TWI374749B - Medical products,food composition and external disinfectant preparation containing ampelopsis brevipedunculata (maxim.) trautv. extract and use thereof in prevention and treatment of enterovirus 71 infection - Google Patents

Medical products,food composition and external disinfectant preparation containing ampelopsis brevipedunculata (maxim.) trautv. extract and use thereof in prevention and treatment of enterovirus 71 infection Download PDF

Info

Publication number
TWI374749B
TWI374749B TW98111599A TW98111599A TWI374749B TW I374749 B TWI374749 B TW I374749B TW 98111599 A TW98111599 A TW 98111599A TW 98111599 A TW98111599 A TW 98111599A TW I374749 B TWI374749 B TW I374749B
Authority
TW
Taiwan
Prior art keywords
dabenshan
grape
extract
enterovirus
solvent
Prior art date
Application number
TW98111599A
Other languages
Chinese (zh)
Other versions
TW201036622A (en
Inventor
Yuan Chuen Wang
Hsing Wen Hsu
Original Assignee
Nat Univ Chung Hsing
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nat Univ Chung Hsing filed Critical Nat Univ Chung Hsing
Priority to TW98111599A priority Critical patent/TWI374749B/en
Publication of TW201036622A publication Critical patent/TW201036622A/en
Application granted granted Critical
Publication of TWI374749B publication Critical patent/TWI374749B/en

Links

Landscapes

  • Medicines Containing Plant Substances (AREA)

