTMORENCY櫻桃含有花色素苷花青素-3-龍膽二糖苷及花青素-3 -芸香糖苷（Li，K.C.等人，J·Publishinh. New York 1 3 9-1 5 3 (199 0)) 〇 Early research shows that MONTMORENCY cherry contains anthocyanins anthocyanins-3-gentiobiside and anthocyanin-3 -rutinoside (Li, KC et al., J.

Am.Chem.Soc.78:979-980(1956))。亦於七種酸櫻桃（ sour cherry)品種中之六種發現有花青素-3-葡萄糖基芸香糖苷（Harborne，J.B.等人，植物化學 3:4 5 3 -4 6 3 ( 1 9 64 ) )。D e k az 〇 s (D ek az 〇 s，E · D .，J · F 〇〇 d Sci.35:237-241(197 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁)Am. Chem. Soc. 78: 979-980 (1956)). Anthocyanin-3-glucosylrutinoside was also found in six of the seven sour cherry varieties (Harborne, JB et al. Phytochemistry 3: 4 5 3 -4 6 3 (1 9 64) ). Dek az 〇s (D ek az 〇s, E · D., J · F 〇〇d Sci. 35: 237-241 (197 This paper size applies to China National Standard (CNS) A4 specifications (210 X 297 mm ) (Please read the notes on the back before filling this page)

12239821223982

五、發明說明（〇3) 〇))記載MONTMORENCY櫻桃中之花色素苷顏料，其係為芍藥素-3 -芸香糖苷、芍藥素及花青素形式，同時有花青素-3-槐糖苷、花青素-3_芸香糖苷及花青素_3_葡萄糖苷。然而，花青素-3-葡萄糖基芸香糖苷及花青素葡萄糖苷、花青素-3-槐糖苷及花青素芸香糖苷已被確定為酸櫻桃中主要之顏料。Chandra等人使用HPLC滯留時間值（Chandra，A.等人，J.Agric.Food.Chem.40: 967-9 69(1 992))記載花青素-3-槐糖苷及花青素葡萄糖苷個別係為密西根州種植之MONTMORENCY櫻桃之主要及次要花色素苷。相同地，花青素-3 -木糖基芸香糖苷已被測出係為MONTMORENCY櫻桃中之次要顏料（Shrinkhande，A.j •及 F.J.Francis，J.Food.Sci.38:6 4.9-65 1(1973))。先前技藝界中，自BALATON及MONTMONRENCY 櫻桃果汁製造純花色素苷（圖1之化合物卜3 )係先使該顏料吸附於 AMBERLITE XAD-2(Singma Chemicals)管柱上（ Chandra，A.等人，J.agric.Food Chem.41 :1062-1065 (1 9 9 3 ))。該管柱以水洗滌至洗提液之p h約7 . 〇。所吸附之顏料與其他酚類一起被甲醇提出。形成之粗製花色素苷被分級，而個別藉C-1 8 MPLC及HPLC純化，以產生供光諸研究使用之純花色素苷。自AMBERLI丁E XAD-2純化500 毫克粗製MONTMORENCY花色素苷產生60毫克純花色素苷1-3，相對地BALATON係為3 9 1.4 3毫克。此項研究顯示自XAD-2所得之MONTMORENCY粗製花色素苷含有高百分比之其他有機化合物。酚類與花色素苷之粗製混合物本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁)V. Description of the invention (〇3) 〇)) describes the anthocyanin pigment in MONTMORENCY cherry, which is in the form of paeonin-3-rutin, paeoniflorin and anthocyanin, and anthocyanin-3-sophoroside , Anthocyanin-3_rutin and anthocyanin_3_glucoside. However, anthocyanin-3-glucosinorutin and anthocyanin glucoside, anthocyanin-3- sophoroside and anthocyanin-rutin have been identified as the main pigments in sour cherries. Chandra et al. Used HPLC retention time values (Chandra, A. et al., J. Agric. Food. Chem. 40: 967-9 69 (1 992)) to document anthocyanin-3-sophoroside and anthocyanin glucoside Some are the major and minor anthocyanins of MONTMORENCY cherries grown in Michigan. Similarly, anthocyanin-3 -xylosylrutin has been detected as a secondary pigment in MONTMORENCY cherry (Shrinkhande, Aj • and FJ Francis, J. Food. Sci. 38: 6 4.9-65 1 ( 1973)). In the prior art industry, the production of pure anthocyanins from BALATON and MONTMONRENCY cherry juice (compound Bu 3 of Fig. 1) was carried out by first adsorbing the pigment on an AMBERLITE XAD-2 (Singma Chemicals) column (Chandra, A. et al., J.agric.Food Chem. 41: 1062-1065 (1 9 9 3)). The column was washed with water until the pH of the eluent was about 7.0. The adsorbed pigment is extracted by methanol along with other phenols. The resulting crude anthocyanins were fractionated and individually purified by C-1 8 MPLC and HPLC to produce pure anthocyanins for use in light studies. Purification of 500 mg of crude MONTMORENCY anthocyanins from AMBERLI D E XAD-2 yielded 60 mg of pure anthocyanins 1-3, compared with 3 9 1.4 3 mg for the BALATON line. This study showed that MONTMORENCY crude anthocyanins from XAD-2 contained a high percentage of other organic compounds. Crude mixture of phenols and anthocyanins This paper size is applicable to Chinese National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back before filling this page)

--------訂---------線I 經濟部智慧財產局員工消費合作社印製 1223982 經濟部智慧財產局員工消費合作社印製 Α7 Β7 五、發明說明（〇4) 尚未使用於任何目的。Garbuti之美國專利第5，，Η 5 號及人之第 5，6 6 5，7 8 3 號及 M〇zaffari 第5，8 17，3 5 4號描述各式各樣之吸附劑樹脂及其用於單離不相關產物的料。此等專利僅說明技藝界使用吸附劑樹脂之一般狀態。-------- Order --------- Line I Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1223982 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 Β7 V. Description of the invention (〇4 ) Has not been used for any purpose. Garbuti's U.S. Patent No. 5, Η5 and others No. 5,6 6 5,7 8 3 and Mozaffari No. 5,8 17,3 5 4 describe a variety of adsorbent resins and their Used to separate irrelevant products. These patents merely describe the general state of use of adsorbent resins in the art world.