Description

1374749 2012年9月11日修正替換頁 六、發明說明： -—--- 【發明所屬之技術領域】 本發明係有關於一種用於預防及對抗腸病毒感染之保 , 健食品組成物，其包含大本山葡萄萃取.物。本發明亦有關 於將大本山葡萄萃取物使用於製備供用於預防及對抗腸病 ' 毒感染的醫藥品。本發明亦有關於一種用於抑制腸病毒活性 之外用消毒劑。本發明亦有關於一種活體外抑制腸病毒活性 之方法。 【先前技術】 • 腸病毒是一大類病毒的總稱’屬於小核糖核酸病毒 (Picornaviruses)中的腸病毒屬（五nierov/rw·? sp.)，為正單股 (positive single strain)之RNA病毒，小型、球狀、無套膜， 呈立體對稱型（或二十面體型）’大小約20至30 nm (劉， 2003)，其具有對熱及酸穩定性之特性，於感染人體時，可 於口咽部進行初步的複製，之後可以承受胃部的強酸，並進 而進入腸道進行大量的複製，因此在學術上統稱之為腸病 毒。 目前人類腸病毒被分離出來的有70多種血清型，其中 包括：三型小兒麻痒病毒（poliovirus) : 1型（type_i)、2型 、 （type-2)、3型（type-3);二十三型克沙奇病毒A群 ' (coxsackievirus A) : A1-A22以及A24型；六型克沙奇病毒 B群（coxsackievirus B): B1至B6型；三十一型伊科病毒 (echovims):除了 10型办，10)及28型（听卜28)以外的^ 至33型；以及四型腸病毒（enterovirus): 68至71型 J· L.，以α/·，1996)。其中腸病毒71型是為最晚發現的腸病 3 1374749 2012年9月11曰修正替換頁 毒，其整體病毒核酸序列已經完全了解，全Ϊ為7,408 — 普酸（nucleotides) (Brown α/., 1995)。 1998年，台灣地區爆發腸病毒大流行，造成全台恐慌。 , 依衛生署之被動監測系統定點醫師之報告統計，於約九萬個 報告病歷中’約有320例為重症病患，其臨床表現以併發腦 膜炎及腦炎等為主’其中至少有55例為死亡病歷。Ho等人 的研究報告指出，手足口病及療性咽夹炎之重症案例中分離 出腸病毒71型者佔92 %，其中之75%證實為腸病毒71型 所造成（Ho, M.，a/” 1999)。此外，台大醫院檢體分離之結 • 果’顯示出腸病毒71型佔29.01%，克沙奇病毒 (coxsackievirus) A24 佔 22.14%，其他病毒佔 48.85%，另外 台大醫院自彰基醫院取得檢體之分離結果，以腸病毒71型 佔多數，由屏基醫院取得檢體亦以腸病毒71型為主（Ka〇 d·，1998)。林口長庚兒童醫院之分離結果顯示出，腸病毒 71型佔40°/。，其他為克沙奇病毒A16、A7、B5、B6等型及 伊科病毒 6、7 型（ech〇virus type -6, -7) (Ning βί α/.，1998)。 關於腸病毒之流行病學的調查結果顯示，腸病毒71型 谷易知:犯15歲以下的孩童，產生症狀的感染主要發生在6 歲以下的兒童，年紀愈小的兒童出現嚴查症狀的機會愈大， 、甚至在病發後的2至4天内急速死亡，致死率大約為ι〇〇〇 .至1〇000分之一。在台灣全年都可能有腸病毒感染發生，而 以5、6月及9、1〇月為兩個流行的高峰（呂等，Μ的；葉等 1999)。 ’、， 腸病毒71型主要引起一些輕微症狀，如發燒、上呼吸 道疾病及斑疹丘等。部分病患會出現典型的咽頰炎或手足口 1374749 2012年9月11日修正替換頁 病，有少數會合併神經系統的併發症》此外、偶而尚fXJj- 較不尋㊉的症狀’如心肌炎、多發性神經炎等（Shindarou ei α/.’ 1979;呂等’ 1999;葉等，1999)。但是在台灣日本香 港、澳洲維多利亞及馬來西亞，同時出現手足口病及嚴重中 框^經系統疾病（呂等，1999;葉等，1999)。腸病毒71型是 目别已知除了小兒麻痒病毒外，也會引起肢體麻痺之腸病毒 (Gilbert ei α/_，1988; Lu ei α/.，2000)。根據文獻報告指出腸 病f 71型進入中樞神經後會侵入神經元，並複製而造成神 經疋的破壞，主要是侵犯腦幹、橋腦、延腦後方，嚴重者.會 侵犯頸椎脊隨（Shen以β/ , 2〇〇〇)，因此感染腸病毒71型引 起的症狀主要依據其侵犯的位置而定。至於為何腸病毒？！ 型侵犯人體後會造成嚴重的中樞神經病變甚至死亡，至今尚 無答案（Yan ei αΛ，2001)。 除腸病毒71型之外，克沙奇Β3型每年在世界各地均會 造成流行（Morens and Pallancsh，1995)。美國從 1983 到 2005 年，感染之案例一共有1943件，其中超過6〇%為低於一歲 之 4c·幼兒’平均致死率為 5.4% (Khetsuriani，α/.，2006)。 台灣分別在1996年及2005年爆發流行，前者之流行造成小 嬰兒敗血症候群等嚴重症狀，死亡率高達11% (Hsieh Wd.， 1997);後者之臨床案例中，克沙奇B3型佔了 29.2% ,重症 比例為3.18% (行政院衛生署疾病管制局，2006)。克沙奇B3 型感染新生兒及嬰幼兒會造成嚴重臨床症狀，甚至死亡，而 於成人，則多數無症狀（Kim ei α/.，2001)。克沙奇B3型亦曾 造成子宮内胎兒的感染（Ouellet ei α/.，2004)。 克沙奇Β3型主要侵犯心臟組織，繼而衍生成為嚴重之 5 1374749 2012年9月11日修正替換頁 心臟疾病。感染克沙奇B3型首先造成急性^ 治癒，則成為慢性心肌炎，進而造成心肌擴張、心臟收縮力 減弱，嚴重者，病患唯有接受心臟移植一途（κ&，以d v 2〇〇1; Chau，d «’.，2007; Groarke and Pevear，1999)，。克沙奇’ • B3型造成心肌炎之原因，主要為病毒攻擊心臟組織，造成 細胞凋亡，以致引起内外之心肌發炎（〇Uvetti以d，1997)。 此外克&奇Β3型會在騰臟器官進行病毒複製，造成外分 泌之器官受損（Henke w α/.，2〇〇1)。 關於腸病毒感染之治療及預防，以腸病毒71型及克沙 .奇Β3型之治療為例’其治療方法目前主要為症狀治療及支 •持性療法，有效的抗病毒藥物仍處於研究階段。腸病毒之疫 苗除了三型小兒麻痒疫苗被開發出來外，其餘的腸病毒均未 開發出預防的疫苗，腸病毒71型及克沙奇Β3型之疫苗亦在 研究階段。而每年引起腸病毒流行之病毒可能都不相同，無 ，得過-次就終身免疫，因此目前為止最有效的預防方式即 是於流行的季節勤洗手、戴口罩以及避免進人公共場所，以 避免受到感染。 關於抗腸病毒71型及克沙奇Β3型藥物之研究，國家衛 生研究院及長庚大學基礎醫學研究所研發出一系列吡啶基 咪唑啉酮（Pyndyl imidaz〇Udin〇nes)抗腸病毒藥物，其中包括 Z 194其具有抑制腸病毒71型早期複製之活性，抑 制機制為抑制病毒吸附於宿主細胞之接受器，並且可以藉由 抑制病毒的脫殼（uncoating)作用，因而抑制病毒對人類細胞 之感染能力（Shih，…/·，2004)。Pyridyl及 其相關衍生物’除可抑制腸病毒71型亦能抑制腸病毒Μ 1374749 2012年9月11日修正替換頁 ^24型及克沙^ 型、伊科病毒9型、克沙奇病毒A9、A1〇、 病毒 B1 型等（Shia ，2002)。 ▲，外’部分研究係企圖由已知天然材料尋找合適的抗腸 病秦藥物。長庚大學基礎醫學研究所探討蒸蛋白抗腸病毒71 聖之活14，研究結果指出從螺旋藻（办卜⑷分離 之異藻膽藍素（allophycocyanin)具有抑制腸病毒71型早期 複製之活性，以及抑制腸病毒71型造成的細胞凋亡及延鍰 病毒RNA的合成（Shih w β/·，2〇〇3)β九層塔之熊果酸（urs〇Hc acid) ’具有抑制腸病毒71型之感染及感染後之複製作用， ED50 為 0.5 μ§/ιη1 (鄭，2〇〇3)。乳鐵蛋白（lact〇ferrin)具有抑制 腸病毒71型及克沙奇病毒A16型吸附細胞之功效（Linei β/.， 2002),乳鐵蛋白之；[Cw為34.5 pg/ml，_且在病毒感染後加 入大於IC%的乳鐵蛋白依然可以降低病毒的效價（翁， 2004)。彰化仁愛中醫醫院對於手足口病之幼兒（平均2〜3 歲）’給予中藥方劑如銀翹散、導赤散及涼膈散，中藥如板 藍根、黃芩、葛根、黃連、甘草、金銀花、柴胡、連翹、石 膏、滑石、烏梅、土伏苓、蛇退、王不留行及大黃等，依病 情輕重按中醫理論加減給予，結果顯示出，經中藥處理後， 病童均在3至4天内痊癒（張，1998)。 此外，華紫金牛（Jr山ϋ chwewsb Benth)水萃取物（Su α/.，2006)及包括大丘藤（Gwawi 山’《e «Sargewioiioxa)等 6 種藥 草之沸水萃取物（Guo以β/.，2006)對克沙奇B3型均顯示出 抑制活性。由鶴掌柴分離之三萜類 (triterpenoids)及由雞三樹c/ypearz'a)葉片分 離之黃烷類（flavans)亦對克沙奇B3型具抑制作用（Li ei «/.， 1374749 2012年9月11日修正替換頁 2006; Li βί α/.，2007)。合成之化合 ^ 如高 ~— (homoisoflavonoids)、arbidol 及[(二苯氧基）丙基]異噁唑 {[(biphenyloxy)propyl]isoxazole}衍生物等，均有不錯之抑制 v 克沙奇 B3 型活性（Tait ei α/., 2006; Makarov ei α/.，2005)。 由於利用化學合成藥物可能會造成非所欲的副作用，且 製作的成本相對較高，若能自具食用安全性且產量豐富、容 易取得的食品原料中萃取出具有抗腸病毒活性之成分，將可 有效降低其開發製成飲料、膠囊、鍵劑或粉末或其他相關之 保健食品之成本。 【發明内容】 基於現有技術的不足，以及此技術領域對於自具食用安 全性且產量豐富、容易取得的食品原料中萃取出具有抗腸病 毒活性之成分之需求，申請人致力於由天然食品原料中研發 出具有抗腸病毒活性之材料。 申請人於一系列之先導試驗中，針對8〇餘種台灣本土 性藥用植物，以中和試驗法（neutralizati〇ntesting)進行抗腸 病，活性之篩選，結果顯示包括『大本山葡萄』的數藥草具 有高度之抗腸病毒71型作用。 大本山葡萄⑽cw/aia (Maxj 又稱漢氏山葡萄，為葡萄的.近親，分佈於台讀 地至海拔超過蘭公尺之山區，為台灣原住民及漢:省 使用之藥草，老莖及根部為治療内傷及筋骨酸痛重要之黄 藥’亦用於利尿、消炎、止血、治療慢性腎炎及肝炎^ 齋毒等（行政院衛生署中醫藥委員會，·)。大本山葡萄 為行政院衛生署列為『可供食品使用原料』之第—類，且 8 1374749 2012年9月11日修正替換頁 相當尚之安全性，毋需進行相關之毒性研究逕行以其 藥材本身或其萃取物製成飲料、膠囊、錠劑或粉末。申請人 由反覆缜密的研究證實大本山葡萄具有相當優異的抗腸 病毒活性，更確認其極具開發性，可供作為抗腸病毒之藥物 及保健食品等》 有研究指出，大本山葡萄之乙醇萃取物能抑制由自由基 造成之肝臟損傷（Yabe and Matsui，1997)以及降低由鐵促進 之肝臟損傷（Yabe，心/.，1998)。大本山葡萄之萃取物具有中 度抗苦酮酸（P— aeidH|起之突變作用^ ^ 1988)。大本山葡萄之甲醇萃取物具有抵抗亞麻油酸。_ a acid)及DNA質體氧化作用（Wu，❹/，2〇〇4)的功效。依據申 請人所知此技術領域中尚未有任何報導指出大本山葡萄具 有抗腸病毒相關之活性。 因此本發明致力於研究大本山葡萄萃取物於抗腸病毒 之應用’並且證實依據本發明之大本山葡萄萃取物介具有 有效的抗腸病毒活性。 種如别述之用於預防及對抗腸 其包含一有效數量的大本山葡 釋劑。 基於上述，本發明提供一 病毒感染之保健食品組成物， 萄萃取物以及適合的載劑或稀 力m ’本發明亦提供 徑將大本山葡萄萃取物使用 於製備㈣預防及對抗腸病毒感染之醫藥品的用途。 =-方面’本發明亦提供一種用於抑制腸病毒活性之 外用消毒劑，其包含一有效數詈的 畀效歎量的大本山蔔萄萃取物以及適 合的载劑或稀釋劑。 ^ ^ ^ 再另一方面，本發明亦提供—種 活體外抑制腸病毒 活性 9 丄J/4749 2012年9月11日修正替換頁 :方：，其包括：將一種如前所述之外用消^劑與腸病毒接—-觸’错以抑制腸病毒之活性。 主依據本發明，前述的保健食品细成物、醫藥品外用消 v =劑以及活體外抑制腸病毒活性之方法係可供用於預防及 、k腸病毒感染，特別是用於預防及對抗腸病毒（⑽r〇virus) ，69型、7G型至71型或克沙奇病毒b群之病毒感染。 發明的-較佳的具體例中，該腸病毒係屬於腸病毒71 型或克沙奇B3型。 生依據本發明’用語「預防」係指降低宿主細胞感染腸 ； 機率其包括干擾腸病毒進入宿主細胞而降低腸病 毒感染。 依據本發明’用語「對抗」係指抑制腸病毒之活性， 其包括減低腸病毒於宿主内之複製能力、減少腸病毒重複 感杂伯主之機率以及舒緩腸病毒引起之症狀。 u$據本發明，前述大本山葡萄萃取物係藉由下列步驟 =製得將經均質化之大本山葡萄與一溶劑相混合，其中 該大本山葡萄係為大本山葡萄之根、莖、葉、果實、種子、 全株或其等之組合，且該溶劑之極性指數（P，）介於二氯甲烷 與水之間，以及利用該溶劑於4<)(：至95β(：萃取經均質化之 該大本山葡萄，藉以獲取大本山葡萄萃取物。 較佳的疋，該萃取步驟進一步包括將溶劑去除，藉以 取付刖述的大本山葡萄萃取物，其中該大本山葡萄萃取物 l括但不限於：呈一濃物縮物或乾燥粉末的形式。 在本發明的一較佳的實施例中，該部分的大本山葡萄係 為大本山葡萄之根、莖、葉、果實、種子、全株或其等之組 丄j/斗/4y 合。較佳的為根、贫或苴笼人 匕12年9月11日修 分的大本山5 °依據本發明，所述的部 處理所得之魏但不㈣.利用切割以及研磨方式 組合所研製的於末小塊或粉末。較佳的是經乾燥根、莖或其 月的較佳的實施例中，前述溶劑係可為極性指 ;丨於二1ή與水之間之溶劑，其包括但不限於水、 &級綱、低級醇、低級垸以及低級烧基自旨。在本發明的一較 佳的實^例巾’該溶#Η㈣自於由下㈣構成之群組：水、 丙嗣虱甲烷、二氣曱烷、95%乙醇以及乙酸乙酯。較佳的， s ^是丙_、氰甲⑥、95%乙醇、乙酸乙酯及二氯甲烷。 更佳的，該溶劑是丙酮。 在本發明的一較佳的實施例中，大本山葡萄與溶劑係 以-以溶劑為基礎介於1%(w/v)至遍(w/v)之間的比例混 在本發月的較佳的實施例中，前述適當的條件包括， 仁不限於以，文〉貝、攪拌、震盪、超音波、索氏熱回流或其 等之組合進行》 依據本發明’前述適當的條件包括於一介於0。(：至100 °C之間的溫度下進行萃取；較佳的，介於斗它至8(rc之間； 以及更佳的是’於室溫下，即大約介於2〇〇c至3 〇〇c之間。 在本發明的一較佳的具體實施例中，該適當的條件係包括於 室溫下以攪拌、震盪、超音波或95°c索氏（soxhlet)萃取予以 進行。 依據本發明’所述的醫藥品包括但不限於口服、非經 腸道途徑予以投藥，其中非經腸道途徑可以包括但不限 於：皮下注射以及肌肉注射。 11 1374749 ⑶以年9月11日修正替換頁 依據本發明，所述的醫藥品可 L--—_____ ^ ^ 带σστ以為，但不限於：散劑、 錠劑、膠囊、懸浮液、凝膠、乳 .夜粉末、泡沬、微乳化劑 或噴霧劑型.；所述的保健食品組成 取物可以為，但不限於：散 、錠劑、膠囊、懸浮液、凝膠、微乳化劑或喷霧劑型；以 及所述的外用消毒劑可以為，但不限於：㈣乳液粉末、 泡沬、微乳化劑、喷霧或懸浮液劑型。 依據本發明’所述的醫藥品、保健食品組成物、外用消 毒劑之pH值不限於特定阳值，亦即其可以介於U "， 而較佳的是介於6至8之間。 在本發明用於活體外抑制腸病毒活性方法的較佳實施 例令’在彳外用消毒劑與腸病毒接觸的步財，係進一步包 括下列步驟：將外用消毒劑喷麗於可能遭受腸病毒污染之物 件上令腸病毒與該外用消,毒劑接觸而降低腸病毒之毒 性。 【實施方式】 本發明將由下列的實施例做為進一步說明，這些實施 例並不限制本發明前面所揭示的内容。熟習本發明之技藝 者，可以做些許之改良與修飾，但不脫離本發明知範疇。 實施例 一般實驗材料及方法 大本山葡萄(Maxim.)1374749 revised list on September 11, 2012. Description of the invention: -----Technical field of the invention The present invention relates to a health food composition for preventing and combating enterovirus infection. Contains Dabenshan grape extract. The present invention is also directed to the use of Dabenshan grape extract for the preparation of a medicament for the prevention and prevention of enteropathic infections. The invention also relates to a disinfectant for use in inhibiting enterovirus activity. The invention also relates to a method of inhibiting enterovirus activity in vitro. [Prior Art] • Enterovirus is a generic term for a large class of viruses. It belongs to the genus Enterovirus (five nierov/rw·? sp.) in Picornaviruses and is a positive single strain of RNA virus. Small, spherical, without a sleeve, stereotactic (or icosahedral) 'size about 20 to 30 nm (Liu, 2003), which has the characteristics of heat and acid stability, when infecting human body, It can be initially replicated in the oropharynx, and then can withstand the strong acid in the stomach, and then enter the intestine for a large amount of replication, so it is academically referred to as enterovirus. At present, human enteroviruses have been isolated from more than 70 serotypes, including: poliovirus: type 1 (type_i), type 2, (type-2), type 3 (type-3); Type 23: Coxsackievirus A: A1-A22 and A24; Coxsackievirus B: Types B1 to B6; 31-type Icovirus (echovims) ): In addition to Type 10, Types 10) and 28 (Listing 28), and Types to 33; and Enterovirus: Types 68 to 71, J.L., α/·, 1996). Among them, enterovirus 71 is the latest found intestinal disease 3 1374749 September 11, 2012 revised replacement page poison, its overall viral nucleic acid sequence is fully understood, the total Ϊ is 7,408 - nucleotides (Brown α /. , 1995). In 1998, a pandemic virus outbreak broke out in Taiwan, causing panic in Taiwan. According to the report of the designated doctors of the Department of Health's passive monitoring system, there are about 320 cases of critically ill patients in about 90,000 reported medical records, and their clinical manifestations are mainly complicated by meningitis and encephalitis. An example is a death medical record. Ho et al.'s research report pointed out that 92% of cases of enterovirus 71 were isolated from severe cases of hand, foot and mouth disease and therapeutic pharyngitis, and 75% of them were confirmed to be caused by enterovirus 71 (Ho, M., a/” 1999). In addition, the results of the sample separation of the National Taiwan University Hospital showed that the enterovirus 71 accounted for 29.01%, the coxsackievirus A24 accounted for 22.14%, and other viruses accounted for 48.85%. The results of the segregation of the specimens were obtained by Zhangji Hospital, and the type of enterovirus 71 was the majority. The specimens obtained by Pingji Hospital were mainly type 71 (Ka〇d·, 1998). The results of the separation of Linkou Chang Geng Children's Hospital showed Outbreak, enterovirus 71 accounted for 40°/. Others were Crohn's virus A16, A7, B5, B6, and Ikevirus type 6, 7 (ech〇virus type -6, -7) (Ning βί α /., 1998) The results of the epidemiological investigation of enterovirus showed that enterovirus 71 type Gu Yizhi: children under the age of 15 have symptoms of infection mainly in children under 6 years old, younger children appear The greater the chance of scrutinizing the symptoms, and even the rapid death within 2 to 4 days after the onset of illness, The fatality rate is about ι〇〇〇. To one thousandth. In the whole year of Taiwan, there may be enterovirus infection, and in May, June and September, the first two peaks (Lv et al. , Μ; 叶等1999). ',, EV71 mainly causes some mild symptoms, such as fever, upper respiratory disease and spotted mound. Some patients may have typical pharyngitis or hand and foot 1374749 2012 On September 11th, the replacement page disease was corrected, and there were a few complications that would be complicated with the nervous system. In addition, occasionally fXJj- is less likely to find symptoms of 'such as myocarditis, polyneuritis, etc. (Shindarou ei α/.' 1979; Lu Etc. '1999; Ye et al., 1999). However, in Taiwan, Japan, Hong Kong, Victoria, Australia and Malaysia, there are both hand, foot and mouth disease and severe mesenteric system diseases (Lv et al., 1999; Ye et al., 1999). Enterovirus 71 It is known that in addition to poliovirus, it also causes intestinal paralysis of the enterovirus (Gilbert ei α/_, 1988; Lu ei α/., 2000). According to the literature report, enteric disease f 71 enters the central nervous system. Will invade the neurons and replicate and cause neural crest Bad, mainly invading the brainstem, bridge brain, and rearward of the brain. In severe cases, it will invade the cervical spine (Shen is β/, 2〇〇〇), so the symptoms caused by infection with enterovirus 71 are mainly based on the location of the invasion. As for why the enterovirus?! type invades the human body, it will cause serious central nervous system disease and even death. There is still no answer yet (Yan ei αΛ, 2001). In addition to the enterovirus 71 type, the Keshaqi Β3 type is in the year. It is prevalent throughout the world (Morens and Pallancsh, 1995). From 1983 to 2005, there were 1943 cases of infection in the United States, of which more than 6〇% were 4c·children under the age of one, and the average fatality rate was 5.4% (Khetsuriani, α/., 2006). Taiwan's outbreaks occurred in 1996 and 2005 respectively. The prevalence of the former caused serious symptoms such as small infant sepsis and the mortality rate was as high as 11% (Hsieh Wd., 1997). In the latter case, the Keshaqi B3 accounted for 29.2%, the critical proportion is 3.18% (Department of Disease Control of the Department of Health, 2006). Newborns and infants infected with Crox B3 can cause severe clinical symptoms and even death, while adults are mostly asymptomatic (Kim ei α/., 2001). The Keshaqi B3 has also caused infection in the womb (Ouellet ei α/., 2004). The saxaki Β3 type mainly invades the heart tissue, which in turn is severely affected. 5 1374749 September 11, 2012 Revision Replacement Page Heart disease. Infection with Keshaqi B3 first causes acute ^ cure, which becomes chronic myocarditis, which causes myocardial dilatation and weakened cardiac contraction. In severe cases, patients only receive heart transplantation (κ & to dv 2〇〇1; Chau , d «'., 2007; Groarke and Pevear, 1999),. Keshaqi's • B3 causes myocarditis, mainly because the virus attacks heart tissue, causing apoptosis, causing inflammation of the heart muscle inside and outside (〇Uvetti to d, 1997). In addition, the gram & michael type 3 will replicate the virus in the organs of the sputum, causing damage to the organs of the external secretion (Henke w α/., 2〇〇1). Regarding the treatment and prevention of enterovirus infection, the treatment of enterovirus 71 and Kesha. Qishen 3 is an example. The treatment methods are mainly symptomatic treatment and supportive therapy. Effective antiviral drugs are still in the research stage. . In addition to the development of the three-type pediatric vaccination vaccine, the enteroviruses have not developed a preventive vaccine, and the vaccines of enterovirus 71 and kesaki Β3 are also in the research stage. The virus that causes the enterovirus epidemic every year may be different. No, it will be life-time immunization. Therefore, the most effective prevention method so far is to wash hands, wear masks and avoid entering public places in the popular season. Avoid getting infected. The National Institutes of Health and the Institute of Basic Medicine of Chang Gung University have developed a series of pyridyl imidaz 〇 Udin〇nes anti-enteric virus drugs for the study of anti-intestinal virus type 71 and kesaki Β type 3 drugs. Including Z 194, which has the activity of inhibiting the early replication of enterovirus 71, the inhibition mechanism is a receptor for inhibiting virus adsorption to host cells, and can inhibit the infection of human cells by inhibiting the uncoating effect of the virus. Ability (Shih,.../·, 2004). Pyridyl and its related derivatives can inhibit enterovirus 除 in addition to inhibiting enterovirus 71. 1374749 Modified on September 11, 2012. Replacement type 24-24 and Kesha type, Iko virus type 9, oxavirus A9 , A1 〇, virus B1 type, etc. (Shia, 2002). ▲, the external part of the research department is trying to find suitable anti-intestinal Qin drugs from known natural materials. The Institute of Basic Medical Sciences of Chang Gung University investigated steaming protein anti-enteric virus 71 Shengzhihu14, and the results indicated that allophycocyanin isolated from spirulina (4) has the activity of inhibiting the early replication of enterovirus 71, and Inhibition of apoptosis caused by enterovirus 71 and synthesis of delayed prion RNA (Shih w β/·, 2〇〇3) β ursolic acid (urs〇Hc acid) has inhibition of enterovirus 71 infection And the replication after infection, ED50 is 0.5 μ§/ιη1 (Zheng, 2〇〇3). Lactoferrin (lact〇ferrin) has the effect of inhibiting the adsorption of enterovirus 71 and oxavirus A16 (Linei) β/., 2002), lactoferrin; [Cw is 34.5 pg/ml, _ and adding more than IC% of lactoferrin after viral infection can still reduce the titer of the virus (Weng, 2004). Changhua Renai Chinese Medicine In the hospital for children with hand, foot and mouth disease (average 2 to 3 years old), Chinese herbal medicines such as Yinqiaosan, Zhichisan and Liangxiesan are given. Traditional Chinese medicines such as Banlangen, Astragalus, Radix Puerariae, Rhizoma Coptidis, Licorice, Honeysuckle, Bupleurum, Forsythia , gypsum, talc, ebony, earthy scorpion, snake retreat Wang does not stay in the line and rhubarb, etc., according to the severity of the disease according to the theory of Chinese medicine plus or minus, the results show that after treatment with Chinese medicine, the sick children are cured within 3 to 4 days (Zhang, 1998). In addition, Hua Zi Jinniu (Jr Hawthorn chwewsb Benth) water extract (Su α/., 2006) and boiling water extracts of 6 herbs including Gwawi Mountain (e «Sargewioiioxa) (Guo is β/., 2006) The singular B3 type showed inhibitory activity. The triterpenoids isolated from the crane palm and the flavans isolated from the leaves of the chicken three trees c/ypearz'a also inhibited the sacchar B3. (Li ei «/., 1374749 Revised Replacement Page 2006, September 11, 2012; Li βί α/., 2007). Synthetic compound ^ such as high ~ - (homoisoflavonoids), arcidol and [(diphenoxy) propyl] isoxazole {[(biphenyloxy) propyl]isoxazole} derivatives, etc., have a good suppression v ksac B3 Type activity (Tait ei α/., 2006; Makarov ei α/., 2005). Since the use of chemically synthesized drugs may cause undesired side effects, and the cost of production is relatively high, if ingredients having anti-enteric activity are extracted from food materials that are safe to eat and are abundant in yield and easy to obtain, It can effectively reduce the cost of developing beverages, capsules, keys or powders or other related health foods. SUMMARY OF THE INVENTION Based on the deficiencies of the prior art, and the technical field for the extraction of components having anti-enteric activity activity from food materials having edible safety and abundant yield and easy availability, the applicant is committed to natural food materials. Developed materials with anti-enteric activity. In a series of pilot tests, the applicant conducted anti-intestinal disease and activity screening for more than 8 kinds of Taiwanese native medicinal plants by neutralization test (neutralizati〇ntesting), and the results showed that "Dabenshan Grape" was included. Several herbs have a high anti-intestinal virus type 71 effect. Dabenshan grape (10) cw/aia (Maxj is also known as the Han's mountain grape, a close relative of the grape, distributed in the reading area to the mountain range above the sea level, for the Taiwanese aborigines and the Han: the herb used in the province, the old stem and root The xanthate that is important for the treatment of internal injuries and muscle aches and pains is also used for diuresis, anti-inflammatory, hemostasis, treatment of chronic nephritis and hepatitis ^ Zhaidu (Chinese Medicine Council of the Executive Yuan, etc.). Listed as “Materials for Food Use”, and 8 1374749 The revised replacement page on September 11, 2012 is quite safe, and it is not necessary to carry out related toxicity studies to make beverages from the herbs themselves or their extracts. , capsules, lozenges or powders. Applicants have confirmed that the Dabenshan grapes have excellent anti-enteric activity, and they are highly developed, and can be used as anti-enteric drugs and health foods. Studies have shown that ethanol extracts from Dabenshan grapes can inhibit liver damage caused by free radicals (Yabe and Matsui, 1997) and reduce liver damage induced by iron (Yabe, heart/., 1998). The extract of Dabenshan grape has moderate resistance to ketoacid (the mutation effect of P-aeidH| ^ 1988). The methanol extract of Dabenshan grape has resistance to linoleic acid. _ a acid) and DNA The effect of body oxidation (Wu, ❹ /, 2〇〇4). To the best of the applicant's knowledge, there have been no reports in this technical field that the Dabenshan grape has anti-enteric virus-related activity. The present invention is therefore directed to the study of the application of the Dabenshan grape extract to the anti-intestinal virus' and demonstrates that the Dabenshan grape extract according to the present invention has potent anti-enteric activity. As used to prevent and combat intestinal tract, it contains an effective amount of Dabenshan's release agent. Based on the above, the present invention provides a virus-infected health food composition, extract, and a suitable carrier or diluent. The present invention also provides for the use of Dabenshan grape extract for the preparation of (4) prevention and combating enterovirus infection. The use of pharmaceuticals. The invention also provides an external disinfectant for inhibiting enterovirus activity comprising an effective amount of 畀 畀 量 的 大 大 大 以及 以及 以及 extract and a suitable carrier or diluent. ^ ^ ^ On the other hand, the present invention also provides an in vitro inhibition of enterovirus activity 9 丄 J/4749 September 11, 2012 Revision Replacement page: Party:, which includes: The agent is connected to the enterovirus to touch the wrong to inhibit the activity of the enterovirus. According to the present invention, the aforementioned health food fines, pharmaceutical external use v = agents and in vitro inhibition of enterovirus activity are available for prevention and infection of k enteroviruses, particularly for preventing and combating enteroviruses. ((10)r〇virus), type 69, type 7G to type 71 or virus infection of group b. In a preferred embodiment of the invention, the enterovirus line belongs to the enterovirus 71 type or the saxie B3 type. The term "prevention" in accordance with the present invention means reducing the infection of a host cell into the intestine; the probability of which involves interfering with enterovirus entry into the host cell and reducing intestinal infection. The term "confrontation" as used in the present invention refers to inhibition of enterovirus activity, which includes reducing the ability of the enterovirus to replicate in a host, reducing the chance of intestinal virus repetitive sensation, and soothing symptoms caused by enterovirus. U$ According to the present invention, the aforementioned Dabenshan grape extract is obtained by mixing the homogenized Dabenshan grape with a solvent by the following steps: wherein the Dabenshan grape is the root, stem and leaf of the Dabenshan grape a combination of fruit, seed, whole plant or the like, and the polarity index (P,) of the solvent is between methylene chloride and water, and the solvent is used at 4 <) (: to 95β (: extraction is homogenized) The Dabenshan grape is obtained to obtain the Dabenshan grape extract. Preferably, the extracting step further comprises removing the solvent to obtain the Dabenshan grape extract, wherein the Dabenshan grape extract is included. Not limited to: in the form of a concentrate or a dry powder. In a preferred embodiment of the invention, the portion of the Dabenshan grape line is the root, stem, leaf, fruit, seed, and whole of the Dabenshan grape. a strain or a group thereof, 丄j/dou/4y. Preferably, the root, the poor or the scorpion scorpion, the Dabenshan 5° which was repaired on September 11, 12, according to the present invention, Wei but not (4). Using cutting and grinding methods In a preferred embodiment of the dried cake, the powder, preferably the dried root, the stem or the month thereof, the solvent may be a polar one; the solvent between the two and the water, including However, it is not limited to water, & grades, lower alcohols, lower grades, and lower grades. In a preferred embodiment of the present invention, the solution is a group of the following (four): water , propylene methane, dioxane, 95% ethanol, and ethyl acetate. Preferably, s ^ is C-, cyanamide 6, 95% ethanol, ethyl acetate, and dichloromethane. More preferably, The solvent is acetone. In a preferred embodiment of the invention, the Dabenshan grape and the solvent are mixed in a solvent-based ratio between 1% (w/v) and pass (w/v). In a preferred embodiment of the lunar month, the foregoing suitable conditions include that the present invention is not limited to the combination of the present invention, the stirring, the oscillating, the ultrasonic, the Soxhlet heat reflow, or the like. Conditions include extraction at a temperature between 0 and 100 ° C; preferably, between the buckets and 8 (rc; and better) It is 'at room temperature, i.e., between about 2 〇〇 c and 3 〇〇 c. In a preferred embodiment of the invention, the appropriate conditions are included at room temperature with stirring and shaking. , ultrasonic or 95 ° c soxhlet extraction is carried out. The pharmaceutical products according to the invention include, but are not limited to, oral, parenteral routes, wherein the parenteral route may include but is not limited to Subcutaneous injection and intramuscular injection. 11 1374749 (3) Modified replacement page on September 11, according to the present invention, the pharmaceutical product may be L---_____ ^ ^ with σστ, but not limited to: powder, lozenge, capsule , suspension, gel, milk, night powder, foam, micro-emulsifier or spray type; the health food composition can be, but not limited to: loose, lozenge, capsule, suspension, gel And a microemulsifier or spray formulation; and the external disinfectant can be, but is not limited to: (iv) an emulsion powder, a foam, a microemulsifier, a spray or a suspension dosage form. The pH of the pharmaceutical, health food composition, and external disinfectant according to the present invention is not limited to a specific positive value, that is, it may be between U ", and preferably between 6 and 8. In a preferred embodiment of the method for inhibiting enterovirus activity in vitro, the step of contacting the external disinfectant with the enterovirus further comprises the steps of: spraying the external disinfectant with the possibility of suffering from enterovirus contamination. The article causes the enterovirus to contact with the external use and the poison to reduce the toxicity of the enterovirus. [Embodiment] The present invention will be further illustrated by the following examples, which are not intended to limit the invention. A person skilled in the art can make some modifications and modifications without departing from the scope of the invention. EXAMPLES General Experimental Materials and Methods Dabenshan Grape (Maxim.)

Trautv.]之乾燥的根莖係購自當地草藥店，並研製成粉末備 用。 B.宿主細胞 人類肌肉纖維母細胞（human embry0nai rhabd〇myosarcoma， 12 1374749 ___ 2012年9月11日修正替換頁 RD; BCRC 60113)及綠猴腎上細胞（African green monkey kidney, Vero; BCRC 60013)購自生物資源保存及研究中心 (Bioresources Collection and Research Center, HsinChu, Taiwan)。 C.病毒株 測試之勝病毒病毒株，包括腸病毒71型標準株（EV71 ATCC VR-784)及臨床株（EV71 CMUH 527-1 及 EV71 CMUH V4079)，來自於中國醫藥大學及高雄醫藥大學。克沙奇B3 型臨床株（CVB3 KMUH 92225、CVB3 KMUH 034189、CVB3 KMUH K516以及CVB3 KMUH 99168)，來自於高雄醫學大 學。 'D.細胞株及培養 1 ml細胞於25T培養瓶中，加入3ml含有10%胎牛血 清（Fetal Bovine Serum, FBS; Biological Industries, Isreal). 之杜氏改良英格爾培養基（Dulbecco’s modified Eagle’s minimum essential, DMEM; Gibco，NY)，於 37°C 含有 5%二 氧化碳之環境培養2天，細胞長滿90%。 吸除培養基，以0.004M磷酸鹽緩衝溶液（PBS, pH 7.4) 沖洗2次，加入1 ml 0.25% trypsin-EDTA與細胞作用20 * 秒，吸除多餘胰蛋白酶-EDTA(trypsin-EDTA)，靜置1分鐘 . 使細胞脫落，以含有10% FBS之DMEM培養基稀釋成為3 xlO5 cell/ml進行各項之試驗。 E.腸病毒之增殖及病毒斑測定 腸病毒71型及克沙奇B3型分別與RD及Vero細胞培 養進行增殖，其方法簡述如下。10 ml(3xl〇5 cell/ml)細胞培 13 1374749 2012年9月11曰修正替換頁 養於含有10%FBS之DMEM的75T培養瓶於37°C含有- 50/〇二氧化碳環境中培養24小時《吸除培養基，以pB8沖 洗兩次，加入2 ml(5xl05 PFU/ ml)病毒液，於37。〇含有5〇/〇 二氧化碳環境中感染1小時，加入含有2〇/〇FBS之DMEM， 於37 °C含有5%二氧化碳環境中培養，至8〇 %細胞產生病 變。3次冷凍（_80t:)及解凍（37r解凍）循環，以使細胞破裂 而釋出病毒，於402 g離心5分鐘，去除細胞碎片，收集上 清液，即為病毒液，於_80°c儲存備用。 腸病毒力價以病毒斑測定。於6孔盤中，每孔加入2 ml 細胞懸浮液（3xl〇5 cell/ml) ’於5%二氧化碳環境中37它培 養24小時，吸除培養基，以pBS沖洗.2次，加入〇 $⑹ 以2% FBS之DMEM十倍續列稀釋之病毒液，以不加入病 毒液者為控制組，於環境中感染丨小時（每15分鐘搖晃 一次）’吸除多餘病毒液，加入覆蓋培養基[含2% FBs及 〇.26%瓊脂糖（agarose)i DMEM]，於5%二氧化碳之環境中 37C培養3天，吸除覆蓋培養基，加入2mi之福馬林， 固疋、-田胞3 0刀鐘，吸除福馬林，加入^ %結晶紫染色細胞$ 刀鐘以自來水沖洗多餘結晶紫，計數病毒斑數目（plaque forming unit，PFU)。 實施例1以不同萃取溶劑所萃取之大本山葡萄萃取 物對抑制腸病毒活性之影響（一） 1-1大本山葡萄萃取物之製備 以磁石授拌萃取。30克大本山葡萄粉末（預先過篩’收 集通過6〇mesh篩網之粉末）加入水、丙網' 95%乙醇、乙 酸乙醋或正己貌等不同極性之溶劑200 W，於室溫磁石攪 1374749 2012年9月11日修正替換頁 拌1小時，於4°C以12300 g離心15分鐘，收集上清f- ㈣以200 ml溶劑重複萃取2次’合併上清液於赋減 壓濃縮至乾’以取得—大本山葡萄萃取物濃縮物，秤重， 計算固形物收率。固形物收率（％，w/w)為濃縮物乾重與大本 山葡萄粉末重量比值之百分率。 大本山葡萄以不同極性的溶劑萃取，分別為水、丙酮、 95/。乙醇、乙酸乙醋及正己烧，固形物收率之結果如表一所 示'奋劑之極性愈尚，萃取物之固形物含量越高，五種萃 取♦劑中，水具有最大之固形物收率（7 73%，w/w)，95%乙 醇及丙_居中’分別為7 〇3%及4 Q1%，乙酸乙自旨及正己烧 最低，分別為0.90%及0.3%。 衣一大本山蜀萄不 同溶劑萃取物之收率 溶劑 固形物收率 (Extraction yield) (%，w/w) 水 7.73 丙酮 4.01 95%乙醇 7.03 乙酸乙酯 0.90 正己烷 0.30 1-2大本山葡萄萃取物對於不同病毒株之5〇%抑制濃 度（50% inhibitory concentration，ICs〇)測定 於ό孔盤中每孔加入2 ml細胞懸浮液（3xi〇5 ceu/mi)， 於5%二氧化碳環境中37°C培養24小時，吸除培養基，以 PBS沖洗2次，加入〇.5 ml含1〇〇 PFU之病毒的不同濃度 萃取物為實驗組，僅加入病毒液為病毒控制組，於環境 中感染1小時（每15分鐘搖晃一次），吸除多餘病毒液，加 入含有相同濃度萃取物之覆蓋培養基（含2%FBS及0.26%瓊 15 1374749 2012年9月11曰修正替換頁 脂糖之DMEM)，於5%二氧化碳之環境中37°C培養3天， 吸除覆蓋培養基，加入2 ml之10%福馬林，固定細胞30 分鐘，吸除福馬林，加入1 %結晶紫染色細胞5分鐘，以自 來水沖洗多餘結晶紫，計數病毒斑數目，結果以抑制率表 示。 抑制率以下列公式計算： 抑制率（Inhibition) (%) =(病毒控制組之病毒斑數目-實驗組病毒斑數目）X 1 0 0 % /控制組病毒斑數 並以萃取物濃度為橫軸，各測試萃取物對各病毒株之 抑制率為縱軸做線性曲線，計算出各萃取物對各病毒株之 IC50。 < 結果 表二為4種溶劑之大本山葡萄萃取物（水、丙酮、95%乙 醇及乙酸乙酯萃取物）對3株腸病毒71型及4株克沙奇B3 型之IC5Q，IC5G值愈低表示抑制腸病毒活性愈高。 表二以病毒斑減少分析法測定大本山葡萄不同溶劑萃取物對 腸病毒71型及克沙奇B3型之50%抑制濃度 腸病毒株 IC50 (μ g/ml) ' 水 丙嗣 95%乙醇 乙酸乙酯 EV71 VR784 151.61±9.03a 26·17±2·51 66.40 ±2.65 25.31 ±2.03 CMUH 527-1 75.04±3·33 18.52 ±0.42 33.77±0·31 41.71 ±0.26 CMUH V4079 54.63 ±2.93 39.98 ±0.45 46.91 ±3.37 45_11±2.51 CVB3 KMUH 92225 67.10±5.30 29_75士 3.43 57.69 ±1.80 47.56士 1.98 KMUH 034189 73.61 ±4.19 62.81 ±4.34 68·35±3·19 119.42 ±0.96 KMUHK516 113·54±1·47 31.86±2·27 49.87 ±0.18 75.49 ±0.69 KMUH 99168 129.48 ±1.09 54.34±4.19 30.53 ±1.01 54.55 ±0.28 每組數據由三次不同實驗每次以三重複進行所得的平均值，以平均值土標準偏差表示。 在4種萃取物中，丙酮萃取物表現出最高之抑制腸病毒 活性，對3株腸病毒71型及4株克沙奇B3型之IC5Q範圍 16 1374749 2012年9月11日修正替換頁 為18.52土0.42至 62 81±4 34 叫/1111;其次▲ 95% 乙醇萃取~— 物’ ％〇範圍為30.53±1.〇1至队則19叫/M ;再次為乙 酸乙3曰萃取物’1(：5()範圍為25.31±2.03至119.42士0.96 “g/ml ;水萃取物對抑制腸病毒活性最低，IC5。範圍為 曰 至151·6士9.30 μδ/ιη1 ;正己烷萃取物則在CC50劑 里時’未表現出抗腸病毒71型及沙奇幻型之活性（結果未 顯示）。綜合表二之結果顯示出，大本山葡萄之丙酮萃取物 具有最好之抑制腸病毒活性，無論對3株腸病毒71型及4 株克沙奇B3型均表現出最低之％。，其次為抓乙醇萃取 物，對腸病毒亦有良好的抑制效果。 實施例2以不同萃取溶劑所萃取之大本山葡萄萃取 物對抑制腸病毒活性之影響（二） 本試驗中以水、氰甲烷、95%乙醇Λ丙酮、乙酸乙酯、 二氯甲烷及正己烷等不同極性之溶劑為萃取溶劑，以前述 之磁石擾拌方法萃取，並以如前所述之方法試驗萃取物之 抗腸病毒活性。 結果 表二顯示大本山葡萄之水、氰甲烷、丙酮、乙酸乙酯、 95%乙醇、二氯甲烷及正己烷等溶劑萃取物之固形物收率及 對 EV71 ATCC VR-784 及 CVB3 KMUH 92225 之 Ic 。 17 1374749The dried rhizome of Trautv.] was purchased from a local herbal shop and developed into a powder preparation. B. Host cells Human muscle fibroblasts (human embry0nai rhabd〇myosarcoma, 12 1374749 ___ September 11, 2012 revised replacement page RD; BCRC 60113) and green monkey kidney cells (African green monkey kidney, Vero; BCRC 60013) Purchased from the Bioresources Collection and Research Center (HsinChu, Taiwan). C. Viral strain The tested viral strains, including Enterovirus 71 standard strain (EV71 ATCC VR-784) and clinical strains (EV71 CMUH 527-1 and EV71 CMUH V4079), were obtained from China Medical University and Kaohsiung Medical University. The Keshaqi B3 clinical strain (CVB3 KMUH 92225, CVB3 KMUH 034189, CVB3 KMUH K516, and CVB3 KMUH 99168) was obtained from Kaohsiung Medical University. 'D. Cell line and culture 1 ml of cells in a 25T culture flask, and add 3 ml of Dulbecco's modified Eagle's minimum essential containing 10% fetal bovine serum (Fetal Bovine Serum, FBS; Biological Industries, Isreal). , DMEM; Gibco, NY), cultured in an environment containing 5% carbon dioxide at 37 ° C for 2 days, the cells were over 90%. The medium was aspirated, washed twice with 0.004 M phosphate buffer solution (PBS, pH 7.4), and 1 ml of 0.25% trypsin-EDTA was added to the cells for 20 * seconds to remove excess trypsin-EDTA (trypsin-EDTA). The cells were detached, and the cells were detached, and diluted to 3 x 10 5 cells/ml in DMEM medium containing 10% FBS for each test. E. Proliferation of Enterovirus and Determination of Viral Spots Enterovirus 71 and Keshaqi B3 were propagated with RD and Vero cells, respectively, and the methods are briefly described below. 10 ml (3xl〇5 cell/ml) cell culture 13 1374749 September 11, 2012 Correction replacement page 75T culture flasks in DMEM containing 10% FBS were cultured for 24 hours at 37 ° C containing - 50 / 〇 carbon dioxide "Aspirate the medium, rinse twice with pB8, and add 2 ml (5 x 105 PFU/ml) of virus solution at 37. 〇 Infected with 5 〇/〇 in a carbon dioxide atmosphere for 1 hour, DMEM containing 2 〇/〇FBS was added, and cultured at 37 ° C in a 5% carbon dioxide atmosphere, and 8 〇 % cells were mutated. 3 times of freezing (_80t:) and thawing (37r thawing) cycle, in order to rupture the cells and release the virus, centrifuge at 402 g for 5 minutes, remove cell debris, collect the supernatant, which is the virus solution, at _80 °c Store for backup. The enterovirus force price was determined by plaque. In a 6-well plate, add 2 ml of cell suspension (3xl〇5 cell/ml) per well. In a 5% carbon dioxide environment, it was cultured for 24 hours, aspirate the medium, rinse with pBS. 2 times, add 〇$(6) The diluted virus solution was re-listed with DMEM of 2% FBS ten times, and the virus-free liquid was used as the control group. In the environment, the infection was 丨 hours (shake every 15 minutes), the excess virus solution was aspirated, and the cover medium was added. 2% FBs and 〇.26% agarose i (dame) DMEM], cultured in 37 ° C for 3 days in a 5% carbon dioxide environment, aspirate the culture medium, add 2 mi of fumarin, solid sputum, - field cell 30 knives , remove the formalin, add ^% crystal violet stained cells $ knife clock to wash excess crystal violet with tap water, count the number of plaque forming units (PFU). Example 1 Effect of Dabenshan Grape Extract Extracted by Different Extraction Solvents on Inhibition of Enterovirus Activity (I) Preparation of 1-1 Dabenshan Grape Extract Extracted by magnetite. 30 grams of Dabenshan grape powder (pre-sifted 'collected through 6 〇mesh sieve powder) to add 200 W of different polar solvents such as water, propyl mesh '95% ethanol, ethyl acetate or positive appearance, stir at room temperature magnet 1374749 On September 11, 2012, the replacement page was mixed for 1 hour, centrifuged at 12300 g for 15 minutes at 4 ° C, and the supernatant was collected. f- (4) Repeated extraction twice with 200 ml of solvent. The combined supernatant was concentrated under reduced pressure. Dry 'to obtain - Dabenshan grape extract concentrate, weigh, calculate the solids yield. The solids yield (%, w/w) is the percentage of the dry weight of the concentrate to the weight of the Dabenshan grape powder. Dabenshan grapes are extracted with solvents of different polarities, namely water, acetone, 95/. Ethanol, ethyl acetate and hexose, the yield of solids is shown in Table 1. The more polar the agent is, the higher the solid content of the extract. Among the five extracts, water has the largest solid content. The yields (7 73%, w/w), 95% ethanol and C-centers were 7 〇 3% and 4 Q1%, respectively, and the lowest values of acetic acid B were 0.90% and 0.3%, respectively. Yield of different solvent extracts of a large amount of yam yam. Extract yield (%, w/w) Water 7.73 Acetone 4.01 95% Ethanol 7.03 Ethyl acetate 0.90 hexane 0.30 1-2 Big Benshan Grape The extract was tested for 5% inhibitory concentration (ICs〇) of different strains. 2 ml of cell suspension (3xi〇5 ceu/mi) was added to each well of the pupil dish in a 5% carbon dioxide environment. Incubate at 37 °C for 24 hours, aspirate the medium, rinse twice with PBS, add 5.5 ml of different concentrations of extract containing 1 〇〇PFU virus to the experimental group, add only the virus solution to the virus control group, in the environment Infected for 1 hour (shake every 15 minutes), remove excess virus solution, add cover medium containing the same concentration of extract (containing 2% FBS and 0.26% Joan 15 1374749 September 11, 2012 modified DMEM for replacement of sucrose) Incubate for 3 days at 37 ° C in a 5% carbon dioxide atmosphere, aspirate the culture medium, add 2 ml of 10% fumarin, fix the cells for 30 minutes, aspirate the formalin, and add 1% crystal violet to stain the cells for 5 minutes. Rinse excess crystal violet with tap water Counting the number of plaques, inhibition rate results are shown in Table. The inhibition rate is calculated by the following formula: Inhibition (%) = (number of plaques in the virus control group - number of plaques in the experimental group) X 1 0 0 % / control group plaque number and extract concentration as the horizontal axis The inhibition rate of each test extract against each virus strain was linearly plotted on the vertical axis, and the IC50 of each extract against each virus strain was calculated. <Results Table 2 shows IC5Q and IC5G values of three strains of enterovirus 71 and four strains of Keshaqi B3 in four solvents: Dabenshan grape extract (water, acetone, 95% ethanol and ethyl acetate extract) The lower the expression, the higher the inhibition of enterovirus activity. Table 2 Determination of the 50% inhibitory concentration of Enterovirus 71 and Keshaqi B3 against the enterovirus 71 (μg/ml) of different solvent extracts of Dabenshan grape by plaque reduction assay. Ethyl ester EV71 VR784 151.61±9.03a 26·17±2·51 66.40 ±2.65 25.31 ±2.03 CMUH 527-1 75.04±3·33 18.52 ±0.42 33.77±0·31 41.71 ±0.26 CMUH V4079 54.63 ±2.93 39.98 ±0.45 46.91 ±3.37 45_11±2.51 CVB3 KMUH 92225 67.10±5.30 29_75士3.43 57.69 ±1.80 47.56士1.98 KMUH 034189 73.61 ±4.19 62.81 ±4.34 68·35±3·19 119.42 ±0.96 KMUHK516 113·54±1·47 31.86±2· 27 49.87 ±0.18 75.49 ±0.69 KMUH 99168 129.48 ±1.09 54.34±4.19 30.53 ±1.01 54.55 ±0.28 The average of each set of data from three different experiments in each of three replicates, expressed as the mean soil standard deviation. Among the four extracts, the acetone extract showed the highest inhibition of enterovirus activity, and the IC5Q range of 3 strains of Enterovirus 71 and 4 Keshaqi B3 was 16 1374749. The revised page on September 11, 2012 was 18.52. Soil 0.42 to 62 81 ± 4 34 is called /1111; secondly ▲ 95% ethanol extraction ~ - '% 〇 range is 30.53 ± 1. 〇 1 to the team is 19 / M; again is acetic acid B 3 extract '1 (:5() ranged from 25.31±2.03 to 119.42±0.96” g/ml; water extract had the lowest inhibitory activity against enterovirus, IC5. The range was 151 to 151.6±9.30 μδ/ιη1; the n-hexane extract was in When CC50 was used, it did not show activity against EV71 and Saqi illusion (results not shown). The results of Table 2 show that the acetone extract of Dabenshan grape has the best inhibitory activity against enterovirus, regardless of 3 strains of enterovirus 71 and 4 strains of Keshaqi B3 showed the lowest %. Secondly, the ethanol extract was used, which also had a good inhibitory effect on enterovirus. Example 2 was extracted with different extraction solvents. Effect of Dabenshan grape extract on inhibition of enterovirus activity (II) Water and cyanide in this experiment The solvent of different polarity such as alkane, 95% ethanol, acetone, ethyl acetate, dichloromethane and n-hexane is used as an extraction solvent, and is extracted by the above-mentioned magnet scramble method, and the anti-intestine of the extract is tested by the method as described above. Viral activity. Table 2 shows the solids yields of solvent extracts from Dabenshan grape water, cyanamide, acetone, ethyl acetate, 95% ethanol, dichloromethane and n-hexane, and for EV71 ATCC VR-784 and CVB3. Ic of KMUH 92225. 17 1374749