Pleva之美國專利第5，5〇3，δ67號描述整體磨碎之櫻桃=燕麥麩於絞肉中之用途。櫻桃用量係為丨〇至1 $重 ϊ百分比，而相信添加燕麥麩係用以補償櫻桃汁。任何情況了，該櫻桃皆明確地於肉品上賦予風味，而產品之可口性無法被廣泛地接受。此專利未描述營養療效或植物療效。近來針對使用櫻桃組織使低脂牛絞肉安定化之研究顯示此種植物來源含有強效抗氧化劑，不僅抑制脂質過氧化，亦抑制=油炸過程巾形成雜環芳族胺及膽固醇氧化產物（ G〇maa^ 人，1FT Abstracts Ν〇·6δΕ- 7 ( 1 996)。用以說明此〃觀祭之理淪係為聚酚諸如類黃酮、花色素苷及花色素常被發現存在於高等植物之空胞中，諸如櫻桃具有此種氧化效果。仍需要一種天然櫻桃衍生組成物，使用為（尤其是）食品補充劑/營養治療性或食品添加劑。發明概述本發明有關一種於生命哺乳類體内提供營養療效或植物療效之方法，包括抗氧化劑療效，其包括： ^於該哺乳類身上餵食一種自櫻桃單離之組成物，選自花色素苷、生物類育酮、酚類及其混合物，用量係於該哺 Ψ 一 -V . --------^---------線 (請先閱讀背面之注意事項再填寫本頁) 1223982 五、發明說明（〇5) 乳類體内提供營養療效或植物療效。本發明有關一種於有需要> +人、、 j而要义生命生物物質中抑制e 之方法，JL向括· A今吐入L J氣化 (請先閱讀背面之注意事項再填寫本頁) A ^ /、括·於生f卩生物物質上提供得自櫻棑而、强自化色素苷、生物類黃酮、紛3¾ 、曰酌頒及其混合物足組成物，用 Ϊ係於該生命生物物質中抑制氧化。用生命生物物質"意指於動物或人類體外或體内而處於培養狀態下的活組織。 Ί ’’花色素苷’’包括包含於櫻桃中之發色性化合物。本發明中’其包括糖苷配基花青素。又生物類黃酮π意指櫻桃中所包含之異類黃酮及類普酉同化合物。 ^ 酉分類’意指具有苯基且具有一或多個輕基之化合物。 π營養療效"意指對於具有生命之哺乳類具有益處，以維生素形式影響哺乳類健康。 ’’植物療效’’意指自植物衍生之產物，提供營養治療性質。 .、發明目的經濟部智慧財產局員工消費合作社印製因此，本發明目的係提出一種天然來源櫻桃組成物，其可使用於食品中或作為飲食補充劑或營養療劑，作為於有生命之組織中防止氧化之抗氧化劑。因此參照以下描述及圖式可進一步明瞭此等及其他目的。圖式簡單說明圖1顯示選擇花色素苷（著色劑）之結構，自B A L Α-TON及MONTMORENCY櫻桃單離。該糖苷配基花青素之本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1223982 A7 五、發明說明（〇6) 位置3上具有羥基。圖2及3係為顯示自樓圖桃所單離之主要生物類黃酮之圖4係顯示自苦櫻桃單離之選擇酚麺圖5係顯示自櫻桃單離花色素苷、類之方法的步驟。生物類黃酮、及圖6係為顯示可使用於圖5之方法中之設備的示意圖經濟部智慧財產局員工消費合作社印製圖7係為顯示花色素苷及，r ^ ^ , 帀。抗氧化劑於脂質體系統中頰不柷虱化效果之圖。氧化伤… 、… Μ 礼化係猎耆添加亞鐵離子而起始。於5式私化合物存在下，勞弁 > 者忠兀 < 哀減連率降低。對照試樣未添加F e 2♦，而F e2+不添加續路a入^ ^ 】w A騙化合物。其他試樣係含有 F e -加2微莫耳濃度試驗化合物。圖8係為顯不所單離化合物丨_ 4、7及8及市售抗氧化劑T B H Q及Β Η T於1 0微莫耳濃度下之抗氧化活性之圖。數據顯示兩次實驗之平均值。化合物}係為柚配基4，，5, 7_ 三羧基黃烷酮（RrOH、R4-〇H&R6-〇h;R2、R3、R5及R7 -Η )。化合物2係為染料木黃酮（圖3 )。化合物3係為綠原酸。化合物4係為懈皮酮· 3 -鼠李糖苷（圖2 )。化合物7係為染料木黃酮-7 -葡萄糖苷（圖3 )。化合物8係為6，7 ·二甲氧基-5,8,4f-三羥基黃酮（圖2)。圖9係為顯示所單離之化合物1、3及4 (圖4 )及部分市售抗氧化劑於2 0微莫耳濃度下之抗氧化活性之圖。化♦ 物2之抗氧化活性係於1 〇〇微莫耳濃度下測量。過氧彳匕^ -9- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱） ---------1---· 11-----訂--------- ^一^^" · (請先閱讀背面之注意事項再填寫本頁) 1223^2 經濟部智慧財產局員工消費合作社印製 A7 五、發明說明（07) 率係藉著以時間求教㈡数表示之螢光強度降低而偵測。相對強度係表不於特定昧pE1 ^^ a、 ^ 、〶下 < 螢光強度除以檢定開始時之原始強度。數值係為二次測量之平均值。較佳具體實例描述自樓桃產生g各^么 , ,, σ化色素苷、生物類黃酮、酚類之組成物形式混合物的較佳女乂住万法係包括自櫻桃提供含有該花色素苷、生物類黃酮、齡来、々4足水落液；自該水溶液將該花色素苷、生物類黃酮、酚来士瓦、々〜&取於一樹脂表面上；使用洗提劑洗提该樹脂表面，以白二女自咸树知表面去除該花色素苷、生物類黃酮、S分類；自該芯&本# 化色素普、生物類黃酮、酚類分離該洗提劑。自櫻桃產製組成物形式之花色素苦、生物類黃酮、酚類的較佳方法係包括提供第一批櫻桃，其中該樓桃係新鮮或急速冷凍並解凍；搗碎該樓如八 _ + S检桃分離果肉及果汁；自該果肉知琢化色素普'生物類黃_、酚類萃取於一水溶液中. 自孩果肉所分離之含有該花色Μ、生物類黃_ 1，水溶液中的該花色素誓、生物類黃酮、齡類攝取於吸附性樹脂粒子上；使用低烷醇晶圓該樹脂粒子，以自子取出該花色素苦、生物類黃酮、齡類;自該花：素“; 生物類黃酮、齡類分離該烷醇使用第― 」矛一批楗桃以所分離之燒醇及已取出該花色素苷、生物類咅離子重複前述步驟。 J岛奴樹脂粒此外，使用於該方法中之較佳可消耗組成物係包下物質之摻合物：自櫻桃單離之花色素: I 王物類黃酮、 -10- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐 (請先閱讀背面之注意事項再填寫本頁)US Patent No. 5,503, δ67 of Pleva describes the use of whole ground cherries = oat bran in ground meat. The amount of cherry is from 0 to 1% by weight, and it is believed that adding oat bran to compensate for cherry juice. In any case, the cherries clearly impart flavor to the meat, and the palatability of the product is not widely accepted. This patent does not describe nutritional or botanical efficacy. Recent studies on the use of cherry tissue to stabilize low-fat beef ground meat have shown that this plant source contains powerful antioxidants that not only inhibit lipid peroxidation, but also inhibit the formation of heterocyclic aromatic amines and cholesterol oxidation products during the frying process ( G〇maa ^ Human, 1FT Abstracts Ν〇 · 6δΕ-7 (1 996). It is used to explain that the principle of this observatory is reduced to polyphenols such as flavonoids, anthocyanins and anthocyanins, which are often found in higher plants. Among the air cells, such as cherries have such an oxidizing effect. There is still a need for a natural cherry-derived composition for use as, among other things, food supplements / nutritive therapeutics or food additives. SUMMARY OF THE INVENTION The present invention relates to a body in a living mammal. A method for providing a nutritional or botanical effect, including an antioxidant effect, comprising: ^ feeding the mammal a composition isolated from cherry, selected from anthocyanins, biological tocopherols, phenols, and mixtures thereof, the dosage It is tied to this feeding Ψ-V. -------- ^ --------- line (please read the precautions on the back before filling this page) 1223982 V. Description of the invention (〇5) Milk body The present invention provides nutritional or botanical effects. The present invention relates to a method for inhibiting e in biological materials that are in need of + human, j, and JL to spit into LJ gasification (please read the back Note: Please fill in this page again.) A ^ /, Included in the raw materials of biological raw materials are derived from Sakura 棑, strong self-chemical pigment glycosides, bioflavonoids, 33¾, 酌 and its mixture foot composition, It is used to inhibit oxidation in the living biological substance. Using the living biological substance means living tissue that is in a culture state in vitro or in vivo of an animal or a human. ΊAnthocyanins are included in cherry It is a chromogenic compound. In the present invention, 'it includes aglycone anthocyanins. Bioflavonoids π means isoflavonoids and pu-like compounds contained in cherries. ^' Classification 'means having a phenyl group and Compounds with one or more light bases. Π Nutritional efficacy " means beneficial to mammals with life, and affects mammalian health in the form of vitamins. `` Plant efficacy '' means a product derived from a plant. For nutritional treatment .. Purpose of the invention. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economics. Therefore, the purpose of the present invention is to propose a cherry composition of natural origin, which can be used in food or as a dietary supplement or nutritional therapy. Antioxidants that prevent oxidation in living tissues. Therefore, these and other purposes can be further understood with reference to the following description and drawings. Brief Description of the Drawings Figure 1 shows the structure of anthocyanins (colorants) selected from BAL Α- TON and MONTMORENCY cherries are isolated. The paper size of this aglycone anthocyanin is applicable to Chinese National Standard (CNS) A4 (210 X 297 mm) 1223982 A7 5. Description of the invention (〇6) There is a hydroxyl group at position 3. Figures 2 and 3 are diagrams showing the main bioflavonoids isolated from Taotu peach. Figure 4 is a diagram showing the selection of phenols isolated from bitter cherry. Figure 5 is a diagram showing the steps of the method of anthocyanins and separations from cherry. . Bioflavonoids, and Figure 6 are schematic diagrams showing the equipment that can be used in the method of Figure 5. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Figure 7 is a diagram showing anthocyanins and, r ^ ^, 帀. Antioxidant in the liposome system. Oxidative injury ..., Μ The etiquette was added by ferrous ions to start. In the presence of the type 5 private compound, the labor force > the loyalty < The control sample did not add F e 2 ♦, and F e 2+ did not add the continuum a to ^ ^ w A cheat compounds. The other samples contained Fe-plus 2 micromolar test compounds. Fig. 8 is a graph showing the antioxidant activity of compounds that are not easily isolated, 4, 7, and 8 and commercially available antioxidants T B H Q and B Η T at a concentration of 10 micromolar. The data show the average of two experiments. Compound} is naringenin 4,5,7-tricarboxyflavanone (RrOH, R4-OH &R6-Oh; R2, R3, R5, and R7-7). Compound 2 is genistein (Figure 3). Compound 3 is chlorogenic acid. Compound 4 is quercetin · 3-rhamnoside (Fig. 2). Compound 7 is genistein-7-glucoside (Figure 3). Compound 8 is 6,7 · dimethoxy-5,8,4f-trihydroxyflavone (Figure 2). Figure 9 is a graph showing the antioxidant activity of isolated compounds 1, 3, and 4 (Figure 4) and some commercially available antioxidants at a concentration of 20 micromolar. The antioxidant activity of Chemical 2 was measured at a concentration of 1000 micromolar. Peroxide dagger ^ -9- This paper size applies to China National Standard (CNS) A4 (210 X 297 public love) --------- 1 --- · 11 ----- Order- ------- ^ 一 ^^ " · (Please read the notes on the back before filling in this page) 1223 ^ 2 Printed by the Consumers ’Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 V. Description of Invention (07) Detected by the decrease in fluorescence intensity as a function of time to ask for advice. The relative intensity is not equal to the specific pE1 ^^ a, ^, His Majesty < Fluorescence intensity divided by the original intensity at the beginning of the test. Values are the average of two measurements. A preferred specific example describes the production of a variety of compounds from G. spp., Sigmatized glycosides, bioflavonoids, and phenols in the form of a mixture of compounds. The preferred method is to provide the anthocyanins from cherry. , Bioflavonoids, linglai, 々4foot water drop; from the aqueous solution, the anthocyanins, bioflavonoids, phenol leswas, 々 ~ & were taken on a resin surface; using an eluent to elute the On the resin surface, the anthocyanins, bioflavonoids, and S were removed from the surface of the salty tree by the white daughter; the eluent was separated from the core & this pigment, bioflavonoids, and phenols. The preferred method of producing anthocyanin, bioflavonoids, and phenols from a composition produced from cherry includes providing the first batch of cherries, in which the peach series is fresh or rapidly frozen and thawed; mashing the building such as eight_ + S test peach to separate the pulp and fruit juice; from the pulp, the biochemical yellow pigments and phenols are extracted in an aqueous solution. The color M, the biological yellow pigment_1, and The anthocyanin, bioflavonoids, and ages are ingested on the adsorbent resin particles; the resin particles are wafered using a low alkanol to take out the anthocyanin, bioflavonoids, and ages from the child; "; Biological flavonoids, age-based separation of the alkanol use the first" "batch of Prunus persica, with the separated burning alcohol and the anthocyanins, biological sulfonium ions have been taken out to repeat the foregoing steps. J island slave resin granules In addition, the preferred consumable composition used in this method is a blend of substances: from the flower of the cherry isolated from the pigment: I king flavonoids, -10- This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm (please read the precautions on the back before filling this page)

1223982 A7 B7 五、發明說明（08) 77邊的乾壤混合物；及食品級載體，其中該混合物相對於。亥载體之重量比係介於約0 . 1比丨〇〇及.丨〇〇比〇 ·丨之間。最後，提出一種於哺乳類體内抑制氧化之較佳方法，其包括銀食該哺乳類以下物質之消耗性組成物，其包含以下物質之摻合物：自櫻桃單離之花色素苦、生物類黃酮及酉分類的乾燥混合物；及食品級載體，其中該混合物相對於孩載體之重量比係介於約〇. i比1〇〇及1〇〇比〇.丨之間。較佳係該組成物含有至少一部分乾燥櫻桃果肉。1223982 A7 B7 V. Description of the invention (08) 77 side dry soil mixture; and food-grade carrier, where the mixture is relative to. The weight ratio of the carrier is between about 0.1 to 1.00 and. 丨 00 to 〇 · 丨. Finally, a better method for inhibiting oxidation in mammals is proposed, which includes silver food, a consumable composition of the following substances, which contains a blend of the following substances: anthocyanin from cherry isolated, bioflavonoids And a dry mixture classified by 酉; and a food-grade carrier, wherein the weight ratio of the mixture to the child carrier is between about 0.1 to 100 and 100 to 0.1. More preferably, the composition contains at least a portion of dried cherry pulp.

本發明所使用之櫻桃可為甜或酸櫻桃。酸櫻桃除了構成苦櫻桃之酸味的其他有機外亦含有高濃度蘋果酸。該方法單離含有糖之蘋果酸及其他有機酸，其可於食品中提供 I味及風味。最佳係為BALAT〇N&M〇NTM〇RENCY 桃。 .文該花色素苷、生物類黃酮及酚類之單離混合物可作為天然營養治療/食品補充劑。就此言之，所單離之混合物 "T為私末液租、或固體形式。例如，該混合物可為可復哼粉末組成物，當使用例如水、+乳或某些其他類似液體復原時，可提供一飲料。或該混合物可為固體形式諸如錠劑、凝膠覆帽、軟凝膠等。此外，該混合物可摻入食物中〇週¥可k供I形式中該混合物含量係介於重之重量百分比至約重量百分比範圍^以約0 • 1重T百分比至約3 〇重量百分比為佳，而約〇 . 5重量百分比至約2 5重量百分比更佳。 -11- C請先閱讀背面之注意事項再填寫本頁) 1¾--------訂---------線康經濟部智慧財產局員工消費合作社印製本紙張尺度適用中國國家標準(CNS)A4 ϋΤΓίΟ X 2心"¥釐) 1223982 經濟部智慧財產局員工消費合作社印製 A7 五、發明說明（〇9) 例如，當該混合物係為鍵劑形式時， 0 . 1毫克至3 ο 〇毫克之花色辛 P J可提供約 ^ 巴京甘及生物類咅酮乏R十丨b 期望其為1至2 〇〇毫克，以6 η Ί ^ I，土 2UU毛兑以⑼-1 00毫克之日劑量為估百（1〇0)顆櫻桃提供60至1〇〇毫> 為佳。一、η、，兄（化色素誓。酚麵f FI j、足曰劑量可為0· 1至50毫克用量。一 ”（圖4) 克齡類。該花色辛誓百果摘楗供“。毫 … 類黃酮、及酚類之用量可藉薯早離:固別化合物並摻合彼者而調整。於一具體實例中：用涊化色素苷、生物類黃酮、及酚類之天然混合物。嗜組成物斫可於含有同等劑量之液體形式下提供。人、、該樹脂具有吸附該花色素苦、生物類黃酮、及酉分類之表囬。較佳吸附性樹脂係為聚合物交聯樹脂，包含苯乙烯及二乙烯基苯，.諸如例如AMBERLITE系列樹脂，例如 AM.BERLITE XAD-4 及 AMBERLITE XAD-16，購自The cherries used in the present invention may be sweet or sour cherries. Tart cherries contain high concentrations of malic acid in addition to other organics that make the bitter cherries. This method isolates malic acid and other organic acids containing sugar, which can provide I taste and flavor in food. The best line is BALATON & MONTOMRENCY peach. . Text This anisocyanin, bioflavonoid and phenolic isolated mixture can be used as a natural nutrition therapy / food supplement. In this connection, the isolated mixture " T is in the form of a private liquid, or a solid form. For example, the mixture may be a reconstituted powder composition that provides a beverage when reconstituted using, for example, water, milk, or some other similar liquid. Or the mixture may be in a solid form such as a lozenge, a gel cap, a soft gel, and the like. In addition, the mixture can be incorporated into foods. The content of the mixture in the form of I is in the range of weight percent to about weight percent ^ preferably from about 0 • 1 weight percent T to about 30 weight percent , And about 0.5 weight percent to about 25 weight percent is more preferred. -11- C Please read the precautions on the back before filling out this page) 1¾ -------- Order --------- Printed by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs Applicable to Chinese National Standard (CNS) A4 ϋΤΓίΟ X 2 heart " ¥ li) 1223982 Printed by A7, Consumer Cooperatives, Intellectual Property Bureau, Ministry of Economic Affairs, 5. A description of the invention (〇9) For example, when the mixture is in bond form, 1 mg to 3 ο mg mg of flavocin PJ can provide about ^ Barkingan and biological acetophenone depletion R 10b. It is expected that it will be 1 to 2000 mg, with 6 η Ί I, soil 2UU Maowei It is better to provide 60 to 100 milligrams of the estimated hundred (100) cherries at a daily dose of ⑼-100 mg. One, η ,, brother (chemical pigment oath. The phenolic surface f FI j, the full dose can be from 0.1 to 50 mg. One "(Figure 4) gram age category. The color scheme is full of oaths for" The amount of flavonoids and phenols can be adjusted by early separation of potatoes: solid compounds and blending them. In a specific example: a natural mixture of tritiated pigment glycosides, bioflavonoids, and phenols 。Eosinophils can be provided in a liquid form containing the same dosage. Human,, and the resin have the surface classification of adsorption of anthocyanin, bioflavonoids, and hydrazones. The preferred adsorbent resin is polymer cross-linking. Resins, including styrene and divinylbenzene, such as, for example, AMBERLITE series resins, such as AM.BERLITE XAD-4 and AMBERLITE XAD-16, purchased from

Hohm & Haas Co·，Philadelphia，PA。其他適用於本發明之聚合物交聯苯乙晞及二乙烯基苯吸附性樹脂係為密西根之道氏化學公司所製造之XFS-4257、XFS-4022、 XUS-4 0 3 2 3 及 XUS-403 22 等。較佳係使用市售F D A認證之苯乙烯-二乙烯基苯（ SDVB)交聯共聚物樹脂（例如AMBERLITE XAD-16)。因此’於較佳具體實例中，AMBERLITE XAD- 1 6購自 Rohm and Haas COmpany，描述於美國專利第 4，297， 22 0號，係作為樹脂。AMBERLITE XAD-16具有粗網結構’同時具有連續聚合物相及連續微孔相。於特佳具體實例中，本發明所使用之樹脂具有由1 〇〇 - 2 0 0微米之粒徑。 -12- ---------L-----------^--------- (請先閱讀背面之注咅？事項再填寫本頁) · 本紙張尺度適用.中國國家標準（CNS)A4規格（210 X 297公釐） 1223982 A7 _______ B7 五、發明說明（10) (請先閱讀背面之注意事項再填寫本頁) 其他吸附劑諸如AMBERLITE XAD吸附劑系列…含有疏水性粗孔樹脂粒，粒徑為1 0 0 -2 0 0微米…亦可使用於本發明方法中。而且，不同Α Μ B E R l I T E S諸如 AMERCHROM CG系列吸附劑，粒徑範圍！ 0〇-2〇〇微米，亦適用於本發明。AMBERLITE XAD-16因為可重複使用夕’人（超過1 0 0次），故較佳。然而，就食品而言，本發明中經政府認證樹脂之使用被視為重要且/或期望。任何溶劑皆可用以去除經吸附之花色素苷、生物類黃酮、及酚類。較佳係為含有1至4個碳原子之底烷醇類，最佳係為乙醇（乙基醇），因其已證明可使用於食品。該乙醇般係與水共’滞；然而’可使用無水乙醇。樓桃中含有 %果酸及糖之水通經該管柱。收集此等化合物，可於食品中作為調味劑。經濟部智慧財產局員工消費合作社印製該花色素苷、生物類黃酮、及酚類較佳係得自 BALATON及MONTMORENCY櫻桃。該櫻桃之組成物部分列示於1997年2月12曰申請之美國申請案第08/799，7 88號及1998年12月11日申請之美國申請案第60/111，9 4 5號及1 9 9 9年2月1 6日申請之第6 0 / 1 2 0，1 7 8號。如此等申請案所描述，該Montmorency(歐洲酸櫻桃）品種佔有密西根及美國中苦櫻桃種植之9 5百分比以上。然而，已種植Balaton苦櫻桃（歐洲酸櫻桃）--一種新穎之苦櫻桃植株--取代數個密西根果園中之Μ ο n t m 〇 r e n c y。此種櫻桃具有較高之花色素苷含量，而被視為較佳品種。 Montmorency及Balaton苦櫻桃之花色素苷含量已有記載 -13- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1223982 A7 -- B7 五、發明說明（11 ) (Wang等人，；i9 9 7;Chandra等人，1 9 9 3 )。然而，之前尚未 (請先閱讀背面之注意事項再填寫本頁) 針對B a 1 a t ο η苦櫻桃中之其他酚類化合物進行詳細評估。早期研究顯示MONTMORENCY櫻桃含有花青素-3-龍膽二糖苷及花青素-3 -芸惱糖苦（l i，K · C ·等人，美國化學協會期刊78:979-980(1956))。亦於四種酸櫻桃品種之六種中發現花青素-3-葡萄糖基芸香糖苷（Harb〇ne，J.B·等人，植物化學3:453-463(1964))。Dekazos(Dekazos，E.D.，J •Food Sci.35:237-241(1970))記載 MONTMORENCY 櫻桃中之花色素苷顏料，其係為芍藥素_ 3 -芸香糖苷、芍藥素及花青素形式’同時有花青素-3 -槐糖苷、花青素_ 3 ·芸香糖苷及花青素-3-葡萄糖苷。然而，花青素_3_葡萄糖基芸香糖苷及花青素-3 -葡萄糖苷、花青素_ 3 —槐糖苷及花青素-3 _芸香糖苷已被確定為酸櫻桃中主要之顏料。Hohm & Haas Co., Philadelphia, PA. Other polymer crosslinked acetophenone and divinylbenzene adsorptive resins are XFS-4257, XFS-4022, XUS-4 0 3 2 3 and XUS manufactured by Dow Chemical Company of Michigan -403 22 etc. Preferably, a commercially available F D A certified styrene-divinylbenzene (SDVB) cross-linked copolymer resin (for example, AMBERLITE XAD-16) is used. Therefore, in a preferred embodiment, AMBERLITE XAD-16 is purchased from Rohm and Haas COmpany and described in U.S. Patent No. 4,297,220 as a resin. AMBERLITE XAD-16 has a coarse network structure and has both a continuous polymer phase and a continuous microporous phase. In a particularly preferred embodiment, the resin used in the present invention has a particle size ranging from 1000 to 2000 microns. -12- --------- L ----------- ^ --------- (Please read the note on the back? Matters before filling out this page) · This paper size is applicable. Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1223982 A7 _______ B7 V. Description of the invention (10) (Please read the precautions on the back before filling this page) Other adsorbents such as AMBERLITE XAD Adsorbent series ... Contains hydrophobic coarse-pored resin particles with a particle size of 100-200 microns ... It can also be used in the method of the present invention. Moreover, different AMB E R l I T E S such as AMERCHROM CG series adsorbents, particle size range! 0-200 microns is also suitable for the present invention. AMBERLITE XAD-16 is better because it can be used repeatedly (over 100 times). For food, however, the use of government-certified resins in the present invention is considered important and / or desirable. Any solvent can be used to remove adsorbed anthocyanins, bioflavonoids, and phenols. Alkanols containing 1 to 4 carbon atoms are preferred, and ethanol (ethyl alcohol) is the most preferred because they have been proven to be useful in foods. The ethanol is generally 'stagnate' with water; however, absolute ethanol can be used. The water containing% fruit acid and sugar passed through the column. These compounds are collected and used as flavoring agents in food. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. The anthocyanins, bioflavonoids, and phenols are preferably obtained from BALATON and MONTMORENCY cherries. The components of this cherry are listed in U.S. Application No. 08/799, 7 88, filed on February 12, 1997, and U.S. Application No. 60/111, 9 4 5, filed on December 11, 1998 and No. 60/1 2 0, 1 7 8 of the application on February 16, 1999. As described in these applications, this Montmorency variety accounts for more than 95% of the bitter cherry cultivation in Michigan and the United States. However, Balaton bitter cherries (European tart cherries), a novel plant of bitter cherries, have been planted to replace M o n t m 0 r en c y in several Michigan orchards. This cherry has a higher anthocyanin content and is considered a better variety. Montmorency and Balaton bitter cherry anthocyanin content have been recorded -13- This paper size applies Chinese National Standard (CNS) A4 (210 X 297 mm) 1223982 A7-B7 V. Description of the invention (11) (Wang et al.) People ,; i9 9 7; Chandra et al., 1 9 9 3). However, it has not been done before (please read the notes on the back before filling out this page) for a detailed evaluation of other phenolic compounds in B a 1 a t ο η bitter cherry. Early research shows that MONTMORENCY cherries contain anthocyanin-3-gentiobiside and anthocyanin-3-annoying sugar bitter (li, K.C. et al., Journal of the American Chemical Society 78: 979-980 (1956)) . Anthocyanin-3-glucosylrutinoside has also been found in six of the four sour cherry varieties (Harbone, J.B. et al. Phytochemistry 3: 453-463 (1964)). Dekazos (Dekazos, ED, J. Food Sci. 35: 237-241 (1970)) describes anthocyanin pigments in MONTMORENCY cherries, which are in the form of paeoniflorin 3-rutin, paeoniflorin, and anthocyanins. There are anthocyanin-3-sophoroside, anthocyanin-3-rutin, and anthocyanin-3-glucoside. However, anthocyanin_3_glucosylrutin and anthocyanin-3_glucoside, anthocyanin_3_sophorinoside and anthocyanin_3_rutin have been identified as the main pigments in sour cherries.

Chandra等人使用HPLC滯留時間值（Chandra，Α·等人，J. Agric_Food.Chem.40: 967-9 69( 1 992 ))記載花青素槐經濟部智慧財產局員工消費合作社印製糖苷及花青素-3 -葡萄糖苷個別係為密西根州種植之 MONTMORENCY櫻桃之主要及次要花色素苷。相同地，花青素_ 3 -木糖基芸香糖苷已被測出係為 MONTMORENCY櫻桃中之次要顏料（Shrinkhande a 了及 F.J.Francis"· Food.Sci.38:64 9- 6 5 1 ( 1 9 73 ))。 ’’載體’’或.”增量劑”意指一種組成物，其係添加以增加得自認櫻桃之純組成物的組成物體積。較佳係乾燥之p 桃果肉。此等包括任何含有可食用澱粉之材料、 : | 主白質諸如脫脂奶粉。例如麵粉、糖、大豆粉、麥芽糖糊精及各種 -14- 適度尺張紙本 Μ 公 97 12 X 10 2 I格規 Α4 S) Ν (C 準標家國國 1223982 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（12) 佐料諸如鹽、胡椒、香料及藥草。增量劑較佳用量以介於約該混合物重量之1 〇 ·6及1 〇 6份數為佳。茲組成物可導入一食品中，用量係介於約〇 ·丨及丨〇毫克/克該食品之活性成分之間。該用量之選擇係不影響該食品之風味，而產生最有益之結果。該食品可為高（潮溼）或低溼度（乾燥），如熟習此技藝者所已知。當作為食品補充劑時，該錠劑係含有介於〇· i至i毫克間之活性成分。特定食品係為經烹煮肉品及其他調理食品，其中該組成物提供抗氧化性質及選擇性顏色於該食品。該組成物可於調理食品上作為佐料，以提供營養治療性或植物治療益處。已發展萃取及單離植物化學品（c h a n d r a，A •等人，J ·Chandra et al. Used HPLC retention time values (Chandra, A. et al., J. Agric_Food.Chem. 40: 967-9 69 (1 992)) to record the anthocyanin and glycosides printed by employees ’cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs of the Anthocyanin Group. Anthocyanins-3 -glucosides are the major and minor anthocyanins of MONTMORENCY cherries grown in Michigan. Similarly, anthocyanins_ 3 -xylosylrutinoside has been detected as the secondary pigment in MONTMORENCY cherry (Shrinkhande a and FJFrancis " · Food.Sci. 38:64 9- 6 5 1 (1 9 73)). "'Carrier'" or. "Bulking agent" means a composition which is added to increase the composition volume of a pure composition obtained from a recognized cherry. It is preferably dried p peach flesh. These include any materials containing edible starch,: | white matter such as skim milk powder. For example, flour, sugar, soy flour, maltodextrin and various -14- Moderate sheets of paper M public 97 12 X 10 2 I standard A4 S) Ν (C quasi-standard home country 1232982 Ministry of Economic Affairs Intellectual Property Bureau employee consumption Cooperative printed A7 B7 5. Description of the invention (12) Condiments such as salt, pepper, spices, and herbs. The preferred amount of extender is between about 10.0 and 10 parts by weight of the mixture. The composition can be introduced into a food, and the dosage is between about 0.00 mg / g and the active ingredient of the food. The selection of the dosage does not affect the flavor of the food, and produces the most beneficial results. The food can be high (humid) or low humidity (dry), as known to those skilled in the art. When used as a food supplement, the lozenge contains active ingredients between 0.1 mg and i mg. Specific Food is cooked meat and other prepared foods, wherein the composition provides antioxidant properties and selective color to the food. The composition can be used as an adjunct to the prepared foods to provide nutritional or phytotherapy benefits Developed And isolated phytochemicals (c h a n d r a, A • et al., J ·

Agr ic .Food. Chem.40: 9 6 7-9 6 9 ( 1 9 92); Wang，H.等人丨 Agridood Chem.45:2 5 5 6-2 5 6 0 ( 1 9 9 7))及迅速篩選抗氧劑活性之方法（Arora，A·及 G.M.’Stuobuij. Amer.Agr ic .Food. Chem. 40: 9 6 7-9 6 9 (1 9 92); Wang, H. et al 丨 Agridood Chem. 45: 2 5 5 6-2 5 6 0 (1 9 9 7)) And rapid screening of antioxidant activity (Arora, A · and GM'Stuobuij. Amer.