EV71 ATCC VR-784 及 山葡萄不同溶劑萃取物對 溶劑 固形物收率 (%，w/w) EV71 VR784 a (μ§/ιη1) 水 氰曱烷 95%乙醇 丙_ 乙酸乙酯 二氧曱烷 正己烷 7.73 1.47 7.03 4.01 0.90 0.76 0.30 151.61 ±9.30逆 19.07 ± 1.26 Β 66.40 ± 2.65 C 26.17 ±2.51 Β 25.31 ± 2.03 Β ^-8^0.34 aEV71 ATCC VR-784 and different solvent extracts of mountain grapes to solvent solids yield (%, w/w) EV71 VR784 a (μ§/ιη1) water cyanide 95% ethanol c ethyl acetate dioxane Alkane 7.73 1.47 7.03 4.01 0.90 0.76 0.30 151.61 ±9.30 inverse 19.07 ± 1.26 Β 66.40 ± 2.65 C 26.17 ±2.51 Β 25.31 ± 2.03 Β ^-8^0.34 a

CVB3 KMUH-92225 67.10 ± 5.30 E 35.09 ± 1.98 B 57.69 ± 1.80 D 29.75 ± 3.43 A 47.56 ± 1.98 C 46.08 ± 1.18 C 每組數據由三次不同實驗每次以三行所得 _ 未偵測到 棚均值伴隨不同大寫字母者之間具有顯著差異平均值士標準偏差表示 不同萃取溶劑之固形物收率之处 果顯示出，水及95%乙 醇有最高之收率（分別為7.73及7.03。/、 。）’丙綱次之（4.01%)， 再次為氰甲炫>(1.47%);乙酸乙酿（〇 9〇 0/、 U/°} ' 二氣甲烷（0.76%) 及正己烷（0.30%)之收率均低於ι〇/〇為。 Α本山葡萄萃取物對 EV71 VR784抑制活性（IC5〇)之結果 千 + a ^ , 顯不出’二氯曱烷萃取物 有最低之 IC5〇(10.81±〇.34 pg/ml)，复-a * ★ Z、-人為氰曱炫、乙酸乙酯 及丙酮萃取物（ic50介於19.07±1.26 $ a 土 26.17 + 2.51 pg/ml 間）’再次為95%乙醇萃取物（64 〇2+i .—^1 Kg/ml)，水萃取物 最高（151.61±9.3〇 jug/ml);正己烷 平取物不具有抗EV71 ATCC VR-784之活性。大本山葡萄萃 平取物對CVB3 KMUH 92225抑制活性之結果顯示，丙酮萃取抓 切有最低之IC5Q(29.75 ±3·43 pg/ml)，其次為氰甲烷萃取物f 初（35·〇9±1.98 pg/ml)，再 次為二氯甲烷（46.08±1.18 祕 唆乙酯（47·56±1·98 pg/ml)萃取物’ 95%乙醇及水萃取物啬 取你*(1(^5。分別為60.49± 18 1374749 2012年9月11日修正替換頁 4.60 及 67.10 ± 5.30 pg/mi)，正己烷萃取物 ~— KMUH-92225.無抑制活性。 综合表三之結果’除正己烷萃取物外，其餘之6種溶劑CVB3 KMUH-92225 67.10 ± 5.30 E 35.09 ± 1.98 B 57.69 ± 1.80 D 29.75 ± 3.43 A 47.56 ± 1.98 C 46.08 ± 1.18 C Each set of data is obtained from three different experiments each time in three rows _ No shed average is detected There is a significant difference between the uppercase letters. The mean standard deviation indicates the solids yield of different extraction solvents. The results show that water and 95% ethanol have the highest yields (7.73 and 7.03, respectively.). The second order of C-class (4.01%), the second time is cyanamide [1.47%]; the acetic acid is brewed (〇9〇0/, U/°} 'di-methane (0.76%) and n-hexane (0.30%) The yields were lower than ι〇/〇. The results of the inhibitory activity of Α71 grape extract on EV71 VR784 (IC5〇) were thousands + a ^ , which showed that the 'dichlorodecane extract had the lowest IC5 〇 (10.81). ±〇.34 pg/ml), complex-a* ★ Z,-man-made cyanide, ethyl acetate and acetone extract (ic50 between 19.07±1.26 $ a soil 26.17 + 2.51 pg/ml) 95% ethanol extract (64 〇2+i .—^1 Kg/ml), the highest water extract (151.61±9.3〇jug/ml); n-hexane extract does not have anti-EV71 ATC The activity of C VR-784. The results of the inhibitory activity of Dabenshan grape extract on CVB3 KMUH 92225 showed that the acetone extraction had the lowest IC5Q (29.75 ±3·43 pg/ml), followed by the cyanide extract f. (35·〇9±1.98 pg/ml), again for dichloromethane (46.08±1.18 唆 ethyl ester (47·56±1·98 pg/ml) extract '95% ethanol and water extract to take you *(1(^5. 60.49±18 1374749 revised on September 11, 2012, revised page 4.60 and 67.10 ± 5.30 pg/mi), n-hexane extract ~- KMUH-92225. No inhibitory activity. Results 'The remaining 6 solvents except the n-hexane extract