Oil Chem.S〇C.74:1 0 3 1 - 1 04 0 ( 1 9 9 7 ))。此等方法係用以確逐、定性及測試得自B A L A T〇N及Μ〇N T Μ〇R E N C Y樓桃之化合物。打汁之櫻桃組織隨之使用己烷、乙酸乙酯及甲醇萃取。甲醇及乙酸乙酯兩提份皆於篩選檢定中顯示強抗氧劑活性。該乙酸乙酯提份再以矽膠真空液體層析純化 ’以產生四種次提份；該次提份再藉製備用逆相HPLC分成四個提份。圖2及3顯示自BALA TON櫻桃單離之生物類黃驗。因此該苦櫻桃中有數種相同或同質化合物。確認兩種新穎之酚類化合物： 1) 1 - ( 3 ·，4 f -二羥基肉桂醯）-2，3 -二羥基環戊烷，及 -15- 本紙張尺度_中國國家標準（CNS)A4規格⑽χ挪公^^ ---------L-----------訂---------線義 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1223982 A7 ____B7 五、發明說明（13 ) II) 1 - ( 3，4、二經基肉桂醯）-2，5 -二經基環戊境。其他自櫻桃汁之乙酸乙酯萃取物單離而藉光譜方法定性之化合物包括：1-(3，_甲氧基，4，-甲氧基肉桂醯）雞衲酸、2-¾基- 3- (2f -經苯基）丙酸、2 -經基-3-(2’-輕笨基）丙酸甲酯、D( +卜蘋果酸、石-穀脂醇及穀脂醇葡萄糖苷。圖4係顯示所單離之部分酚類。得自果汁提份之花色素誓成分亦已經確認並完全定性（Chandra，A.等人，J.Agric Fo〇d.Chem.41:i〇62(1992);Wang，H.等人， J.Agric. Food Chem.4 5 :2 5 5 6-2 5 6 0 ( 1 9 9 7 ));結果顯示此等化合物含有強效抗氧化活性。如圖4所示，該化合物Oil Chem. Soc. 74: 1 0 3 1-1 04 0 (1 9 7)). These methods are used to identify, characterize, and test compounds obtained from B A L A TON and MO N T MOR EN C Y. The beaten cherry tissue was then extracted with hexane, ethyl acetate and methanol. Both methanol and ethyl acetate showed strong antioxidant activity in screening tests. The ethyl acetate fraction was purified by silica gel liquid chromatography to produce four fractions; the fraction was further separated into four fractions by reverse-phase HPLC. Figures 2 and 3 show the biological test results of the single cherries isolated from BALA TON. There are therefore several identical or homogeneous compounds in this bitter cherry. Identification of two novel phenolic compounds: 1) 1-(3 ·, 4 f -dihydroxycinnamic acid) -2,3 -dihydroxycyclopentane, and -15- Paper size_Chinese National Standard (CNS) A4 specifications ⑽χ Norwegian ^^ --------- L ----------- Order --------- line meaning (Please read the precautions on the back before filling (This page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1223982 A7 ____B7 V. Description of the Invention (13) II) 1-(3,4, Nicotine Cinnamon 醯) -2,5-Nicotine Cyclopentate. Other compounds that are isolated from the ethyl acetate extract of cherry juice and characterized by spectroscopic methods include: 1- (3, _methoxy, 4, -methoxycinnamic acid) gallic acid, 2-¾yl-3 -(2f-transphenyl) propionic acid, 2-transyl-3- (2'-light benzyl) propionic acid methyl ester, D (+ methyl malic acid, stone-sitosterol and glucosinolate. Figure 4 shows the isolated phenols. The anthocyanin components obtained from juice extracts have also been identified and fully characterized (Chandra, A. et al., J. Agric Fo.d. Chem. 41: i〇62 (1992); Wang, H. et al., J. Agric. Food Chem. 4 5: 2 5 5 6-2 5 6 0 (19 9 7)); the results show that these compounds contain potent antioxidant activity. As shown in Figure 4, the compound

可為異構物形式或為純物質形式，其中R !及R 2係選自經基及氫，而R ,及R2中之一係為羥基。該化合物可係為前述化合物之異構物，其中R ι係為羥基且R 2係為氫。特定之化合物異構物係為3,，4，-二羥基肉桂醯）-環戊-2 ，5 -二醇’其可自苦櫻桃單離成純化合物形式。該化合物亦可為前述化合物之異構物，其中R !係為氫且r2係為羥基。此種特定之化合物異構物係為1 _(3，，4，-二羥基肉桂醯 )-環戊-2 ’ 3 -二醇，其係自苦櫻桃單離成純化合物形式。 -16- 本紙張尺度適用中國國豕標準（CNS)A4規格（21〇 X 297公爱） I ,- -—--------訂---------線 c請先閱讀背面之注意事項再填寫本頁) 1223982 A7 B7 五、發明說明（14) 本發明亦有關一種於有需方法，立包括接板* 一 2 ^礼類體内抑制氧化之 ”已祜知供自苦櫻桃單離 ^ 合物及純物質：式化合物或并構物混It may be in the form of an isomer or in the form of a pure substance, wherein R! And R2 are selected from the group consisting of hydrogen and hydrogen, and one of R, and R2 is a hydroxyl group. The compound may be an isomer of the aforementioned compound, wherein R 1 is a hydroxyl group and R 2 is a hydrogen. A specific compound isomer is 3,4, -dihydroxycinnamonene) -cyclopentane-2,5-diol ', which can be isolated from bitter cherry to a pure compound form. This compound may also be an isomer of the aforementioned compound, wherein R! Is hydrogen and r2 is hydroxy. This particular compound isomer is 1- (3,4, -dihydroxycinnamic acid) -cyclopentane-2'3-diol, which is isolated from bitter cherry into pure compound form. -16- This paper size applies to China National Standard (CNS) A4 (21〇X 297 public love) I,---------- Order --------- line c please (Read the precautions on the back before filling this page) 1223982 A7 B7 V. Description of the invention (14) The present invention also relates to a method in need, including a connection board *-2 ^ Inhibition of oxidation in vivo "has been known For self-bittering cherries isolated compounds and pure substances: Compounds of formula or mixed compounds

(請先閱讀背面之注意事項再填寫本頁) 其中R,及I係選自羥基或氫，基’該物質用量係抑制該材料之1 I中：-係為輕方法係使用前述化合物之異構物，复。-具體貫例中’話係為氫。此等特別之化合物：：Ri係為搜基且R: ,,. 傅初係為1 - f 3 · 4 * -二辦其内桂醯）-環戊-2 , 5-二醇，其可自（，尨基肉式。於另一具體實例中，該方口料異構物，其中R1係為氯且,2係為超：使二述化合物之的異構物係為1-(3，，4，-二羥基肉桂醯卜環戊j别 < 化合物經濟部智慧財產局員工消費合作社印製 -二醇禮實例中，其可自苦櫻桃單離成純化合物形式。另一且 ’ 方法係包括使用該異構物之混合物。本發明之一具體實例中，該組成物係包今。單離之1 - ( 3 ’，4、二羥基肉桂醯）_環戊_ 2，3 _ _ J自吉櫻物或與1-(3’，4、二羥基肉桂醯）·環戊_2，％了醇花純化〜醇之滿八 ;及一種供該化合物使用之無毒性載體或增量剖口另一具體實例中，該組成物係包含可自苦庐: 本發 &姚卓離之；I _ < -17-本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1223982 A7 B7 15 五、發明說明（，4 ’ -二技基肉桂醯）-環戍· 2，5 -二醇之純化合物或與1 - ( 3" ，4 ’ -二基肉桂醯）-環戍_ 2，3 _二醇之混合物；及一種供該化合物使用之無毒性載體或增量劑。該組成物可另外含有添加於該純化合物中而得自櫻桃之其他抗氧化劑，諸如花色素誓、類叉酮、或其他酚類。此外，該組成物所使用之載體或增量劑係為適於動物或人類使用之載體。遠組成物係由本發明抗氧化劑及微粒可食用增量劑所組成’該增量劑用量為每份本發明物質介於約〇 ·丨至3 〇份間’孩產物與一可氧化材料同時導入時，抑制該材料之氧化。含有本發明抗氧化劑之組成物可形成任何適當之固體或液體劑型，而作為營養治療劑/食品補充劑。所選擇之劑型通常提供日劑量之1 · ( 3，，4 L二羥基肉桂醯卜環戊_ 2， 5 -二醇或1 - ( 3 ’，4 ’ -二羥基肉桂醯卜環戍_ 2，3 ·二醇之純化 3物 3有兩兴構物之摻合物、或含有其他抗氧化劑化合物（摻合物。本發明或為組成物、混合物、或純物質形式，可作為抗發炎劑，或用以治療慢性疾病，或預防疾病，舒緩疾病狀況，或刺激免疫反應以更有效地對抗疾病。本發明組成物、混合物、或純物質形式尤其可用以預防或治療各種疾病，其係部分或完全由自由基所致。如前文所述，本發明組成物可另外包括一或多種抗氧化劑，選自花色素苷、生物類黃酮、酚類及其混合物。該 k桃I性化色素苷、生物類黃酮、及酚類較佳係自櫻桃單離，尤其是BALATON及MONTMORENCY櫻桃。較佳具體實例中，該花色素苷係選自花青素_3_2，，-〇_ -------—rl — ΛΨ (請先閱讀背面之注意事項再填寫本頁) 訂---------線| 經濟部智慧財產局員工消費合作社印製(Please read the precautions on the back before filling this page) where R, and I are selected from hydroxyl or hydrogen, and the amount of this substance is to inhibit the material from 1 I:-is a light method and uses the difference of the aforementioned compounds Structure, complex. -In the specific example, the phrase 'is hydrogen. These special compounds :: Ri is Suji and R: ,, .. Fu Chu is 1-f 3 · 4 *-Erbanqiuigui)-cyclopentan-2, 5-diol, which can Since (, fluorene-based meat formula. In another specific example, the castrate isomers, wherein R1 is chlorine and, 2 is super: the isomer of the second-order compound is 1- (3 In the example printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs and the Intellectual Property Bureau of the Ministry of Compound Economy, it was isolated from the bitter cherry into a pure compound form. Another and ' The method includes using a mixture of the isomers. In a specific embodiment of the present invention, the composition is included in the present invention. 1-(3 ', 4, dihydroxycinnamic acid) _cyclopentane_ 2,3_ _ J Ziji Yingwu or 1- (3 ', 4, dihydroxycinnamon tincture) · cyclopentane_2,% purified alcohol flower ~ full of alcohol; and a non-toxic carrier or additive for the compound In another specific example of the measurement profile, the composition contains self-biting: Benfa & Yao Zhuozhi; I _ < -17- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 male ) 1223982 A7 B7 15 V. Description of the invention (, 4'-Dialkylcinnadium 醯) -Cyclopentamidine · 2,5-diol pure compound or with 1-(3 ", 4'-Diylcinnamon 醯)- A mixture of cyclohexyl-2,3-diols; and a non-toxic carrier or extender for use with the compound. The composition may additionally contain other antioxidants, such as flowers, from cherries added to the pure compound Pigment vows, fork-like ketones, or other phenols. In addition, the carrier or extender used in the composition is a carrier suitable for animal or human use. The distant composition is an edible extender made of the antioxidants and microparticles of the present invention. The amount of the bulking agent is between about 0.00 to 30 parts of the substance of the present invention when the product is introduced with an oxidizable material at the same time, which inhibits the oxidation of the material. The composition can be formed into any suitable solid or liquid dosage form as a nutritional therapeutic / food supplement. The selected dosage form usually provides a daily dose of 1 · (3,4 L dihydroxycinnamic acid cyclopentaline _ 2, 5 -Diol or 1-(3 ', 4'- Hydroxycinnamic acid, bucyclamidine, 2,3 • Purification of diols, 3, 3 Blends with two compounds, or containing other antioxidant compounds (blends. The present invention may be a composition, mixture, or pure Material form, can be used as anti-inflammatory agent, or to treat chronic disease, or prevent disease, soothe disease condition, or stimulate immune response to fight disease more effectively. The composition, mixture, or pure material form of the present invention is especially useful for prevention Or treat various diseases, which are caused partially or completely by free radicals. As mentioned above, the composition of the present invention may further include one or more antioxidants selected from anthocyanins, bioflavonoids, phenols, and mixtures thereof. The k peach I sexual pigments, bioflavonoids, and phenols are preferably isolated from cherries, especially BALATON and MONTMORENCY cherries. In a preferred embodiment, the anthocyanin is selected from the group of anthocyanins_3_2, -〇_ --------- rl — ΛΨ (Please read the notes on the back before filling this page) Order- ------- line | Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs

經濟部智慧財產局員工消費合作社印製 1223982Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1223982