萃取物均具有抗腸病毒EV71 ATCC VR-784及CVB3 KMUH 92225活性；其中以丙酮萃取物之活性最佳。 實施例3利用時間歷程分析（time course analysis)探 討大本山葡萄丙酮萃取物抑制腸病毒活性可能之機制 為了探討大本山葡萄丙酮萃取物抑制EV71及CV3可能 機制’在本實施例中係利用時間歷程分析（time c〇urse analysis) 了解於不同時間點大本山葡萄丙酮萃取物對於腸病 毒株EV71及CV3對細胞感染之抑制作用，其中時間歷程分 析係利用下列方法進行。 ML盤之每孔加入2ml細胞懸浮液（3xl〇5cell/ml)，置 於5%二氧化碳環境中37t培養24小時。吸除培養基，以 PBS沖洗2次’加入〇,5 ml之1〇〇 pFU病毒液，於代環境 中感染1小時（每15分鐘搖晃一次），吸除多餘病毒液，於 病毒感染前1、2小時、病毒感染時及病毒感染後1、2、4、 8、12、16、20 及 24 小時（分別以 _1、_2、〇、i、2、4、8、 I2 16、2〇及24 h表示），加入不同濃度之大本山葡萄萃取 物（20 pg/nU、40 pg/m 卜 62·5 pg/ml 及 100 μ§/ιη1)，以僅加 入病毒液為病毒控制組。加入含有相同濃度萃取物之覆蓋 培養基（含2%FBS及〇.26°/。瓊脂糖之DMEM)。於5%二氧化 碳之環境中37t培養3天。吸除覆蓋培養基，加入2ml之 100/〇福馬林’固定細胞3〇分鐘，吸除福馬林，加入結晶 紫染色細胞5分鐘，以自來水沖洗多餘結晶紫，計數病毒 1374749 2012年9月11日修正替換頁 斑數目，結果以抑制率表示。 結果 大本山葡萄丙酮萃取物對EV71 ATCC VR-784之抑制 作用之時間歷程分析之結果係如下面表四所示。 表四大本山葡萄丙酮萃取物對腸病毒71型VR784之效應的 時間歷程分析結果 感染 時間點 抑制率（％)a 20 pg/ml 40 pg/ml 62.5 μ^πύ 100 μ^ηύ -2h c 72.42 土 0.95 A cb73.54±0.28AB ab 77.89 ±3.15 A a 79.29 士 4.46 A -1 h b 71.16 ±0.23 A ab77.53 ±4.48A ab 77.53 ±3.87 A a 80.84 士 5.71 A Oh b 65.32 ±4.08 A a76.83±2.79A a73_05±4.75A a 77.45 ±3.80 A 1 h c 49.36 ± 2.18 B b66.44±3.13BC a 74.75 ±0.50 A a 78.21 ±0.40 A 2h b 47.06 ± 8.45 B a60.52± 10.19C a 62_02 士 4.07 B a 73.26 ± 7.50 AB 4h c 14.18 ± 7.48 C b47.28±4.09D ab 61.18 ± 11.87 B a 66.44 ± 9.97 B 8h 0 0 0 0 a每組數據由三次不同實驗每次以三重複進行所得的平均值，以平均值士標準偏差表示。各欄内 平均值伴隨不同大寫字母者之間具有顯著差異(P &lt;〇.〇5)。各列内平均值伴隨不同小寫字母者之 間具有顯著差異仏&lt;0.05)。 於病毒感染前1小時及2小時加入萃取物（參見-2 h及-1 h)，結果顯示出20至100 pg/ml之濃度均有相當好之抑制作 用，抑制率達71.16±0.23至80·84±5.71%，隨作用劑量增高， 其抑制率顯著增加（ρ&lt;〇.05)，此結果顯示出大本山葡萄丙酮 萃取物對腸病毒71型VR784具預防感染之效果。當萃取物 及病毒液同時加入（參見0 h)細胞中，20 pg/ml之濃度抑制率 為65.32±4_08 %，當濃度增加至40至100 pg/ml時，抑制率 貝1J 顯著的增加達 73.05±4.75 至 77·45±3.80ο/〇 (ρ&lt;0·05)，40 至 1 00 pg/ml劑量間無顯著性差異〇&lt;0.05)，此結果顯示出大本 山葡萄丙酮萃取物具有直接殺死腸病毒71型VR784或防止 20 1374749 2012年9月11日修正替換頁 病母附著細胞之效果。於病毒感染細胞i 2小時後加~]^~~~~~ 萃取物（參見1 h及2 h)’抑制率間無顯著性差異&amp;&lt;〇 〇5)， 2〇至100 pg/ml之抑制率分別為47 〇6±8 45至78 2卜 0·4〇%，顯示作用濃度越高，抑制率越高；62 5至ι〇〇 “/Μ 間無差異（p&lt;0.05)。當於病毒感染細胞4小時後加入萃取物 (參見4h)，則抑制率快速下降(^〈005)，2〇至⑽盹—濃 度之抑制率為14.18±7.48 i 66.44±9.97%，顯示作用劑量越 下降幅度越大；62.5至1〇〇μ§/ιη1間無差異㈣〇5)。 备於病毒感染細胞8小時後加入萃取物（參見8 h)，各濃度 均無抑制作用（〇〇/〇)。 又 綜合表四之結果可見，大本山葡萄丙酮萃取物對腸病毒 71型VR784之作用機制包括預防及直接抑制病毒感染，以 及抑制病毒早期之複製。抑制率隨細胞感染病毒時間增加而 下降’作用劑量越高抑制效果越好，4乍用方式為劑量依賴 用劑置间於62·5 μ§/ιη1時，則抑制率不再 〇&lt;0.05)。 用二 抑㈣ 21 1374749 2012年9月11日修正替換頁 表五大本山葡萄丙酮萃取物對CVB3 KMUH 92225之效應的 時間歷程分析結果The extracts have anti-enteric EV71 ATCC VR-784 and CVB3 KMUH 92225 activities; among them, the acetone extract has the best activity. Example 3: Using time course analysis to explore the possible mechanism of inhibition of enterovirus activity by acetone extract of Dabenshan grape in order to explore the possible mechanism of inhibition of EV71 and CV3 by acetone extract of Dabenshan grape in this example. Time c〇urse analysis To understand the inhibitory effect of acetone extract of Dabenshan grape on cell infection of enterovirus strains EV71 and CV3 at different time points, the time history analysis was carried out by the following methods. 2 ml of a cell suspension (3 x 10 〇 5 cells/ml) was added to each well of the ML disk, and cultured at 37 t for 24 hours in a 5% carbon dioxide atmosphere. Absorb the medium, rinse twice with PBS, add 〇, 5 ml of 1 〇〇pFU virus solution, infect the environment for 1 hour (shake every 15 minutes), and remove the excess virus solution before the virus infection. 2 hours, virus infection, and 1, 2, 4, 8, 12, 16, 20, and 24 hours after virus infection (_1, _2, 〇, i, 2, 4, 8, I2 16, 2, and 24 h)), different concentrations of Dabenshan grape extract (20 pg/nU, 40 pg/m Bu 62·5 pg/ml and 100 μ§/ιη1) were added to add only the virus solution to the virus control group. A cover medium (2% FBS and 〇.26 °/. agarose DMEM) containing the same concentration of extract was added. Incubate for 3 days at 37t in a 5% carbon dioxide environment. Aspirate the culture medium, add 2 ml of 100/〇福马林's fixed cells for 3 minutes, aspirate the formalin, add crystal violet to stain the cells for 5 minutes, rinse excess crystal violet with tap water, count the virus 1374749 revised September 11, 2012 The number of page spots is replaced and the result is expressed as the inhibition rate. Results The results of the time course analysis of the inhibitory effect of the acetone extract of Dabenshan grape on EV71 ATCC VR-784 are shown in Table 4 below. Table 4 Time history analysis of the effect of acetone extract of Dabenshan grape on enterovirus 71 VR784 results Infection time inhibition rate (%) a 20 pg/ml 40 pg/ml 62.5 μ^πύ 100 μ^ηύ -2h c 72.42 Soil 0.95 A cb73.54±0.28AB ab 77.89 ±3.15 A a 79.29 ± 4.46 A -1 hb 71.16 ±0.23 A ab77.53 ±4.48A ab 77.53 ±3.87 A a 80.84 ± 5.71 A Oh b 65.32 ±4.08 A a76. 83±2.79A a73_05±4.75A a 77.45 ±3.80 A 1 hc 49.36 ± 2.18 B b66.44±3.13BC a 74.75 ±0.50 A a 78.21 ±0.40 A 2h b 47.06 ± 8.45 B a60.52± 10.19C a 62_02 4.07 B a 73.26 ± 7.50 AB 4h c 14.18 ± 7.48 C b47.28±4.09D ab 61.18 ± 11.87 B a 66.44 ± 9.97 B 8h 0 0 0 0 a Each set of data is obtained from three different experiments each time with three replicates. The average value is expressed as the mean standard deviation. The average value in each column is significantly different from the different capital letters (P &lt; 〇.〇5). The average value in each column is significantly different from the different lowercase letters 仏 &lt; 0.05). The extract was added 1 hour and 2 hours before the virus infection (see -2 h and -1 h). The results showed that the concentration of 20 to 100 pg/ml had a good inhibitory effect, and the inhibition rate reached 71.16±0.23 to 80. · 84±5.71%, with the increase of the dose, the inhibition rate increased significantly (ρ&lt;〇.05). This result showed that the acetone extract of Dabenshan grape had the effect of preventing infection of Enterovirus 71 VR784. When the extract and virus solution were added simultaneously (see 0 h), the inhibition rate of 20 pg/ml was 65.32±4_08%. When the concentration was increased to 40-100 pg/ml, the inhibition rate increased significantly. 73.05±4.75 to 77·45±3.80ο/〇(ρ&lt;0·05), there was no significant difference between doses of 40 to 100 pg/ml 〇&lt;0.05), which showed that the acetone extract of Dabenshan grape had Directly kill enterovirus type 71 VR784 or prevent 20 1374749 on September 11, 2012 to correct the effect of the replacement page on the attached cells. After 2 hours of virus-infected cells, add ~]^~~~~~ extract (see 1 h and 2 h). There was no significant difference between the inhibition rates &&lt;〇〇5), 2〇 to 100 pg/ The inhibition rate of ml was 47 〇6±8 45 to 78 2 Bu 0·4〇%, indicating that the higher the concentration, the higher the inhibition rate; 62 5 to ι〇〇 “/Μ no difference (p&lt;0.05) When the extract was added to the virus for 4 hours (see 4h), the inhibition rate decreased rapidly (^<005), and the inhibition rate of 2〇 to (10)盹-concentration was 14.18±7.48 i 66.44±9.97%. The decrease in dose was greater; there was no difference between 62.5 and 1〇〇μ§/ιη1 (4)〇5). The extract was added to the virus-infected cells for 8 hours (see 8 h), and there was no inhibition at each concentration (〇〇 The results of Table 4 show that the mechanism of action of Dabenshan grape acetone extract on EV71 is to prevent and directly inhibit viral infection, and to inhibit the early replication of virus. The inhibition rate increases with the time of virus infection. And the lower the dose, the better the inhibition effect, and the 4 乍 method is dose-dependent agent placement at 62·5 μ§/ιη1 , The inhibition rate is no longer square &lt;. 0.05) (iv) with diethyl suppression 21 is 1374749 September 11, 2012 amended five alternative page table acetone extract of Vitis present the analysis results of the time course of CVB3 KMUH effects 92225