、發明說明（16) 3 τλ 比喃葡糖基-6”-〇-V-L -鼠李糖基喃葡糖香、 —匕月素-3 - 6 - 〇 - 3 - L -鼠李糖基-3 · D - 口比喃葡糖苷、花青素-° Ί D - u比喃葡糖苷及其混合物。於較隹具體實例中，該生物類黃酮係為7-甲氧基-5，8，4，-三羥基黃酮。含有本發明抗氧化劑或本發明化合物之組成物可添加於食品’以作為一般抗氧化劑食品添加劑。該食品添加劑尤其可包括1-(3，，4，-二羥基肉桂醯）_環戊-2，3-二醇或ΙΟ ’ 4 - 一無基肉桂 Si ) - 環戊 · 2 ， 5 - 二醇之純化合物或含有兩并構物之混合物。該食品添加劑可另外包括其他抗氧化 ^ ’選自花色素苷、花青素、生物類黃鹼、酚類及其混合物。較佳具體實例中，該花色素苷係選自花青素 D - p比喃葡糖基-6 - V - L -鼠李糖基· 3 - d · ρ比喃葡糖苷、花同素-j - 6 - 〇 - V - L -鼠李糖基-3 - D -吡喃葡糖苷、花青素 • 3 - 3 - D _吡喃葡糖苷及其混合物。於較佳具體實例中，該生物類頁酮係為7 -甲氧基-5，8，4 ’ -三輕基黃酮。該花色素苷、花青素、生物類黃鹼及酚類較佳係自櫻桃單離，尤其係自BALATON及MONTMORENCY櫻桃。含有本發明抗氧化劑或本發明化合物之組成物可為添加於食品之一般抗氧化劑食品添加劑，該食品或高（潮溼）或低溼度（乾燥&) 、新鮮或未經烹調、經熟化或經烹煮。添加於含^脂質之食品時，該抗氧化劑化合物或組成物抑制該脂質，而抑制腐壞。於烹煮前添加該組成物於肉品，扣… 抑制雜裱芳族胺（ HHA)之形成，HHA係於烹煮期間產生，^l、且顯不有致癌性 -19- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁)Description of the invention (16) 3 τλ glucopyranosyl-6 "-〇-VL -rhamnosyl glucosamine,-Dangerin-3-6-0-3-L -rhamnosyl- 3 · D-glucopyranoside, anthocyanins-° Ί D-u glucopyranoside and mixtures thereof. In a more specific example, the bioflavonoid is 7-methoxy-5,8, 4, -trihydroxyflavone. The composition containing the antioxidant or the compound of the present invention may be added to foods as a general antioxidant food additive. The food additive may include 1- (3,4, -dihydroxycinnamon especially醯) _Cyclopentane-2,3-diol or 10 ′ 4 -monobasic cinnamon Si) -Cyclopentane 2,5 -diol A pure compound or a mixture containing a dimer. The food additive may additionally Including other antioxidants ^ 'selected from anthocyanins, anthocyanins, bioflavonoids, phenols, and mixtures thereof. In a preferred embodiment, the anthocyanins are selected from anthocyanins D-p-glucopyranose -6-V-L-rhamnosyl, 3-d, p-glucoside, anthocyanin-j-6-〇-V-L-rhamnosyl-3, D-glucopyranoside , Anthocyanins • 3-3-D glucopyranoside and mixtures thereof. In a preferred embodiment, the biological phytonone is 7-methoxy-5,8,4'-triloylflavonoid. The anthocyanin Anthocyanins, bioflavonoids and phenols are preferably isolated from cherries, especially from BALATON and MONTMORENCY cherries. The composition containing the antioxidant or the compound of the present invention can be a general antioxidant food added to food Additive, the food is either high (wet) or low humidity (dry &), fresh or uncooked, ripened or cooked. When added to foods containing lipids, the antioxidant compound or composition inhibits the lipids Adding this composition to the meat before cooking, buckle ... inhibits the formation of hybrid aromatic amines (HHA), which are produced during cooking, and are not carcinogenic- 19- This paper size is in accordance with China National Standard (CNS) A4 (210 X 297 mm) (Please read the precautions on the back before filling this page)

1223982 A7 ______Β7 ___ 五、發明說明（Π) 實施例1及2 (請先閱讀背面之注意事項再填寫本頁) 如圖5所示，將個別迅速冷凍（"IQF”）之櫻桃（已去核 )解凍，於工業級WARING摻合器.中摻合。該混合物於1 〇，0 0 0轉每分鐘下離心，傾析該果汁。殘留物，果肉以乾酪包布（cheese cloth)擠出任何多餘果汁。經濟部智慧財產局員工消費合作社印製該果肉係於1 5 °C下冷凍乾燥。果汁於A Μ B E R L I T E XAD- 1 6 HP樹脂上處理，以產製櫻桃酸性花色素苷、生物類黃酮、及酚類。該XAD- 1 6樹脂1公斤以乙醇（1 -2升）洗滌，之後以水（6升）洗滌。該XAD樹脂-1 6於水中放置1 小時’之後置入具有棉塞之玻璃管（1〇内徑X9〇厘米長度 )中。經充填之管柱以水（2升）洗滌，之後放置欲分離之果汁。每次純化8 0 0毫升之果汁。該果汁添加於該管表面上，在不流動下使之沉降。之後以水洗提，丟棄先頭1升。收集次2升洗滌液，因其含有因含來自櫻桃之蘋果酸及糖而呈酸性之櫻桃汁。該管柱使用另外4升水洗滌（若為 BALATON)，若為M0NTM〇RENCY櫻桃汁則使用5升。一旦收集該櫻桃汁，則丟棄其餘水洗液。該管柱隨之以乙醇（1·3-1.5升）洗提，收集含有花色素苦、生物類黃酮、及酚類之紅色溶液（70 0_800毫升）。將該管柱放空，以1〇升水洗滌，之後重複該過程多次（超過1〇〇次）。紅色醇溶液於真空下（20耄托耳）蒸發，以去除乙醇及水溶液，使用百萬分之5 0份數之抗壞血酸安定化，於丨〇它下冷凍乾燥。收集紅色粉末並儲存。實施例1結果： -20-本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱）經濟部智慧財產局員工消費合作社印製 1223982 A7 - B7 五、發明說明（18) BALATON ^ ^ JQF櫻桃重量 15.74公斤乾燥果肉重量 605克果汁體積 12.16升花色素苷、生物類黃酮、及酚類之重量（紅色粉末） 31·35克酸副產物之體積（蘋果酸及糖） @35升實施例2結果： MONTMORENCY 櫻桃 IQF櫻桃重量 30.45公斤乾燥果肉重量 895克果汁體積 24.03升花色素苷、生物類黃酮、及酚類之重量（紅色粉末） 47克酸副產物之體積（蘋果酸及糖） @75升實施例1及2之紅色粉末較佳係與乾燥果肉混合以作為載體，製錠成包括該載體之i至1 〇〇〇毫克錠劑（1個成人之日劑量）。可於所單離之花色素苷、生物類黃酮、及齡類中添加各種食品級酸類，以防止分解。以不添加調味劑為佳。以抗壞血酸（維生素C)為佳。該酸可於乾燥該櫻桃化合物之前或之後添加，以於之前添加為佳。小規模處理中使用冷凍乾燥以去除水。大規模產製時，以於空氣循環爐中乾燥為佳。 -21- 本紙張尺度適用中國國豕仏準（CNS)A4規格（21〇 X 297公爱 ---------L·---- --------訂---------線 (請先閱讀背面之注意事項再填寫本頁} 五、發明說明（19) 貫施例3 (請先閱讀背面之注意事項再填寫本頁) 如圖6所示’提供一開放式容器1〇，具有輸入管線" 中：輸出管線12，個別具有闕U及“。於該開放容器10 ::供該樹脂珠粒15。於容器丨〇中導入水，之後經輸出 7 12去除並丟棄。將與實施例丨相同之櫻桃果汁（無果 2或果桂）導入容器10中，使之放置25分鐘。水及果汁之度係介於約2 0 c及3 0 °c之間。該含有蘋果酸及糖之櫻兆2殘留物經輸出管線12取出，而保留作為食物調味劑。谷器中之樹脂15再次以來自輸入管線丨丨之水洗滌，取出，並經輸出管線1 2丟棄。位於該樹脂粒上之花色素苷生物潁κ酿1、及i分類使用經由輸入管線1 1導入之9 5百分比乙醇萃取。自容器丨〇取出含有花色素誓、生物類黃酮及酚類之乙醇。自該花色素苷、生物類黃酮、及酚類取出乙醇，使用閃蒸乾燥於氮下乾燥。形成之粉末以與乾燥樓桃果肉或其他與實施例1相同之載體混合為佳。樹脂粒以水洗滌，之後該樹脂及乙醇係循環多次。實施例4 經濟部智慧財產局員工消費合作社印製得自櫻桃之粗製乙酸乙酯萃取物（含有花色素苷、生物類貝§同、及酚類）係於水溶液中於各種條件下使用螢光檢定試驗抗氧化劑活性。首先描述該螢光檢定。篩選大量化合物或萃取物需要合理表示該化合物本身或於食品產物中之結構及功能特性之模型系統（或系統等）。该试驗亦需靈敏、快速而平價。使用用以評估抗氧化效率而以螢光為底質之檢定（Arora，A.及G.M. Strasburg，J. -22- 本紙張尺度適用中國國家標準（CNS)A4規格（210 x 297公釐） A71223982 A7 ______ Β7 ___ 5. Description of the Invention (Π) Examples 1 and 2 (Please read the precautions on the back before filling out this page) As shown in Figure 5, the individual cherries (" IQF ") which have been frozen quickly (have been removed (Nucleus) Thawed and blended in an industrial-grade WARING blender. The mixture was centrifuged at 10,000 rpm to decant the juice. The residue, pulp was squeezed out with cheese cloth Any excess juice. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. The pulp is freeze-dried at 15 ° C. The juice is processed on A BERLITE XAD-1 6 HP resin to produce cherry acid anthocyanins, bio Flavonoids, and phenols. 1 kg of the XAD-16 resin was washed with ethanol (1-2 liters), and then washed with water (6 liters). The XAD resin-1 6 was placed in water for 1 hour and placed in the A tampon glass tube (10 inner diameter X 90 cm length). After filling the column with water (2 liters), the juice to be separated is placed. 800 ml of juice is purified each time. The juice is added On the surface of the tube, it is allowed to settle without flowing. Elution, discard the first 1 liter. Collect the next 2 liters of washing solution, because it contains cherry juice that is acidic because it contains malic acid and sugar from cherries. The column is washed with another 4 liters of water (if BALATON), if it is 5 litres of MONTM〇RENCY cherry juice was used. Once the cherry juice was collected, the remaining water washing liquid was discarded. The column was subsequently eluted with ethanol (1.3-1.5 liters) to collect anthocyanin, bioflavonoids, And a red solution of phenols (70 0-800 ml). The column was vented, washed with 10 liters of water, and then the process was repeated multiple times (more than 100 times). The red alcohol solution was under vacuum (20 Torr) Evaporate to remove ethanol and water solution. Use 50 parts per million of ascorbic acid to stabilize and freeze dry under it. Collect red powder and store. Example 1 Results: -20- This paper scale applies to China Standard (CNS) A4 specification (210 X 297 public love) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1223982 A7-B7 V. Description of the invention (18) BALATON ^ ^ JQF cherry weight 15.74 kg dry pulp weight 605 g juice volume 1 Weight of 2.16 liters of anthocyanins, bioflavonoids, and phenols (red powder) 31.35 grams of acid by-product volume (malic acid and sugar) @ 35 liters Example 2 Results: MONTMORENCY Cherry IQF Cherry weight 30.45 kg dry Flesh weight 895 g Juice volume 24.03 liters Anthocyanins, bioflavonoids, and phenols (red powder) 47 g acid byproducts (malic acid and sugar) @ 75 liters Jiaxi is mixed with dried pulp as a carrier, and tablets are made into tablets containing i to 1000 mg of the carrier (daily dose for one adult). Various food-grade acids can be added to the isolated anthocyanins, bioflavonoids, and ages to prevent decomposition. It is better not to add flavor. Ascorbic acid (vitamin C) is preferred. The acid may be added before or after the cherry compound is dried, preferably before the addition. Freeze drying is used in small scale processing to remove water. For large-scale production, it is better to dry in an air circulating furnace. -21- This paper size applies to China National Standard (CNS) A4 (21〇X 297 Public Love --------- L · ---- -------- Order- ------- Line (Please read the precautions on the back before filling this page} V. Description of the invention (19) Implementation Example 3 (Please read the precautions on the back before filling out this page) As shown in Figure 6 'Provide an open container 10 with input lines " Medium: output lines 12, each with 阙 U and ". In the open container 10 :: for the resin beads 15. Introduce water into the container, and then It is removed and discarded by output 7 12. The same cherry juice (no fruit 2 or fruit cinnamon) as in Example 丨 is introduced into the container 10 and allowed to stand for 25 minutes. The degree of water and juice is between about 2 0 c and 3 Between 0 ° c. The residue of Sakura trillion 2 containing malic acid and sugar is taken out through the output line 12 and is retained as a food flavoring agent. The resin 15 in the trough is washed again with water from the input line 丨丨 and taken out, It is discarded through the output line 12. The anthocyanin biotin kappa 1, and i that are located on the resin particles are extracted with 95% ethanol introduced through the input line 11 1. Self-capacity Take out ethanol containing anthocyanins, bioflavonoids, and phenols. Take out ethanol from the anthocyanins, bioflavonoids, and phenols and use flash evaporation to dry under nitrogen. The resulting powder is used to dry the floor. Peach pulp or other same carrier as in Example 1 is better to mix. The resin pellets are washed with water, and then the resin and ethanol are circulated multiple times. Example 4 Crude acetic acid printed from cherry by the Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economy Ethyl acetate extract (containing anthocyanins, biological shellfish, and phenols) is used in aqueous solutions under various conditions to test the antioxidant activity using fluorescent assays. The fluorescent assay is described first. Screen a large number of compounds or extracts A model system (or system, etc.) that reasonably represents the structural and functional characteristics of the compound itself or in a food product is required. The test also needs to be sensitive, fast, and affordable. Use fluorescence as a substrate to evaluate antioxidant efficiency Verification (Arora, A. and GM Strasburg, J. -22- This paper size applies to China National Standard (CNS) A4 (210 x 297 mm) A7

^i982 五、發明說明（20 )^ i982 V. Description of Invention (20)

Am.Chem.Soc.1 9 9 6 )。製備由^硬脂醯·2_亞麻油醯-^· =油-3-磷酸膽鹼組成之大型單層泡囊，精確地模擬生物薄腠（過氧化之主要部位）的性質。將螢光探針…丨，6 -二笨=己三烯丙酸—併入該膜中’使得該極性端基將該探針支撐於孩含水界面附近，而疏水性部分則與該脂肪酸鏈平. 行。此探針與過氧化期間所產生之自由基反應，導致營光強度隨時間而降低。使用過氧化起始劑（諸如亞鐵離子或自由基產生劑A A P Η (偶氮基雙_〔眯基丙烷鹽酸鹽〕）以起始該反應，螢光降低之動力學係於欲試驗之抗氧化劑组成物存在或不存在下決定。化合物於特定濃度下之檢定現在僅進行二十一分鐘，僅消耗數微克脂質，而可輕易地使用簡易之螢光計進行。根據 MacDonald 等人（MacDonald，R.C·等人 Biochim.Biophys.Acta 1061:297-303(1991))之方法自更月日醯“-亞麻油龜_ s n _甘油· 3 _磷酸膽驗製備大型單層心義（L U V s)。簡5之，脂質溶於氯仿中，使用旋轉蒸發器乾燥成薄膜。乾燥之薄膜再懸浮於緩衝水溶液中，使用Am.Chem.Soc. 1 9 9 6). A large monolayer vesicle composed of ^ stearylamine · 2_linseed oil 醯-^ · = oil-3-phosphate choline was prepared to accurately simulate the properties of biothin (the main part of peroxidation). Fluorescent probes are incorporated into the membrane, 6-dibenzyl = hexatriallyl acid 'so that the polar end group supports the probe near the aqueous interface, while the hydrophobic part is linked to the fatty acid chain Flat. OK. This probe reacts with free radicals generated during peroxidation, resulting in a decrease in camping light intensity over time. Use a peroxidation initiator (such as ferrous ion or radical generator AAP Η (azobis_ [fluorenylpropane hydrochloride]) to initiate the reaction. The kinetics of fluorescence reduction is based on the The antioxidant composition is determined in the presence or absence. The test of a compound at a specific concentration is now performed for only twenty-one minutes, consumes only a few micrograms of lipid, and can be easily performed using a simple fluorometer. According to MacDonald et al. (MacDonald , RC · et al. Biochim. Biophys. Acta 1061: 297-303 (1991)) method of preparing large-scale single-layered Xinyi (LUV s) from the meniscus "-linseed turtle _ sn _ glycerine 3 _ phosphate bile test ). Jane 5. Lipids are dissolved in chloroform and dried on a rotary evaporator to form a film. The dried film is resuspended in a buffered aqueous solution and used.

Llp〇S〇fast Piposome 擠塑機（Avestin，Inc. Ottawa，Can- )、二由具有i 〇〇爱微米孔徑之聚碳酸s旨滤器重複擠塑。使用冰;東碎裂掃描式電子顯微鏡確認該泡囊之單層性質。该勞光探針―二苯基己三烯-丙酸（DPH-P A )—係於製備期間於1 : 3 5 0莫耳比（探針：脂質）下併入該泡囊中。螢光實驗中’含有 DPH-PA 之 LUVs 懸浮於 100mM NaCl，5〇 mM Tris-HEPES緩衝劑pH7〇中，最終濃度為ι〇〇微莫耳濃 -23- 本紙巧度適用^國家標準規格（210 ;公髮）一 --— --------l·—J—Λ^ (請先閱讀背面之注意事項再填寫本頁) 訂— 經濟部智慧財產局員工消費合作社印製 1223982 A7 五、發明說明（21 度。螢光探針係於3 8 4毫微米下激發，於4 2 3毫微米下偵測發射光。脂質氧化係於該LUVs中藉著添加亞鐵離子或自由基產生劑A A P Η而抑制；該過程係藉著於二十一分鐘内產生之自由基反應導致D Ρ Η - Ρ Α之螢光強度降低而偵測。以時間函數表示之螢光強度降低圖係用以決定脂質氧化之動物學。結果顯示粗製花色素苷萃取物與乙酸乙酯之混合物可抑制氧化。可使用花色素苷、生物類黃酮及酚類之溶劑萃取；然而就▲產物欲作為食品或就價格因素而言，此種情況較為不佳。使用較佳之吸附劑樹脂時，此步驟·非必要。亦可使用層析分離並重組該成分；然而，就本發明目的而言，此情況因為包括高壓液體層析而變得太過昂貴。實施例5 口亥心桃花色素苷使用由Arora及Strasburg所發展之方法檢定抗氧化劑活性。隨著反應之進行，螢光探針降解 ’使螢光強度下降。因此，於抗氧化劑存在下，可降低螢光降解之速率。實驗顯示花色素苷1 - 3及糖苷配基、花青素、之柷氧化活性，較市售抗氧化劑經丁基化羥基甲苯醚及經丁基化之羥基甲苯（BHT)(圖7)優越。而且，此等櫻桃化合物顯示較α _生育酚良好之抗氧化活性。於二毫莫耳濃度下，含有生育酚之試樣與含有Feh而不添加抗氧化劑（圖7)之試樣無法分辨。化色素苷（糖苷配基，花青素，具有較葡萄糖苷高之效率，顯示花色素苷之抗氧化活性係由其糖苷配基所：。 -------------- (請先閱讀背面之注意事項再填寫本頁) i訂---------線 11^· 經濟部智慧財產局員工消費合作社印製 -24- 1223982 A7LlpSOfast Piposome extruder (Avestin, Inc. Ottawa, Can-), two extruded from a polycarbonate filter with a pore size of 100 μm. The monolayer nature of the vesicles was confirmed using an ice fragmentation scanning electron microscope. The luminescent probe, diphenylhexatriene-propionic acid (DPH-PA), was incorporated into the vesicle at a molar ratio (probe: lipid) of 1: 350 during the preparation. In the fluorescence experiment, the LUVs containing DPH-PA were suspended in 100 mM NaCl, 50 mM Tris-HEPES buffer pH 70, and the final concentration was ιΟΟμmolar -23. 210; public hair) I --- -------- l · —J—Λ ^ (Please read the notes on the back before filling this page) Order — Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1223982 A7 V. Description of the invention (21 degrees. Fluorescent probes are excited at 3 8 4 nm, and the emitted light is detected at 4 2 3 nm. Lipid oxidation is added to the LUVs by adding ferrous ions or free Base generation agent AAP Η; this process is detected by the free radical reaction generated within 21 minutes resulting in a decrease in the fluorescence intensity of D P Η-Ρ Α. Graph of fluorescence intensity reduction as a function of time It is used to determine the zoology of lipid oxidation. The results show that a mixture of crude anthocyanin extract and ethyl acetate can inhibit oxidation. Anthocyanins, bioflavonoids and phenols can be used for solvent extraction; This is more the case for food or for price reasons This step is not necessary when using better adsorbent resins. Chromatography can also be used to separate and reconstitute the component; however, for the purposes of the present invention, this situation becomes too expensive because it includes high pressure liquid chromatography Example 5 The antioxidant activity was determined by the method developed by Arora and Strasburg. As the reaction proceeds, the fluorescent probe is degraded to reduce the fluorescence intensity. Therefore, in the presence of antioxidants, Can reduce the rate of fluorescent degradation. Experiments show that anthocyanins 1-3 and aglycone, anthocyanins, hydrazone oxidation activity, compared with commercially available antioxidants butylated hydroxytoluene and butylated hydroxytoluene (BHT) (Figure 7) is superior. Moreover, these cherry compounds show better antioxidant activity than α-tocopherol. At a concentration of two millimolars, the samples containing tocopherol and Feh containing no antioxidant ( Figure 7) The sample cannot be distinguished. The anthocyanins (glycosides, anthocyanins, have higher efficiency than glucosides, showing that the antioxidant activity of anthocyanins is determined by their aglycones: ---- ----- ----- (Please read the notes on the back before filling in this page) i-Order -------- line 11 ^ · Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs and Consumer Affairs Co., Ltd. -24- 1223982 A7

化色素苷1 - 3個別含有3、2、及1個粹$ 多妾-! α # 糖殘基，說明了於祁色素甘1所發現之最低抗氧化活目嵙产於C 3位置之糖殘基對於柷氧化活性似乎極為重要。 — 教 ,,^ 工 < 糖早兀數目命丄 ’柷虱化活性愈高。而且’根據記載，芳氧：^ 響化合物之抗氧化活性，而可產 & '影龙主主、t 」產生乳化強化作用。因此，素《抗氧化活性可視其芳氧基之安定性而定。花甘及花青素之B環中的鄰-二經基取代對於經由該3,及4:、部分產生《自由基的安定化極為重要。而且，花色素中 <都_二#基具有鉗合金屬離子之潛力，&防止由離子所誘發之脂質過氧化。製備花青素時，含有1- 3 ( 5 0 0毫克）之花色素苦混合物於8〇t下與3N鹽酸(2〇毫升)授拌1〇小時。反應混合物如同製備花色素*般地於XAD_4M上純化。將花青素《甲醇溶液蒸乾，產生紅色非晶形粉末（19〇毫克），儲存於-3 0 °C直至使用。抗氧化檢定中，該緩衝劑係儲存於CheUx 1〇〇中，以去除金屬離子。含有5微莫耳濃度之卜硬脂醯_2_亞麻油醞-sn-甘油-3-磷酸膽鹼（Avanti p〇lar Usids, inc.，Aia_ baster，AL)及15微莫耳濃度之螢光探針3_(對_(6_苯基 J 5 己一知基）丰基丙酸（Molecular Probes,Inc.The glycosides 1-3 contain 3, 2, and 1 respectively. 妾多!-! Α # sugar residues, which explains the lowest antioxidant activity of the sugars found at the C 3 position found in Qi pigment Gan 1. Residues seem to be extremely important for tritium oxidation activity. — Teach ,, ^ 工 < Sugar premature number fate 柷 The higher the ticking activity. Moreover, according to the record, the aryl oxide: ^ oxidizes the antioxidant activity of the compound, and can produce " the shadow dragon master, t " Therefore, the antioxidant activity depends on the stability of its aryloxy group. The ortho-dioyl substitution in the B ring of broccoli and anthocyanins is very important for the stabilization of free radicals that are partially generated via the 3, and 4 :. In addition, the <di-## group in anthocyanins has the potential to clamp metal ions, and to prevent lipid peroxidation induced by ions. In the preparation of anthocyanins, an anthocyanin mixture containing 1 to 3 (500 mg) was mixed with 3N hydrochloric acid (20 ml) at 80 t for 10 hours. The reaction mixture was purified on XAD_4M as anthocyanins *. The anthocyanin solution was evaporated to dryness to give a red amorphous powder (190 mg), which was stored at -30 ° C until use. In the antioxidant assay, the buffer is stored in CheUx 100 to remove metal ions. Contains 5 micromoles of stearylamine _2_ flaxseed oil-sn-glycerol-3-phosphate choline (Avanti polar Usids, inc., Aia_ baster, AL) and 15 micromoles of fluorescein Photoprobe 3_ (p -_ (6_phenylJ 5 hexamethylene) fungyl propionic acid (Molecular Probes, Inc.