感染 時間點 抑制率（％)a 31.25 pg/ml 50 pg/ml 62.5 pg/ml 100 μ^ηύ -2h a 1.81 士 0.13 D b 1.63±0_15G a 2.23 ± 0.20 EF a 1.99 ± 0.18 F -1 h a 1.33 ±0.15 D a 1.40 ± 0.10 G a 1.63 ± 0.15 F a 1.37 ± 0.12 F Oh a 23.06 ± 3.66 B a 25.73 ± 1.16 E a 24.82 ± 1.08 D a 25.54 士 1.50 D 1 h c 33.卯 ±4.36 A b 81.56 ±4.01 A a 92.79 ±5.62 A a 100.00 ±0.00 A 2h d 33.62 ±3.45 A c73_92 士 5.30B b 91.74 ±1.96 A a 100.00 ±0.00 A 4h d31.81± 1.29A c 71.78 ±2.86 BC b 91.19 ± 4.80 AB a 100.00 ±0.00 A 8h c 24.84 ± 2.99 B b 66.54 ± 4.66 C a 85.85 ± 4.39 B a93‘07±3.46B 12 h d 15.41 ±2.16 C c 46.34 ± 4.87 D b 65.39 ± 4.84 C a 78.68 ± 4.86 C 16 h c 3.84 ± 0.36 D b 18.58 ± 1.63 F a 24.51 ± 3.51 D a 27.82 ± 0.54 D 20 h b 1.88 ± 1.81 D b 1.33 ± 2.08 G a 7.60 ± 2.26 E a 6.90 ± 3.56 E 24 h a 0.66 ±1.52 D b 0.03 ± 2.08 G a 1.97 ± 0.80 F a 3.10 ± 0.89 F 本由三次2實$每次以三重1進行所均值，以平均值±標準偏差表示。各攔g 者之間具有顯著差異㈣.°5)。各列内平均值伴隨不同小寫字母者之 於病毒感染前1小時加入萃取物（參見i h及2 h)，各濃 度（31.25至1〇〇08/!111)對病毒之抑制率均相當低（2%以下）， 各濃度間之抑制率無差異㈣.〇5)。當萃取物及病毒液同時 加入細胞中（參見〇 h)，31.25至100 _ml濃度之抑制率為 23_06±3.66至25_73 ± 1·16% ’各濃度間沒有顯著差異 (Ρ&lt;〇·〇5广此結果顯示出萃取物有直接殺死部分病毒⑵％) 或防=病毒附著細胞之作用。於病毒感染細胞ι至4小時後 加入萃取物(參見i h至4 h)，抑制率間無顯著性差異 (尸&lt;0.05)，作用劑量越高，抑制率越大㈣州，於a 5㈣⑹ 劑量之抑制率達90%以上，議叫㈤則達ι〇〇%。於 染細胞8至16小時後加人萃取物（參見8h、12h及16^ 22 1374749 _ 2012年9月11日修正替換頁 隨時間增加抑制率急速下降〇&lt;〇.〇5)，各濃度之抑制率由8h 時之 24.84±2.99至 93.07±3.46%之間，降至16 11時之3.84± 0.3 6至27.82±0.54%之間；作用劑量越高抑制率越大，62.5 及1 00 pg/ml間無差異&lt;0.05)。於病毒感染細胞20小時後 加入萃取物（參見20 h及24 h)，抑制率幾降為0。 .綜合表五之結果，大本山葡萄丙酮萃取物對CVB3 KMUH 92225之作用機制包括直接殺死部分病毒或防止病毒 附著細胞之作用以及抑制病毒之複製作用。隨感染時間增加 抑制率下降，作用劑量越高抑制率越大，作用方式為劑量依 賴型；當作用劑量高於62.5 pg/ml時，則抑制率不再增加 (ρ&lt;0·05)。 由表四及表五之結果顯示出，大本山葡萄丙酮萃取物對 EV71 ATCC VR-784 及 CV3 KMUH 92225，均有高度之抑制 作用，但對兩者之抑制機制不全然相同。對EV71 ATCC VR-784具有預防病毒感染及抑制病毒吸附及早期複製之作 用；對CV3 KMUH 92225則主要為抑制病毒的複製作用。大 本山葡萄丙酮萃取物對此二腸病毒株之作用均為劑量依賴 型，劑量越高抑制率越大，62.5 pg/ml劑量可得最大之抑制 率。 * 實施例4 pH對大本山葡萄丙酮萃取物抑制腸病毒活 • 性之影響 大本山葡萄丙酮萃取物中含許多種類之活性成分，而這 些成分可能會因不同的pH環境而造成部分結構修飾，以致 影響其活性。因此於本實施例中首先測試pH值對宿主細胞 之影響；並選擇適合宿主細胞存活之pH環境（pH 6〜8)， 23 1374749 2012年9月11曰修正替換頁 進一步將大本山葡萄丙酮萃取物置此等pH環境中，測定萃 取物抑制腸病毒（EV71 ATCC VR-784及 CVB3 KMUH 92225)之活性，藉以了解大本山葡萄丙酮萃取物抑制腸病毒 之活性，是否會受到環境pH值之影響。 4-1. pH對細胞存活率之影響 * 96孔盤中每孔加入O.lml RD細胞懸浮液 （3x 105 cell/ml)，於5%二氧化碳之環境中37°C培養24小時。吸除 培養基’以PBS沖洗兩次，分別加入0.2 ml以HC1或NaOH 調整為 pH 2、3、4、5、6、7、8 之 2% FBS 之 DMEM，細 胞控制組則加入未調整pH值之2%FBS之DMEM，於5%二 氧化碳之環境中37°C培養3天。吸除培養基，以PBS沖洗， 加入20 μΐ漠度為0.5 mg/ml之MTT，靜置2小時，吸除多 餘MTT，加入1 5 0 μΐ二甲亞.礙（dimethylsulfoxide)溶解甲月 朁結晶，以 ELIS A 讀取儀（SLT Spectra 400 ATX, Salzburg， Austria)，於波長550 nm下測定吸收值，計算細胞存活率 (〇/〇)，存活率（％) = (〇DT/ODc) X 100% ;其中 ODT 及 〇Dc 分別為不同pH值之實驗組及控制組之5 50 nm吸收值。計 算出pH對RD細胞之存活率。 結果 • 首先測試感染病毒之細胞（RD及Vero)於pH 2-8環境 - 中之存活率，結果如表六所示。不論RD或Vero細胞，環 境之pH越低細胞存活率越低（p&lt;0.05)，當pH值小於4以下 (包含pH 4)，兩者細胞之存活率皆在20%以下；於pH 5環 境下，存活率為 34·32±0_71% (RD)及 42.16±3.97% (Vero); 於pH 6環境下，則細胞存活率提高至60%以上[62.41 ±1.89% 24 1374749 2012年9月11日修正替換頁 (RD)及 77·93±6·67% (Vero)];當於 pH 7 至 8 環境時，細胞 存活率更提高至91%以上，RD及Vero之存活率分別為91.48 ±3.31 至 114.80±5.42〇/〇及 103.37±3.84〇/〇至 1 08.38±8·95〇/〇。 表六 RD及Vero細胞於不同環境pH之存活率Infection time point inhibition rate (%) a 31.25 pg/ml 50 pg/ml 62.5 pg/ml 100 μ^ηύ -2h a 1.81 ± 0.13 D b 1.63 ± 0_15G a 2.23 ± 0.20 EF a 1.99 ± 0.18 F -1 ha 1.33 ±0.15 D a 1.40 ± 0.10 G a 1.63 ± 0.15 F a 1.37 ± 0.12 F Oh a 23.06 ± 3.66 B a 25.73 ± 1.16 E a 24.82 ± 1.08 D a 25.54 ± 1.50 D 1 hc 33. 卯 ± 4.36 A b 81.56 ± 4.01 A a 92.79 ±5.62 A a 100.00 ±0.00 A 2h d 33.62 ±3.45 A c73_92 ± 5.30B b 91.74 ±1.96 A a 100.00 ±0.00 A 4h d31.81± 1.29A c 71.78 ±2.86 BC b 91.19 ± 4.80 AB a 100.00 ±0.00 A 8h c 24.84 ± 2.99 B b 66.54 ± 4.66 C a 85.85 ± 4.39 B a93'07±3.46B 12 hd 15.41 ±2.16 C c 46.34 ± 4.87 D b 65.39 ± 4.84 C a 78.68 ± 4.86 C 16 hc 3.84 ± 0.36 D b 18.58 ± 1.63 F a 24.51 ± 3.51 D a 27.82 ± 0.54 D 20 hb 1.88 ± 1.81 D b 1.33 ± 2.08 G a 7.60 ± 2.26 E a 6.90 ± 3.56 E 24 ha 0.66 ±1.52 D b 0.03 ± 2.08 G a 1.97 ± 0.80 F a 3.10 ± 0.89 F The average value is obtained by three times 2 real $ each time with triple 1 and expressed as mean ± standard deviation. There is a significant difference between the barriers (4). °5). The average value in each column was accompanied by different lowercase letters to the extract 1 hour before the virus infection (see ih and 2 h), and the inhibition rate of each concentration (31.25 to 1〇〇08/!111) was quite low ( 2% or less), there is no difference in the inhibition rate between the concentrations (4). 〇 5). When the extract and virus solution were simultaneously added to the cells (see 〇h), the inhibition rate of 31.25 to 100 _ml was 23_06±3.66 to 25_73 ± 1.16%. There was no significant difference between the concentrations (Ρ&lt;〇·〇5广This result shows that the extract directly kills part of the virus (2)%) or prevents the virus from adhering to the cells. After adding the extract to the virus-infected cells for 4 hours (see ih to 4 h), there was no significant difference in inhibition rate (corporate &lt; 0.05). The higher the dose, the greater the inhibition rate. (4) The state, at a 5 (four) (6) dose The inhibition rate is over 90%, and the negotiation (five) is up to ι〇〇%. Add the extract after 8 to 16 hours of staining the cells (see 8h, 12h and 16^22 1374749 _ September 11, 2012, the revised replacement page increases the inhibition rate rapidly over time 〇 &lt;〇.〇5), each concentration The inhibition rate was between 24.84±2.99 and 93.07±3.46% at 8h, and decreased from 3.84±0.3 6 to 27.82±0.54% at 16 11; the higher the dose, the greater the inhibition rate, 62.5 and 100 pg There is no difference between /ml (0.05). After 20 hours of virus-infected cells, the extract was added (see 20 h and 24 h) and the inhibition rate dropped to zero. As a result of the comprehensive table V, the mechanism of action of the acetone extract of Dabenshan grape on CVB3 KMUH 92225 includes directly killing part of the virus or preventing the adhesion of the virus to the cells and inhibiting the replication of the virus. As the infection time increases, the inhibition rate decreases. The higher the dose, the greater the inhibition rate. The mode of action is dose-dependent. When the dose is higher than 62.5 pg/ml, the inhibition rate does not increase (ρ&lt;0.05). The results of Tables 4 and 5 show that the acetone extract of Dabenshan grape has a high inhibitory effect on EV71 ATCC VR-784 and CV3 KMUH 92225, but the inhibition mechanism of the two is not completely the same. EV71 ATCC VR-784 has the effect of preventing viral infection and inhibiting virus adsorption and early replication; for CV3 KMUH 92225 is mainly to inhibit viral replication. The acetone extract of Dabenshan grape has a dose-dependent effect on this enterovirus strain. The higher the dose, the greater the inhibition rate, and the maximum inhibition rate is 62.5 pg/ml. * Example 4 Effect of pH on the inhibition of enterovirus activity by the acetone extract of Dabenshan grape. The acetone extract of Dabenshan grape contains many kinds of active ingredients, and these ingredients may cause partial structural modification due to different pH environments. So that it affects its activity. Therefore, in this embodiment, the effect of pH on the host cell is first tested; and the pH environment suitable for the survival of the host cell (pH 6 to 8) is selected, 23 1374749 September 11th, 2012 revised replacement page to further extract the acetone of Dabenshan grape The activity of the extract against the enterovirus (EV71 ATCC VR-784 and CVB3 KMUH 92225) was determined in such a pH environment to understand whether the acetone extract of Dabenshan grape inhibits the activity of enterovirus and is affected by the environmental pH. 4-1. Effect of pH on cell viability * 0.1 ml of RD cell suspension (3 x 105 cells/ml) was added to each well of a 96-well plate, and cultured at 37 ° C for 24 hours in a 5% carbon dioxide atmosphere. The aspirate medium was washed twice with PBS, and 0.2 ml of DMEM adjusted to pH 2, 3, 4, 5, 6, 7, 8 of 2% FBS with HC1 or NaOH was added, and the cell control group was added with unadjusted pH. The 2% FBS DMEM was cultured for 3 days at 37 ° C in a 5% carbon dioxide atmosphere. Absorb the medium, rinse with PBS, add 20 μM MTT with a gradient of 0.5 mg/ml, let stand for 2 hours, remove excess MTT, and add 150 μM dimethylsulfoxide to dissolve the crystal of methotrexate. The absorbance was measured at an wavelength of 550 nm using an ELIS A reader (SLT Spectra 400 ATX, Salzburg, Austria) to calculate the cell viability (〇/〇), and the survival rate (%) = (〇DT/ODc) X 100 %; where ODT and 〇Dc are the 50 50 nm absorption values of the experimental and control groups of different pH values, respectively. The survival rate of pH to RD cells was calculated. Results • The survival rate of virus-infected cells (RD and Vero) in pH 2-8 environment was first tested. The results are shown in Table 6. Regardless of RD or Vero cells, the lower the pH of the environment, the lower the cell viability (p<0.05). When the pH is less than 4 (including pH 4), the viability of both cells is below 20%; in the environment of pH 5 The survival rate was 34·32±0_71% (RD) and 42.16±3.97% (Vero); in the pH 6 environment, the cell survival rate increased to over 60% [62.41 ±1.89% 24 1374749 September 11, 2012 The daily correction replacement page (RD) and 77·93±6·67% (Vero)]; when pH 7 to 8 environment, the cell survival rate increased to 91% or more, and the survival rates of RD and Vero were 91.48 ± 3.31 to 114.80±5.42〇/〇 and 103.37±3.84〇/〇 to 1 08.38±8·95〇/〇. Table 6 Survival rates of RD and Vero cells at different environmental pH

PH 存活率(％) RD Vero 2 10.29 ± 0.06 F 16.26 ± 1.83 D 3 11.62±Q.79EF 18.67 ± 1.65 D 4 15.62 ± 2.05 E 19.41 ±3.61 D 5 34.32 ± 0.71 D 42.16 ± 3.97 C 6 62.41 ± 1.89 C 77.93 ± 6.67 B 7 91.48 ±3.31 B 103.37 ±3.84 A 8 114.80 ±5.42 A 108.38 ±8.95 A 3每組數據由三次不同實驗每次以三重複進行所得的平均值，以平均值 士標準偏差表示。各糊内平均值伴隨不同大寫字母者之間具有顯著差異 (p &lt;0.05) ° 4-2. pH對大本山葡萄丙酮萃取物抑制腸病毒活性之影 響 由於過低之細胞存活率會影響實驗結果之準確度及精 密度（再現性），因此pH對大本山葡萄丙酮萃取物抑制腸病 毒活性影響之試驗選擇於pH 6至8之環境下進行。 6孔盤之每孔加入2 ml細胞懸浮液（3xl05 cell/ml)，於 5%二氧化碳環境中37°C培養24小時。吸除培養基，以PBS 沖洗2次，加入0.5 ml含有100 PFU病毒之不同濃度萃取 物為實驗組，以僅加入病毒液者為病毒控制組，於4°C環境 中感染1小時（每1 5分鐘搖晃一次），吸除多餘病毒液，加 入以HC1或NaOH調整為pH 6、7、8之覆蓋培養基（含2% FBS及0.26%瓊脂糖之DMEM)，於5%二氧化碳之環境中 3 7°C培養3天。吸除覆蓋培養基，加入2 ml之10%福馬林， 固定細胞30分鐘，吸除福馬林，加入1%結晶紫染色細胞5 25 1374749 2012年9月11日修正替換頁 分鐘，以自來水沖洗多餘結晶紫，計數病毒斑數目，結果 以抑制率表示。 、结果 結果如表七所示，對於EV71 ATCC VR-784及CVB3 KMUH 92225兩者皆測試兩種不同濃度。 表七pH對大本山葡萄丙酮萃取物抗腸病毒活性之影響PH Survival Rate (%) RD Vero 2 10.29 ± 0.06 F 16.26 ± 1.83 D 3 11.62±Q.79EF 18.67 ± 1.65 D 4 15.62 ± 2.05 E 19.41 ±3.61 D 5 34.32 ± 0.71 D 42.16 ± 3.97 C 6 62.41 ± 1.89 C 77.93 ± 6.67 B 7 91.48 ±3.31 B 103.37 ±3.84 A 8 114.80 ±5.42 A 108.38 ±8.95 A 3 The average of each set of data from three different experiments in each of three replicates, expressed as the mean ± standard deviation. The average value of each paste was significantly different between different uppercase letters (p &lt; 0.05) ° 4-2. The effect of pH on the inhibition of enterovirus activity by Dabenshan grape acetone extract due to too low cell survival rate will affect the experiment The accuracy and precision (reproducibility) of the results, so the effect of pH on the inhibition of enterovirus activity by the acetone extract of Dabenshan grape was selected in the environment of pH 6 to 8. 2 ml of the cell suspension (3 x 105 cells/ml) was added to each well of a 6-well plate, and cultured at 37 ° C for 24 hours in a 5% carbon dioxide atmosphere. The medium was aspirated and washed twice with PBS. 0.5 ml of different concentrations of extract containing 100 PFU of virus were added to the experimental group, and only virus-incorporated group was added to the virus control group, and infection was carried out for 1 hour at 4 ° C (every 15). Shake once a minute), remove excess virus solution, and add a cover medium (2% FBS and 0.26% agarose DMEM) adjusted to pH 6, 7, 8 with HC1 or NaOH, in a 5% carbon dioxide environment. Incubate for 3 days at °C. Aspirate the culture medium, add 2 ml of 10% fumarin, fix the cells for 30 minutes, aspirate the formalin, add 1% crystal violet stained cells 5 25 1374749 September 11, 2012 Correction replacement page minutes, rinse excess crystals with tap water Violet, the number of plaques was counted and the results were expressed as inhibition rates. Results The results are shown in Table 7. Two different concentrations were tested for both EV71 ATCC VR-784 and CVB3 KMUH 92225. Effect of pH on the anti-enteric activity of acetone extract of Dabenshan grape

PH 抑制率（％) EV71 ATCC VR-784 CVB3 KMUH 92225 40 μ^πιΐ 100 pg/ml 50 μξ/πύ 62.5 pg/ml 6 33.27 ± 2.62 Ba 46.98 ± 4.41 B 76.74 ±8.84 A 90.25 ±4.60 A 7 59.37 士 1.47 A 70.85 ±0.79 A 77.22 ±4.24 A 87.93 ±5.97 A 8 56.36 ± 3.23 A 68.33 ±0.99 A 72.28 ±3.94 A 80.78 ±5.99 A 3每組數據由三次不同實驗每次以三重複進行所得的平均值，以平均值±標準偏差表示。 各欄内平均值伴隨不同大寫字母者之間具有顯著差異(p&lt;0.05)。。 對於EV71 ATCC VR-784之結果顯示，大本山葡萄丙酮 萃取物於pH 6環境下之抑制率[33·27±2.62% (40 pg/ml); 46·98±4·41°/〇 (100 pg/ml)]低於 pH 7 環境下[59.3 7±1_47% (40 4呂/!111);70.85±0.79%(10〇4§/1111)]及？118環境下[56.36± 3.23% (40 pg/ml) ; 68.33±0·99% (100 pg/ml)]，於 pH 7 及 8 環境下之抑制率無顯著性差異〇&lt;〇.05)。 對於CVB3 KMUH 92225之結果顯示，於pH 6至8間 之抑制率無顯著性差異（^&lt;0.05)，50 pg/ml及62.5 pg/ml濃 度之抑制率分別為72.28±3.94%至77.22±4.24%以及80.78± 5.99%至 90.25±4.60〇/〇。 綜上所述，大本山葡萄丙酮萃取物抗腸病毒（EV71 ATCC VR-784 及 CVB3 KMUH 92225)之活性，於 pH 7 至 8 之環境具有高度之抑制作用，不受環境pH值影響，於pH 6 26 1374749 2012年9月11日修正替換頁 環境中，萃取物掛 EV71 ATCC VR_784 低。 實施例5 大本山葡萄丙酮萃取物對pH之穩定性 本實施例係將大本山葡萄丙酮萃取物於不同pH環境 中處理段時間，測定pH處理對萃取物之穩定性，係以下 述方法進行。 大本山葡萄丙酮萃取物以DMS〇溶解並配製成為5〇〇 mg/mi 之儲存溶液（st〇ck siluti〇n)，再分別以 pH i、2、8 ark &amp; Lubs buffer 及 pH 3-7 之]Vicllvaine buffer 配製成 為50 mg/ml之濃度，於室溫靜置2小時，以培養基稀釋成 為5 mg/ml，備用。 於6孔盤中每孔加入2 ml細胞懸浮液（3χΐ〇5 ceii/mi)， 於5%二氧化碳環境中3rc培養24小時。吸除培聲基，以 PBS沖洗2次，加入〇.5 ml含有1〇〇 pFU病毒之經不同pH 處理之不同濃度萃取物為實驗組，以僅加入病毒液者為病 毒控制組；於4。〇環境中感染1小時（每15分鐘搖晃一次）， 吸除多餘病毒液，加入覆蓋培養基（含2% FBS及〇 26%瓊脂 糖之DMEM) ’於5〇/。二氧化碳之環境中37t：培養3天。吸 除覆蓋培養基，加入2 ml之10%福馬林，固定細胞3〇分鐘， 吸除福馬林，加入1 °/。結晶紫染色細胞5分鐘，以自來水沖 洗多餘結晶紫’計數病毒斑數目，結果以抑制率表示。 以上實驗每次做三重複，重複做三次，實驗結果以平 均值土標準偏差（SD)表示’並以司徒頓t檢定（Student，s t test) (p &lt; 0.0 5 )做統計分析。 結果 27 1374749 2012年9月11日修正替換頁 大本山葡萄為可食用之原料（行政院衛生署食品衛生處， 2005)，進入人體後會在不同pH環境中停留一段時間，因而 影響其活性’因此將大本山葡萄丙酮萃取物置於pH 2至8 緩衝溶液中處理2小時，測定其抑制腸病毒（EV71 ATCC \^-784及（11\^31(^111^ 92225)之活性，結果如表八所示。 表八大本山葡萄丙酮萃取物對pH之穩定性PH inhibition rate (%) EV71 ATCC VR-784 CVB3 KMUH 92225 40 μ^πιΐ 100 pg/ml 50 μξ/πύ 62.5 pg/ml 6 33.27 ± 2.62 Ba 46.98 ± 4.41 B 76.74 ±8.84 A 90.25 ±4.60 A 7 59.37 1.47 A 70.85 ±0.79 A 77.22 ±4.24 A 87.93 ±5.97 A 8 56.36 ± 3.23 A 68.33 ±0.99 A 72.28 ±3.94 A 80.78 ±5.99 A 3 The average of each set of data from three different experiments in each of three replicates, Expressed as mean ± standard deviation. The mean value in each column was significantly different between the different capital letters (p &lt; 0.05). . The results of EV71 ATCC VR-784 showed that the inhibition rate of Dabenshan grape acetone extract in pH 6 environment [33·27±2.62% (40 pg/ml); 46·98±4·41°/〇 (100 Pg/ml)] below pH 7 [59.3 7±1_47% (40 4 Lu/!111); 70.85±0.79% (10〇4§/1111)] and? Under the environment of 118 [56.36±3.23% (40 pg/ml); 68.33±0·99% (100 pg/ml)], there was no significant difference in the inhibition rate under pH 7 and 8 〇&lt;〇.05) . As for the results of CVB3 KMUH 92225, there was no significant difference in the inhibition rate between pH 6 and 8 (^ &lt; 0.05), and the inhibition rates of 50 pg/ml and 62.5 pg/ml were 72.28 ± 3.94% to 77.22 ±, respectively. 4.24% and 80.78 ± 5.99% to 90.25 ± 4.60 〇 / 〇. In summary, the activity of the acetone extract of Dabenshan grape against EV71 (EV71 ATCC VR-784 and CVB3 KMUH 92225) has a high inhibitory effect in the environment of pH 7 to 8, independent of the pH value of the environment, at pH 6 26 1374749 September 11, 2012 Revised replacement page environment, extract EV71 ATCC VR_784 low. Example 5 Stability of Acetone Extract of Dabenshan Grape against pH In this example, the acetone extract of Dabenshan grape was treated in different pH environments for a period of time, and the stability of the pH treatment to the extract was measured by the following method. The acetone extract of Dabenshan grape was dissolved in DMS and prepared into a 5 〇〇mg/mi storage solution (st〇ck siluti〇n), and then pH i, 2, 8 ark &amp; Lubs buffer and pH 3-7, respectively. The Vicllvaine buffer was prepared at a concentration of 50 mg/ml, allowed to stand at room temperature for 2 hours, diluted to 5 mg/ml in the medium, and used. 2 ml of cell suspension (3χΐ〇5 ceii/mi) was added to each well of a 6-well plate, and cultured in 3 rc for 24 hours in a 5% carbon dioxide atmosphere. Aspirate the vocal base, rinse twice with PBS, add 55 ml of different concentrations of extracts containing 1 〇〇pFU virus at different pH treatments to the experimental group, and add only the virus solution to the virus control group; . Infect the environment for 1 hour (shake every 15 minutes), remove the excess virus solution, and add the cover medium (DMEM containing 2% FBS and 〇 26% agarose) at 5〇/. 37t in the environment of carbon dioxide: cultured for 3 days. The cover medium was aspirated, 2 ml of 10% fumarin was added, the cells were fixed for 3 minutes, and the formalin was aspirated and added at 1 °/. The cells were stained with crystal violet for 5 minutes, and the number of virions was counted by washing excess crystal violet with tap water, and the results were expressed as inhibition rates. Each of the above experiments was repeated three times and repeated three times. The results were expressed as mean soil standard deviation (SD) and statistical analysis was performed using Student's t test (p &lt; 0.0 5 ). Result 27 1374749 Modified on September 11, 2012, the Dabenshan grape is an edible raw material (Food Sanitation Division of the Executive Yuan Department of Health, 2005). After entering the human body, it will stay in different pH environments for a period of time, thus affecting its activity' Therefore, the acetone extract of Dabenshan grape was treated in pH 2 to 8 buffer solution for 2 hours, and its activity against EV71 ATCC \^-784 and (11\^31(^111^92225) was determined. Table 8. Table 8 Stability of Acetone Extract of Dabenshan Grape to pH

pH 抑制率（％) EV71 ATCC VR-784 CVB3 KMUH 92225 40 pg/ml 100 pg/ml 50 pg/ml 62.5 pg/ml 2 45.45 ± 3.09 Ca 50.47 ± 3.22 C 44.46 ±5.81 Ca 55.22 ± 1.15 D 3 45.53 ± 3.01 C 53.42 ± 6.34 C 64.84 ± 3.19 B 67.25 ± 5.44 C 4 47.44 ± 1.64 BC 62.84 ± 2.50 B 65.55 ± 1.11 B 67.80 ± 5.55 C 5 51.60 ± 3.58 BC 62.96 ± 3.78 B 71.32 ± 0.45 B 76.80 ± 2.05 B 6 52.55 ± 3.85 B 68.74 ± 2.55 B 83.38 ±4.60 A 90.13 ±6.92 A 7 60.80 ±7.43 A 76.80 ±3.33 A 85.65 ±5.02 A 97.98 ±1.16 A 8 Ά An f 64.72 ±5.10 A 77.08 ±4.12 A 82.69 ±2.20 A 90.36 ±6.51 A 8每組數據由三次不同實驗每次以三重複進行所得的平均值，以平均值±標準偏差表示。 各欄内平均值伴隨不同大寫字母者之間具有顯著差異(p &lt;〇.〇5)。 不論對 EV71 ATCC VR-784 或 CVB3 KMUH 92225，大 本山葡萄丙酮萃取物之抑制率均隨處理之pH下降而降低 (ρ&lt;0·05)。對EV71 ATCC VR_784之結果顯示出，大本山葡 萄丙酮萃取物經pH 7至8處理後之抑制率[60.80±7.43 %至 64.72±5.10% (40 gg/ml) ; 76.80±3.33%至 77.08±4.12 % 〇00 pg/ml)]不受影響，與未經pH處理之萃取物（1 h) [66.44土 3.13% (40 pg/ml)及 78.21±0.40% (1〇〇 pg/ml)]相似（參見表 四）。當萃取物經pH 4至6處理後，其抑制率明顯下降 (/?&lt;0.05) ’ 40 及 1〇〇 pg/ml 之劑量分別由 pH 7 至 8 之 64.72 ±5.10%下降至 47.44±1.64〇/〇及 77.08±4.12%下降至 62.84± 28 1374749 2012年9月11日修正替換頁 2_5〇%，平均約降低15%。萃取物經PH2至3處理後抑^- 更下降至 45·45 ± 3.90% (40 pg/ml)及 50.47 ± 3.22% (1〇〇 gg/ml) (ρ&lt;0·05) ’與pH 7至8比較’平均約降低25%。大本 山葡萄丙酮萃取物經pH 2至8處理後，對CBB3 KMUH 92225之結果顯示出，經pH 6至8處理之抑制率間[82 69± 2.20 至 85.65±5.02% (50 pg/ml) ; 90·13±6.92 至 97.98±1.16〇/〇 (62.5 gg/ml)]無顯著性差異&amp;&lt;〇 〇5)，與未經pH處理之萃 取物（1 h) [81.56±4.01% (50 pg/ml)及 92.79±5.62% (62 5 μ8/πι1)]相似（參見表五）；當處理之pH降低，抑制率隨之下 降，濃度越低下降幅度越大(；?&lt;〇.〇5)，經pH 2至5處理，pH inhibition rate (%) EV71 ATCC VR-784 CVB3 KMUH 92225 40 pg/ml 100 pg/ml 50 pg/ml 62.5 pg/ml 2 45.45 ± 3.09 Ca 50.47 ± 3.22 C 44.46 ±5.81 Ca 55.22 ± 1.15 D 3 45.53 ± 3.01 C 53.42 ± 6.34 C 64.84 ± 3.19 B 67.25 ± 5.44 C 4 47.44 ± 1.64 BC 62.84 ± 2.50 B 65.55 ± 1.11 B 67.80 ± 5.55 C 5 51.60 ± 3.58 BC 62.96 ± 3.78 B 71.32 ± 0.45 B 76.80 ± 2.05 B 6 52.55 ± 3.85 B 68.74 ± 2.55 B 83.38 ± 4.60 A 90.13 ±6.92 A 7 60.80 ±7.43 A 76.80 ±3.33 A 85.65 ±5.02 A 97.98 ±1.16 A 8 Ά An f 64.72 ±5.10 A 77.08 ±4.12 A 82.69 ±2.20 A 90.36 ± 6.51 A8 Each set of data is averaged from three replicates each time in triplicate, expressed as mean ± standard deviation. The average value in each column is significantly different from that of different capital letters (p &lt; 〇.〇5). Regardless of the EV71 ATCC VR-784 or CVB3 KMUH 92225, the inhibition rate of Dabenshan grape acetone extract decreased with the pH of the treatment (ρ&lt;0·05). The results of EV71 ATCC VR_784 showed that the inhibition rate of Dabenshan grape acetone extract after treatment with pH 7 to 8 [60.80±7.43% to 64.72±5.10% (40 gg/ml); 76.80±3.33% to 77.08±4.12 % 〇00 pg/ml)] is not affected, similar to the extract without pH treatment (1 h) [66.44 soil 3.13% (40 pg/ml) and 78.21±0.40% (1〇〇pg/ml)] (See Table 4). When the extract was treated with pH 4 to 6, the inhibition rate decreased significantly (/?&lt;0.05). The doses of '40 and 1〇〇pg/ml decreased from 64.72 ± 5.10% of pH 7 to 8 to 47.44 ± 1.64, respectively. 〇/〇 and 77.08±4.12% dropped to 62.84±28 1374749 On September 11, 2012, the revised replacement page was 2_5〇%, with an average reduction of about 15%. After the extract was treated with PH2 to 3, it decreased to 45.45 ± 3.90% (40 pg/ml) and 50.47 ± 3.22% (1〇〇gg/ml) (ρ&lt;0·05) ' with pH 7 To 8 comparisons, the average is about 25% lower. After the acetone extract of Dabenshan grape was treated with pH 2 to 8, the results of CBB3 KMUH 92225 showed that the inhibition rate was between pH 6 and 8 [82 69 ± 2.20 to 85.65 ± 5.02% (50 pg / ml); 90·13±6.92 to 97.98±1.16〇/〇(62.5 gg/ml)] no significant difference &&lt;〇〇5), with pH-free extract (1 h) [81.56±4.01% ( 50 pg/ml) and 92.79±5.62% (62 5 μ8/πι1)] are similar (see Table 5); when the pH of the treatment is lowered, the inhibition rate decreases, and the lower the concentration, the greater the decrease ((??&lt;〇 .〇5), treated with pH 2 to 5,