Eugene，〇R)之混合物於真空下乾燥。形成之薄膜懸浮於$ 〇〇升緩衝劑（NaCl，0.15 Μ;ΕΙ)ΤΑ 〇」mM; M〇ps 1〇mM )中，於乙醇-乾冰浴中進行丨〇個冷凍-解凍循環。懸浮液使用 LiP〇soFast 擠塑機（AvesUn，Inc.，〇ttawa，Canada) -25- 本紙張尺度適用中國國家標準(CNS)A4規格（210 X 297公^7 (請先閱讀背面之注意事項再填寫本頁) ---------^--------- 經濟部智慧財產局員工消費合作社印製 1223982 A7 五、發明說明（23) (請先閱讀背面之注意事項再填寫本頁) 迥絰具有1 0 〇耄微米孔徑之聚碳酸酯2 9次。形成之脂質體 (2 0 0毛微莫耳濃度）懸浮於2毫升緩衝劑（1 00mM NaCl，50 mM ϋΕΡΕ3，ρΗ7·0)中。藉著添加4毫微莫耳濃度之以2,而起始過氧化。花色素苦、ΒΗΤ、&食子酸丙g旨、及（.生育酉分^准生、素E )係於2微莫耳濃度下丨則試。對照試樣不添加 Fe"或試驗化合物。脂質懸浮液之螢光強度係於使用或不使用試驗化合物下偵測21分鐘，之後立即添加Fe2_，使用 Μ 4800 螢光光度計（SLM InstrUments，Urbana，IL)進 :相對螢光值係、藉著將特定時間下之螢光值除以t = 〇分户\而决足結果係列示於圖7。自苦櫻桃單離之花色 f誓（卜3 —圖1 )及花青素於體外具有相當於市售產物之抗虱：活性。花色素苷卜3及其糖苷配基、《青素於2毫莫耳/辰度了之脂質過氧化的抑制個別係為3 9、7 0、7 5及5 7 百刀比、。於2耄莫耳濃度下，1 _3及花青素之抗氧化活性係相田万、第二丁基1 I經丁基化之羥基甲笨之抗氧化活性’而優於維生素E。實施例6及7 經濟部智慧財產局員工消費合作社印製與實施例5相同地，試驗圖2、3及4所示之選擇化合斗5 k氧化活性。結果係列示於圖8及9。就實施例6而言 ’以下化合物之抗氧化活性係表示於圖8 : 化合物1 ==柚配基-4，，5,7-三羧基黃烷酮化合物2 =染料木黃酮化合物3 =綠原酸化合物4 ：=獬皮酮-3 -鼠李糖苷 -26- 1223982 A7 B7A mixture of Eugene, OR) was dried under vacuum. The formed thin film was suspended in 0.00 liters of buffer (NaCl, 0.15 M; E1) ΤΑΟ ″ mM; Mops 10mM), and subjected to 10 freeze-thaw cycles in an ethanol-dry ice bath. Suspension uses LiP〇SoFast extruder (AvesUn, Inc., 〇ttawa, Canada) -25- This paper size applies to China National Standard (CNS) A4 specification (210 X 297 public ^ 7 (Please read the precautions on the back first) (Fill in this page again) --------- ^ --------- Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1223982 A7 V. Invention Description (23) (Please read the note on the back first Please fill in this page again.) Polycarbonate with a 100 μm pore size was used 29 times. The liposomes (200 micromolar concentration) formed were suspended in 2 ml of buffer (100 mM NaCl, 50 mM). ϋΕΡΕ3, ρΗ7 · 0). Peroxidation was initiated by adding 4 nanomolar concentrations to 2. Anthocyanin, βH, & gallic acid, and (... Bio-E) was tested at a concentration of 2 micromolar. The control sample was not added with Fe " or test compound. The fluorescence intensity of the lipid suspension was detected with or without the test compound for 21 minutes, after which Add Fe2_ immediately, use LM 4800 Fluorometer (SLM InstrUments, Urbana, IL): Relative fluorescence value, by changing the specific time The following fluorescence value is divided by t = 〇分 household \ and the final result series is shown in Figure 7. The self-bittering cherry isolated color f o (Figure 3-Figure 1) and anthocyanins have the equivalent of commercially available in vitro Anti-lice of the product: activity. Anthocyanin 3 and its aglycone, and the inhibition of lipid peroxidation of penicillin at 2 mmol / Chen are individual lines 3, 9, 0, 7 5 and 5 7 At a concentration of 100 kDa, the antioxidant activity of 1_3 and anthocyanins at 2 mol% is the antioxidant activity of Aida and the second butyl 1 I butylated hydroxymethylbenzyl, which is superior to vitamins. E. Examples 6 and 7 The consumer cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs printed the same experiment as in Example 5, and tested the selective chemical combination 5k oxidation activity shown in Figures 2, 3, and 4. The series of results are shown in Figures 8 and 9 For Example 6, the antioxidant activity of the following compounds is shown in Fig. 8: Compound 1 == Naringen-4, 5,7-tricarboxyflavanone compound 2 = Genistein 3 = Green Orthoacid compound 4: = dermatone-3-rhamnoside-26-1223982 A7 B7

五、發明説明（24) 化合物5 =堪#酵-3 -芸香糖苷化合物6 = 3’氧基堪非醇-3-芸香糖苷 (請先閱讀背面之注音？事項再填寫本頁) 化合物7 =染料木只酮-7，葡萄糖苷化合物8 = 5,8-二羥基-6，7·二甲氧基黃酮。實施例7中，以下化合物之抗氧化活性係顯示於圖9 : 化合物1 =綠原酸甲醋化合物2 = 2 -羥基—3 -(鄰-羥基笨基）·丙酸化合物3 = 1-(3’，4、二羥基肉桂醯）-2，5_二羥基環戊烷。化合物4=1-(3，4- 一备基肉桂驢）-2，3 -二經基環戊烷。圖9中，抗氧化劑化合物1、2、3及4 (如圖4所示）係與各種抗氧化劑比較。所試驗之抗氧化劑係為咖啡酸、綠原酸、對-羥基肉桂酸、及市售抗氧化劑第三丁基氫（ TBHQ)及經丁基化之羥基甲苯（BHT)。經濟部智慧財產局員工消費合作社印製圖8及9中所顯示之數據係使用由F e2+誘發之脂質過氧化檢定得到，以偵測抗氧化活性。製備使用於F e2+所誘導之脂質過氧化檢定使用之受質時，含有5微莫耳濃度之} _ 硬脂醯-2-亞麻油醯- sn -甘油-3-磷酸膽鹼（Avanti Polar Lisids，Inc·，Alabaster，AL)及0.015微莫耳濃度之螢光探針3-(對-(6 -苯基）-1，3，5 -己三晞基）苯基丙酸（％〇16(：11-lar ?1*(^6$，111(：.，£1^£1^，〇11)之混合物於真空下乾燥。形成之薄膜懸浮於1 0 0 0微升（實施例6)及5 0 0微升（實施例7) 含有 0.15M NaCl，0.1mM EDTA 及 0.01M4-嗎啉丙項酸（ -27- 本紙張尺度適用中國國家標準（CNS)A4規格（21〇 X 297公釐） 1223982 經濟部智慧財產局員工消費合社印 .製 A7 Β7 五、發明說明（25) Μ〇P S )之緩衝劑中該μ〇P S緩衝劑已預先使用c HE L ΕX 1V. Description of the invention (24) Compound 5 = Kan # zyme-3-rutin compound 6 = 3 'oxycanfinol-3-rutinoside (please read the note on the back? Matters before filling out this page) Compound 7 = Genistein-7, glucoside compound 8 = 5,8-dihydroxy-6,7 · dimethoxyflavone. In Example 7, the antioxidant activity of the following compounds is shown in Fig. 9: Compound 1 = methyl chlorogenic acid vinegar compound 2 = 2-hydroxy-3-(o-hydroxybenzyl) · propionic acid compound 3 = 1- ( 3 ', 4, dihydroxycinnamon 醯) -2,5-dihydroxycyclopentane. Compound 4 = 1- (3,4-mono-Cinnamon donkey) -2,3-Di-Cyclocyclopentane. In Figure 9, the antioxidant compounds 1, 2, 3 and 4 (shown in Figure 4) are compared with various antioxidants. The antioxidants tested were caffeic acid, chlorogenic acid, p-hydroxycinnamic acid, and commercially available antioxidants tertiary butyl hydrogen (TBHQ) and butylated hydroxytoluene (BHT). Printed by the Employees' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs The data shown in Figures 8 and 9 were obtained using a lipid peroxidation assay induced by Fe2 + to detect antioxidant activity. When used for preparation of lipid peroxidation induced by Fe2 +, it contains 5 micromolar concentration} _Stearin 醯 -2-linseed oil 醯 -sn-glycerol-3-phosphate choline (Avanti Polar Lisids , Inc., Alabaster, AL) and 0.015 micromolar fluorescent probe 3- (p- (6-phenyl) -1,3,5-hexanetriamidino) phenylpropionic acid (% 〇16 (: 11-lar? 1 * (^ 6 $, 111 (:., £ 1 ^ £ 1 ^, 〇11)) The mixture was dried under vacuum. The formed film was suspended in 100 microliters (Example 6 ) And 500 microliters (Example 7) containing 0.15M NaCl, 0.1mM EDTA, and 0.01M 4-morpholine propionic acid (-27- This paper size applies the Chinese National Standard (CNS) A4 specification (21〇X 297 (Mm) 1223982 Printed by the Consumers' Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs. Produced by A7 B7 V. Description of the invention (25) MoPS) The μ〇PS buffer has been pre-used with c HE L ΕX 1