Kg/ml劑量之抑制率由71.32±0.45% (pH 5)下降至44.46士 5.81% (pH 2)，及 62.5 pg/ml 劑量之抑制率由 76 8〇±2 〇5% (PH 5)下降至55.22±1.15% (pH 2)。綜合表八之結果大本 山葡萄丙酮萃取物以pH 7至8處理，不影響其抗腸病毒之 活性，當處理之pH下降（pH 2至6)其活性隨之降低。 實施例6以不同萃取方法所萃取之大本山葡萄萃取 物對抑制腸病毒活性之影響 在本實施例中，進一步分析利用不同萃取條件（包括萃 取方法、萃取溶劑、萃取溫度）所萃取之大本山葡萄萃取物 於抗腸病毒活性之差異，藉以評估由大本山葡萄中萃取出 有效分之最適萃取條件。 、索氏及超音波等不同萃取 ，進行大本山葡萄萃取物抗 本試驗以磁石攪拌、震盪 方法，以95%乙醇為萃取溶劑 腸病毒活性之評估。 (1)磁石攪拌萃取 29 丄：W4/49 2012年9月11日修正替換頁 30克大本々葡萄粉末（預先過筛收 師網之粉末）加入95%乙醆7nn 1 ^ Sft 乙％ 200 m卜於室溫磁石攪拌i 時，於4°C以12300 e離、、,c八拉 g離心15分鐘，收集上清液，殘渣 W溶劑錢萃取2次，合併上清液，於帆減壓濃缩 祥重，計算固形物收率。固形物收率（％»為濃縮 物乾重與大本山葡萄粉末重量比值之百分率。 (2)震盪萃取 3〇克大本山葡萄粉束（60 mesh過篩）置於5〇〇 mi三角 瓶中，加入95%乙醇2〇〇爪卜於室溫1〇〇 rpm震盪i小時， ;4 C以12300 g離心15分鐘，收集上清液，殘渣以如 溶劑重複萃取2次，合併上清液，於贼減壓濃縮至乾， 狎重。计异固形物收率。 (3)索氏萃取 v 5克大本山葡萄粉末（預先過篩，收集通過60 mesh筛 網之粉末）放置於索氏迴流萃取器之圓筒濾紙中，受器中加 入95。/。乙醇2〇〇 mi，於95恆溫水槽迴流萃取約I〗小時， 收集全部萃取液，於40°C減壓濃縮至乾，秤重。計算固形 物收率。 (4)超音波萃取 15克大本山葡萄粉末（60 mesh過篩）置於500 ml三角瓶 中’加入95%乙醇2〇〇 ml，於室溫進行超音波萃取1小時， 於4 C以123 00 g離心w分鐘，收集上清液，殘渣以200 ml 溶劑重複萃取2次，合併上清液，於4(TC減壓濃縮至乾， 枰重。計算固形物收率。 30 1374749 &quot; 2012年9月11日修正替換頁 大本山葡萄以攪拌、震盪、索氏及超音波等不同方法所 得之萃取物，對 EV71 ATCC VR-784 及 CVB3 KMUH 92225 之IC5D結果不於表九。 表九以病毒斑減少分析法測定大本山葡萄不同方法萃取物對EV71 ATCC ^_784及 CVB3 KMUH 92225 之 50%抑制濃度 萃取方法 固形物收率 IC50a (pg/ml) (%，w/w) EV71 VR784 CVB3 KMUH 92225 攪拌(stirring) 7.03 66.40 ± 2.65 A 57.69 ±1.80 A 震盪(shaking) 8.13 64.02 ± 1.31 A 60.05 ±2.87 A 索氏(Soxhlet) 8.89 120.61 ± 2.69 C 84.61 ± 0.85 B 超音波(ultrasonic) 8.03 75.36 土 0.62 B 56·85±3_00Α 每组數據由三次不同實驗每次以三重複進行所得的平均值，以平均值土標準偏差表示。各搁内 平均值伴隨不同大寫字母者之間具有顯著差異&lt;〇.〇5)。 對EV71 ATCC VR-784之IC50結果顯示出，4種萃取方 法所得之萃取物中，以攪拌及震盪萃取有最低之IC5()(分別 為66.40±2.+ 65及64.0.2±1.31 pg/ml)，其次為超音波萃取 (75.36±0.62 pg/ml)，再次為索氏萃取（120.61±2.69 pg/ml) (ρ&lt;0·05)。對 CVB3 KMUH 92225 之 IC50 結果顯示出，搜拌、 震盪及超音波萃取物均有相同之抗CVB3 KMUH 92225活性 (IC50 : 56.86±3.00 pg/ml 至 60·05±2·87 pg/ml)，索氏萃取物 之 IC5〇 則高於前三者（84.61±0.85 pg/ml) 〇?&lt;0.05)。 综合表九之結果，大本山葡萄以室溫之攪拌及震盪方法 所得之萃取物，有最佳之抗EV71ATCC VR-784及CVB3 KMUH 92225 活性。 實施例7 以不同溫度所萃取之大本山葡萄萃取物對 抑制腸病毒活性之影響 本試驗係於4°C、25°C、60°C及80°C等不同溫度，以 95%乙醇為萃取溶劑，以前述之震盪方法萃取，並分別以如 前所述之方法試驗萃取物之抗腸病毒活性。 31 1374749 , 2012年9月11日修正替換頁 結果 大本山葡萄不同溫度萃取物對EV71 ATCC VR-784及 CVB3 KMUH 92225之IC5()結果示於表十。 • 表十以病毒斑減少分析法測定大本山葡萄不同溫度萃取物對EV71 ATCCVR-784及 CVB3 KMUH 92225 之 50%抑制濃度The inhibition rate of Kg/ml dose decreased from 71.32±0.45% (pH 5) to 44.46±5.81% (pH 2), and the inhibition rate of 62.5 pg/ml dose decreased from 76 8〇±2 〇5% (PH 5). To 55.22 ± 1.15% (pH 2). Summary of the results of Table 8 Dabenshan grape acetone extract treated with pH 7 to 8 does not affect its anti-intestinal activity, and its activity decreases as the pH of the treatment decreases (pH 2 to 6). Example 6 Effect of Dabenshan Grape Extract Extracted by Different Extraction Methods on Inhibition of Enterovirus Activity In this example, the large extracts extracted by different extraction conditions (including extraction method, extraction solvent, extraction temperature) were further analyzed. The difference in the activity of the mountain grape extract against the enterovirus, in order to evaluate the optimum extraction conditions for extracting the effective fraction from the Dabenshan grape. Different extractions such as Soxhlet and Ultrasonic were carried out to evaluate the activity of Enterovirus in Dabenshan grape extract with magnetic stirring and shaking method and 95% ethanol as extraction solvent. (1) Magnet Stirring Extraction 29 丄: W4/49 September 11, 2012 Revision Replacement Page 30 g of Big Benji Grape Powder (pre-sifted into the powder of the net) Add 95% acetamidine 7nn 1 ^ Sft B% 200 When stirring at room temperature, the magnet is stirred at 12300 °C at 12 ° C for 15 minutes at 4 ° C. The supernatant is collected, and the residue is extracted twice with solvent. The supernatant is combined and reduced. Concentrate the weight and calculate the yield of solids. Solids yield (%» is the ratio of the dry weight of the concentrate to the weight of the Dabenshan grape powder. (2) Oscillation extraction 3 gram of Dabenshan grape powder bundle (60 mesh sieved) placed in a 5 〇〇mi triangle bottle Add 95% ethanol 2 〇〇 claws and shake at room temperature 1 rpm for 1 hour; 4 C centrifuge at 12300 g for 15 minutes, collect the supernatant, and repeat the extraction twice with solvent, and combine the supernatant. The thief is concentrated to dryness under reduced pressure, and the weight is measured. (3) Soxhlet extraction v 5 g of Dabenshan grape powder (pre-sifted, collected through a 60 mesh sieve powder) placed in Soxhlet reflux In the cylindrical filter paper of the extractor, 95% ethanol was added to the receiver, and the mixture was refluxed in a constant temperature water bath for about 1 hour. The whole extract was collected and concentrated to dryness under reduced pressure at 40 ° C. Calculate the yield of solids. (4) Ultrasonic extraction 15 g of Dabenshan grape powder (60 mesh sieved) placed in a 500 ml flask. Add 95% ethanol 2〇〇ml and perform ultrasonic extraction at room temperature. Hour, centrifuge at 123 00 g for 4 minutes at 4 C, collect the supernatant, and repeat the extraction twice with 200 ml of solvent. Combine the supernatant and concentrate at 4 (TC under reduced pressure to dryness, 枰 weight. Calculate the yield of solids. 30 1374749 &quot; Modified on September 11, 2012 to replace the big Benshan grapes for mixing, shaking, Soxhlet and ultrasonic The IC5D results of the EV71 ATCC VR-784 and CVB3 KMUH 92225 are not shown in Table 9. Table 9 shows the different methods of extracting the Dabenshan grape by plaque reduction analysis for EV71 ATCC ^_784 and CVB3 KMUH 92225 50% inhibitory concentration extraction method solids yield IC50a (pg/ml) (%, w/w) EV71 VR784 CVB3 KMUH 92225 stirring (sirring) 7.03 66.40 ± 2.65 A 57.69 ±1.80 A shaking (shaking) 8.13 64.02 ± 1.31 A 60.05 ±2.87 A Soxhlet 8.89 120.61 ± 2.69 C 84.61 ± 0.85 B Ultrasonic 8.03 75.36 Soil 0.62 B 56·85±3_00Α Each set of data is obtained from three different experiments each time with three replicates. The average value is expressed as the mean soil standard deviation. There is a significant difference between the averages in each case with different capital letters &lt;〇.〇5). The IC50 results for EV71 ATCC VR-784 show that 4 extraction methods Income In the extract, the lowest IC5() was extracted by stirring and shaking (66.40±2.+65 and 64.0.2±1.31 pg/ml, respectively), followed by ultrasonic extraction (75.36±0.62 pg/ml), again Soxhlet extraction (120.61 ± 2.69 pg / ml) (ρ &lt; 0. 05). The IC50 results for CVB3 KMUH 92225 showed that the same anti-CVB3 KMUH 92225 activity (IC50: 56.86 ± 3.00 pg/ml to 60·05 ± 2.87 pg/ml) was obtained for the mix, turbulence and ultrasonic extracts. The IC5 索 of Soxhlet extract was higher than the first three (84.61±0.85 pg/ml) 〇?&lt;0.05). Based on the results of Table 9, the extracts of Dabenshan grapes with room temperature stirring and shaking method have the best anti-EV71ATCC VR-784 and CVB3 KMUH 92225 activities. Example 7 Effect of Dabenshan Grape Extract Extracted at Different Temperatures on Inhibition of Enterovirus Activity The test was carried out at 95 °C with different temperatures at 4 °C, 25 °C, 60 °C and 80 °C. The solvent was extracted by the aforementioned oscillating method, and the anti-enteric activity of the extract was tested by the method as described above. 31 1374749 , Revised replacement page on September 11, 2012 Results The IC5() results for different temperature extracts of Dabenshan grapes against EV71 ATCC VR-784 and CVB3 KMUH 92225 are shown in Table 10. • Table 10 uses plaque reduction analysis to determine the 50% inhibitory concentration of different temperature extracts of Dabenshan grapes on EV71 ATCCVR-784 and CVB3 KMUH 92225

溫度 固形物收率 IC50a(pg/ml) (%, w/w) EV71 VR784 CVB3 KMUH 92225 4eC 8.01 81.18 ± 0.95 B 74.99 ± 4.53 B 25〇C 8.13 64.02±1.31 A 60.05 ±2.87 A 60°C 8.36 90.23 士 2.13 C 59.82 士 2.51 A 80°C 9.27 93.41 ±2.55 C 58.06 ±0.69 A a每組數據由三次不同實驗每次以三重複進行所得的平均值，以平均值±標準偏差表示。各攔内 平均值伴隨不同大寫字母者之間具有顯著差異(p&lt;0.05)。 對EV71 ATCC VR-784之IC50結果顯示出，4種不同溫 度所得之萃取物中以25°C萃取物有最低之IC5Q (64.02±1.31 pg/ml)，其次為4°C萃取物（81·18±0·95 pg/ml)，再次為60及 80°C 萃取物（分別為 90.23±2.13 pg/ml 及 93.41±2.55 pg/ml) 〇&lt;0.05)。對 CVB3 KMUH 92225 之 IC50 結果顯示出，25、 60及85°C萃取物有相同之抗CVB3 KMUH-92225活性（IC50: 58.06±0.69. pg/ml 至 60.05±2·87 pg/ml)，其 IC50 均低於 4°C 萃取物（74.99:1:4.53 pg/ml) (;?&lt;0.05)。 綜合表十之結果，大本山葡萄於25 °C萃取所得之萃取 物，有最佳之抗 EV71 ATCC VR-784 及 CVB3 KMUH 92225 活性。 根據本發明可作之不同修正及變化對於熟悉該項技術 者而言均顯然不會偏離本發明的範圍與精神。雖然本發明已 敘述特定的較佳具體事實，必須瞭解的是本發明不應被不當 地限制於該等特定具體事實上。事實上，在實施本發明之已 述模式方面，對於熟習該項技術者而言顯而易知之不同修正 32 1374749 2012年9月11日修正替換頁 亦被涵蓋於下列申請專利範圍之内。 【圖式簡單說明】 (無） . 【主要元件符號說明】 (無） .· 【參考文獻】Temperature solids yield IC50a (pg/ml) (%, w/w) EV71 VR784 CVB3 KMUH 92225 4eC 8.01 81.18 ± 0.95 B 74.99 ± 4.53 B 25〇C 8.13 64.02±1.31 A 60.05 ±2.87 A 60°C 8.36 90.23 ± 2.13 C 59.82 ± 2.51 A 80 ° C 9.27 93.41 ± 2.55 C 58.06 ± 0.69 A a The average of each set of data from three different experiments in each of three replicates, expressed as mean ± standard deviation. The mean values within each block were significantly different between the different capital letters (p &lt; 0.05). The IC50 results for EV71 ATCC VR-784 showed that the extracts obtained at 4 different temperatures had the lowest IC5Q (64.02 ± 1.31 pg/ml) at 25 °C, followed by the 4 °C extract (81· 18±0·95 pg/ml), again 60 and 80 °C extracts (90.23±2.13 pg/ml and 93.41±2.55 pg/ml, respectively) 〇&lt;0.05). The IC50 results for CVB3 KMUH 92225 showed that the 25, 60 and 85 °C extracts had the same anti-CVB3 KMUH-92225 activity (IC50: 58.06 ± 0.69. pg/ml to 60.05 ± 2.87 pg/ml), IC50 was below 4 °C extract (74.99:1:4.53 pg/ml) (;?&lt;0.05). Based on the results of Table 10, the extracts of Dabenshan grapes extracted at 25 °C have the best anti-EV71 ATCC VR-784 and CVB3 KMUH 92225 activities. It is apparent to those skilled in the art that various modifications and variations can be made without departing from the scope and spirit of the invention. Although the present invention has been described in terms of specific preferred embodiments, it should be understood that the invention should not be In fact, in the implementation of the described mode of the present invention, different amendments are readily apparent to those skilled in the art. 32 1374749 The revised replacement page of September 11, 2012 is also covered by the following claims. [Simple description of the diagram] (None) . [Description of main component symbols] (None) .· [References]

Brown, B. A. and Pallanasch, M. A., Virus Res., 39: 195-205 (1995).Brown, B. A. and Pallanasch, M. A., Virus Res., 39: 195-205 (1995).

Chau, D.H.W., Yuan, J., Zhang, H., Cheung, P., Lim, T., Liu, Z., Sail, A., and Yang, D., Apoptosis, 12: 513-524 (2007).Chau, DHW, Yuan, J., Zhang, H., Cheung, P., Lim, T., Liu, Z., Sail, A., and Yang, D., Apoptosis, 12: 513-524 (2007) .

Gilbert, G. L., Dickson, K. E., Waters, M. J., Kennett, M. L., Land, S. A., and Sneddon, M., Pediatr. Infect. Dis. J.,7: 484-488 (1988).Gilbert, G. L., Dickson, K. E., Waters, M. J., Kennett, M. L., Land, S. A., and Sneddon, M., Pediatr. Infect. Dis. J., 7: 484-488 (1988).

Groarke，J.M. and Pevear，D.C.，179, 1538-1541 (1999).Groarke, J.M. and Pevear, D.C., 179, 1538-1541 (1999).

Guo, J.P., Pang, J., Wang, X.W., Shen, Z.Q., Jin, M., and Li, J.W., World J Gastroenterol.^ 12: 4078-4081 (2006).Guo, J.P., Pang, J., Wang, X.W., Shen, Z.Q., Jin, M., and Li, J.W., World J Gastroenterol.^ 12: 4078-4081 (2006).

Henke, A., Nestler, M., Strunze, S., Saluz, H.P., Hortschansky, P., Menzel, B., Martin, U., Zell, R., Stelzner, A. and Munder, T., Virology 289: 15-22 (2001).Henke, A., Nestler, M., Strunze, S., Saluz, HP, Hortschansky, P., Menzel, B., Martin, U., Zell, R., Stelzner, A. and Munder, T., Virology 289: 15-22 (2001).

Ho, M., Chen, E. R., Hsu, K. H., Twu, S. J., Chen, K. T., Tsai, SF., Wang, J. R., and Shih, S. R.,N. Engl J. Med., 341: 929-935 (1999).Ho, M., Chen, ER, Hsu, KH, Twu, SJ, Chen, KT, Tsai, SF., Wang, JR, and Shih, SR, N. Engl J. Med., 341: 929-935 (1999) ).

Hsieh W.S., Yang P.H„ Chu S.M., Lin, T.Y., Ning, H.C., and Yeh, T.F., Pediatr Res., 41: 222A (1997).Hsieh W.S., Yang P.H„ Chu S.M., Lin, T.Y., Ning, H.C., and Yeh, T.F., Pediatr Res., 41: 222A (1997).

Khetsuriani, N·，LaMonte-Fowlkes