〇〇(得自 Sigma Chemicals，St· Louis，MiSS〇uri)於 5 克/1C 〇 t升緩衝劑下處理，以去除任何微量金屬離子。該懸浮液使用乾冰/乙醇浴進行1 〇次冷凍-解凍循環。該脂質-緩衝劑懸浮液隨後使用L i ρ 〇 s 〇 F a s t擠塑機（A v e s t i n，I n c ·, 〇11 a w a，C a n a d a)通經具有1 〇〇毫微米孔徑之聚碳酸酯2 9 次以產製單層脂質體。進行由Fe-诱發之脂質過氧化檢定時，2〇微升之前述脂質體懸浮液於含有lOOmM NaCl，50mM N-2-經基乙基旅啡-N - 2 -乙石買酉父（Η E P E S )緩衝劑ρ η 7 · 0之C Η E L Ε X 1 0 0 中稀釋土 2毫升（貫施例6 )(實施例7為2 0 0 m Μ N a C 1，1 0 0 mM HEPES緩衝劑ΡΗ7·0)，於室溫下培育5分鐘，之後於螢光光度計保持於2 3 °C之恒溫比色杯支架中培育另外5分鐘。於任何試驗化合物不存在或存在下，藉著添加〇 · 5 uM FeCl2以起始過氧化，以得到〇5 uM Fe2，之最終濃度. 。對照試樣不含Fe2+或任何試驗化合物。此等脂質體溶液 I螢光強度於2 1分鐘期間於3 8 4毫微米激光波長下使用勞光光度計（SLM4_，SLM Instruments，Inc.，时㈣，^ •，urbana，imnois)每三分鐘測量一次。相對螢光強度隨時間而變之減量表示過氧化速率。脂質氧化之抑制百分比 (p I)係使用下式計算： 77 PI = {〔 (Fre丨)。1-(Frel)Fe/〔 (Fref)“Fre丨)Fe〕丨 X ^ 其中（F^)P1係為Fe( Π )及試樣於21分鐘時間周期結耒時之相對螢光，（Fref)。係為對照試樣於2丨分鐘時間周^ ^ -28- &紙張尺度適用中國國家標準（CNS)A4規i_(2i〇 x 297公t j--〜----- ---I---ί— I ---I ! I 訂---—I 丨丨丨- (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 ^23982 五、發明說明（26 ) 束時之相對螢光，而（F^)Fe係為含Fe( Π )之試樣於21分鐘時間周期結束時之螢光Arora等4 1,( 1 997)，J. Amer.〇il Chem.Soc.74]031-1 0 40)。貫施例6中，化合物1、2、3、4、7及8係於1 0微莫耳濃度下檢定抗氧化活性。類黃酮對於F e 2 +脂質過氧化之抑制效果係因其甜合F e 2 +而形成無法起始過氧化之惰性錯合物所致（A f a n a s ’ e v e t a 1 ·，1 9 8 9 )。此外，類黃酮之 F e 2 +錯合物被視為保持其自由基掃除活性，故可掃除脂貝過氧化中之自由基中間體。而且，類黃i同可作為自由基掃除劑。如圖8所示，化合物8 ( 5，8，4，-三羥基_ 6，7 _三甲氧基哭酮）之抗氧化活性係優於該1、2、4、及7於1 〇微莫耳濃度下之抗氧化活性。早期έ己載顯示B環上存有鄰·二經基（B〇rs等人，1990 )、C環上位於位置3上之經基（Afana，s等人，1989· M0ra等人，199〇)及位於上之雙鍵與扣合氧基官能基（B 〇 r s等人，1 9 9 〇 )被視為類黃酮有效地掃除自由基所必要。即使化合物8不具有3 -羥基，且僅於B環上具有— 怠基，化合物8之抗氧化活性仍高於懈皮酮_ 3 _鼠李糖苷。懈皮酮-3 -鼠李糖苷除3羥基及位於C 2 - C 3上之雙鍵與4 _合氧基外，另含有鄰-二羥基。化合物8增進之抗氧化活性可能係因位於A環上之羥基及兩甲氧基所致。ΑΓ〇α等人（IQ 9 7 )记載7，8 -二羥基黃酮顯示類似獬皮酮之抗氧化活性，唯其於B環及3 -位置上缺乏任何取代。圖9中，化合物3及4於20微莫耳濃度下於大型單層泡 ___ _29_ 本紙張尺度適ϋ國家標準（CNS)A4規^ (2iQ χ撕^ ---------^ Ϊ—--------tr— (請先閱讀背面之注意事項再填寫本頁) 1223982 A7 五、發明說明（27) 囊中對Fe”秀發之脂質過氧化的抑制活性係約8〇百分比。化合物1顯示約50百分比之抑制活性。然而，化合物2 使於100微莫耳濃度下測試，仍未顯示抗氧化活性。該檢定結果亦顯示對-經基肉桂酸與阿魏酸比較之下係為弱才: 氧化劑。然而，咖啡酸同質物化合物3及4於此檢定中= 示最高之抗氧化活性^ TBHQ及BHT對脂質過氧化之抑制、百分比於20微莫耳濃度下係>9〇百分比。此檢定顯示咖啡酸係為最佳氧化劑’隨後為化合物4、化合物3、綠酸甲酯。 h 實施例8 試驗實施例1及2之組成物的抗氧化性質，發現相當於實施例5。田抗氧化化合物之潛在用途係降低或消除細胞氧化損壞 ’掃除自由基以減少細胞死亡，減少動脈硬化或動脈變硬 ’減少心臟病發作，減少與關節炎及痛風有關之疼痛，及減緩老化。已知别文描述僅用以說明本發明，而本發明僅受限於申請專利範圍。 --------l·.—--------訂--------- {請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 -30- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）〇〇 (available from Sigma Chemicals, St. Louis, MiSSour) was treated under 5 g / 1 liter of buffer to remove any trace metal ions. This suspension was subjected to 10 freeze-thaw cycles using a dry ice / ethanol bath. The lipid-buffer suspension was then passed through a polycarbonate having a pore size of 1,000 nanometers using a Li ρ 〇s 〇F ast extruder (A vestin, Inc., 〇11 awa, Canada). 9 Secondly, monolayer liposomes are produced. For the Fe-induced lipid peroxidation assay, 20 microliters of the aforementioned liposome suspension was contained in 100 mM NaCl, 50 mM N-2-Ethylethyltraphine-N-2-Ethylmethionine ( Η EPES) Buffer ρ η 7 · 0 C Η EL Ε X 1 0 0 2 ml of diluted soil (performed in Example 6) (Example 7 is 2 0 0 m Μ N a C 1, 1 0 0 mM HEPES Buffer (pH 7.0 · 0), incubated at room temperature for 5 minutes, and then incubated in a constant temperature cuvette holder maintained at 23 ° C with a fluorometer for another 5 minutes. In the absence or presence of any test compound, by adding 0.5 uM FeCl2 to initiate peroxidation to obtain a final concentration of 0.05 uM Fe2. The control samples did not contain Fe2 + or any test compounds. The fluorescence intensity of these liposome solutions I was measured at a laser wavelength of 3 8 4 nm over a period of 21 minutes using a labor photometer (SLM4_, SLM Instruments, Inc., Shiji, ^, urbana, imnois) every three minutes. Measure once. The decrease in relative fluorescence intensity over time indicates the rate of peroxidation. Percent inhibition of lipid oxidation (p I) was calculated using the formula: 77 PI = {[(Fre 丨). 1- (Frel) Fe / [(Fref) “Fre 丨) Fe] 丨 X ^ where (F ^) P1 is the relative fluorescence of Fe (Π) and the sample at the 21 minute time period, (Fref ). It is a control sample in the time of 2 丨 minutes. ^ -28- & Paper size applies Chinese National Standard (CNS) A4 Regulation i_ (2i〇x 297 male t j-~ ------ -I --- ί— I --- I! I Order ----- I 丨丨丨-(Please read the precautions on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs ^ 23982 5 2. Description of the invention The relative fluorescence at the time of (26) beam, and (F ^) Fe is the fluorescence at the end of the 21-minute time period of the sample containing Fe (Π). Arora et al. 1, 1, 1997, J Amer. Chem. Soc. 74] 031-1 0 40). In Example 6, compounds 1, 2, 3, 4, 7 and 8 were tested for antioxidant activity at a concentration of 10 micromolar. The inhibitory effect of flavonoids on F 2 + lipid peroxidation is due to the formation of inert complexes that cannot initiate peroxidation due to its sweetness to F 2 + (A fanas' eveta 1 ·, 1 9 8 9). In addition, the F e 2+ complex of flavonoids is considered to maintain its free radical scavenging activity, so it can remove lipids. Free radical intermediates in peroxidation. In addition, flavonoids can also be used as free radical scavengers. As shown in Figure 8, compound 8 (5,8,4, -trihydroxy_6,7_trimethoxy cryonone ) The antioxidant activity is better than those of 1, 2, 4, and 7 at a concentration of 10 micromolar. Earlier, it has been shown that there is an ortho-secondary radical on the B ring (Bors et al.) (1990), the cyclic group at position 3 on the C ring (Afana, s et al., 1989 · Mora et al., 199), and the double bond and the oxo functional group on the ring (B ors et al.) , 199 〇) is considered to be necessary for flavonoids to effectively scavenge free radicals. Even if compound 8 does not have a 3-hydroxy group, and only has a-group on the B ring, the antioxidant activity of compound 8 is still higher than that Ketone _ 3 _ rhamnoside. Retinone-3-rhamnoside, in addition to the 3 hydroxy group and the double bond on C 2-C 3 and 4- oxy group, also contains o-dihydroxy group. Compound 8 enhances it Antioxidant activity may be due to the hydroxyl and dimethoxy groups located on ring A. AΓ〇α et al. (IQ 9 7) states that 7,8-dihydroxyflavones show antioxidant activity similar to that of dermatone. Its in The B ring and 3-position lack any substitutions. In Figure 9, compounds 3 and 4 are in large monolayer bubbles at a concentration of 20 micromolar ___ _29_ This paper is compliant with the national standard (CNS) A4 regulations ^ (2iQ χ Tear ^ --------- ^ Ϊ —-------- tr— (Please read the notes on the back before filling this page) 1223982 A7 V. Description of the invention (27) Fe in the capsule "The inhibitory activity of lipid peroxidation in hair is about 80%. Compound 1 showed about 50% inhibitory activity. However, Compound 2 was tested at a concentration of 100 micromolar and still did not show antioxidant activity. The test results also show that p-based cinnamic acid is weaker than ferulic acid: oxidant. However, caffeic acid compounds 3 and 4 were tested in this test = showed the highest antioxidant activity ^ TBHQ and BHT inhibition of lipid peroxidation, the percentage is> 90% at a concentration of 20 micromolar. This test shows that caffeic acid is the best oxidant 'followed by compound 4, compound 3, and methyl chloroate. h Example 8 The antioxidant properties of the compositions of Examples 1 and 2 were tested and found to be equivalent to that of Example 5. The potential use of antioxidant compounds is to reduce or eliminate oxidative damage to cells ‘sweep free radicals to reduce cell death, reduce arteriosclerosis or hardening of the arteries’, reduce heart attacks, reduce pain associated with arthritis and gout, and slow aging. The other descriptions are known only to illustrate the present invention, and the present invention is limited only by the scope of patent application. -------- l · .—-------- Order --------- {Please read the notes on the back before filling out this page) Employees ’Consumption of Intellectual Property Bureau of the Ministry of Economic Affairs Printed by the cooperative -30- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)

Claims

1223982 A8 第088121631號申請案93年06月01日替正本1223982 A8 Application No. 088121631 Replaced original on June 01, 1993

、申請專利範圍 (01) 1、一種可於具有生命之哺乳類體内.提供抗氧化作用之可食性組成物，該組成物包括由櫻桃中單離出來之混合物’該單離方法包含提供一種櫻桃水性萃取物、將該水性萃取物與一樹脂接觸、及一以洗提液洗提該樹脂之步驟’其中該組成物係含有：具下式之花色素苷：Scope of patent application (01) 1. An edible composition that can provide anti-oxidant effect in a living mammal. The composition includes a mixture isolated from cherries. The isolation method includes providing a cherry Aqueous extract, contacting the aqueous extract with a resin, and a step of eluting the resin with an eluent, wherein the composition contains: anthocyanins having the formula:

r2為 I----------警.！_ 訂---------線 (請先閱讀背S之注意事項再填Κ本頁) 經濟部智慧財產局員工消費合作社印製之式下具r2 is I ---------- Police.! _ Order --------- line (please read the precautions of S before filling in this page) Printed by the Employee Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs

Η 或 Η ο ) 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1223982 A8 B8 C8 D8 申請專利範圍 (02)Η or Η ο) This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm) 1223982 A8 B8 C8 D8 Patent Application Scope (02)

R4 ο 或其中心為㈤，R2為OH, Η或OCH” R3為ΟΗ，Η,鼠李糖或香糖，R4為OH, R5為Η或〇CH3,心為郎或OCH3, '為1!或〇Η,R4 ο or its center is ㈤, R2 is OH, Η or OCH ”R3 is 〇Η, Η, rhamnose or chewing sugar, R4 is OH, R5 is Η or 〇CH3, heart is Lang or OCH3, 'is 1! Or 〇Η,

其中匕為〇Η或OCH 具下式之酚類： .COOCH, 112為011或H, R3為OH或葡萄糖 ΗΟΛ 丫 '、、、ΟΗ ΌΗWhere D is phenol or OCH with the following formula: .COOCH, 112 is 011 or H, R3 is OH or glucose ΗΟΛ Ya ',,, 〇Η ΌΗ

R1 ---1---------------^---^訂---------線麵 (請先閱讀背面之泫意事項再填¾本頁) 經濟部智慧財產局員工消費合作社印製 Ecr^y OH 其中R為H或CH3, :^為郎或H, 112為！1或OH, 及食品級載體。如申請專利範圍第1項之組成物，其中該哺乳類係為人類，而該組成物係為調理食品。如申請專利範圍第1項之組成物，其中該組成物係細乾燥。 32 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） Λ8 Β8 C8 ΠοR1 --- 1 --------------- ^ --- ^ order --------- line surface (please read the intentions on the back before filling in this ¾ Page) Ecr ^ y OH printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economics where R is H or CH3,: ^ is Lang or H, 112 is! 1 or OH, and food-grade carriers. For example, the composition of the scope of patent application No. 1 in which the mammal is a human and the composition is a prepared food. For example, the composition of the scope of patent application No. 1 wherein the composition is finely dried. 32 This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) Λ8 Β8 C8 Πο

(03) 申請專利範圍 4如申清專利範圍第1 、燥樓桃果肉。員《組成物，其中該載體係為乾 5'^ϊ專利範圍第1项之組成物，其中該組成物係含有載組，且組成物相對姊、、、載租《比例係介於0 · 1比1 〇〇及100比0· 1之間。 6、如申請專利範圍第h之组成物，其中該組成物液體形式。 ^ 7、如申請專利範圍第i至6項中任一項之組成物，其中該’·且成物係為花色素苷、生物類黃酮及酚類之混合物。 8如申μ專利範圍第1項之組成物，其中該組成物係為經單離形式苦櫻桃。 … 9如申清專利範圍第1項之組成物，其中該組成物係自甜櫻桃單離。 1 0、如申請專利範圍第丨項之組成物，其中該組成物係自 MONTMORENCY櫻桃單離。 11、如申請專利範圍第丨項之組成物，其中該組成物係自 BALATON櫻桃單離。 ih---------φ^ί, (請先閱tt背面之事項再填冥本頁) 、f J -t— n J · n Bn n n «I I— fen I(03) Scope of patent application 4 For example, the scope of patent application for clearing the first, dry floor peach pulp. "Composition, wherein the carrier is the composition of item 1 of the patent scope of dry 5 '^ ϊ, wherein the composition contains a carrier group, and the composition is relatively small, the ratio is between 0 · Between 1 to 100 and 100 to 0.1. 6. The composition in the range h of the patent application, wherein the composition is in liquid form. ^ 7. The composition according to any one of items i to 6 of the scope of application for a patent, wherein the 'and the product is a mixture of anthocyanins, bioflavonoids and phenols. 8 The composition according to item 1 of the patent application scope, wherein the composition is a bitter cherry in the form of detachment. … 9 The composition of claim 1 in the patent scope, wherein the composition is isolated from sweet cherry. 10. The composition according to item 丨 in the scope of patent application, wherein the composition is isolated from MONTMORENCY cherry. 11. The composition according to item 丨 of the patent application scope, wherein the composition is isolated from BALATON cherry. ih --------- φ ^ ί, (please read the matter on the back of tt before filling this page), f J -t— n J · n Bn n n «I I— fen I

經濟部智慧財產局員工消費合作杜印製Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs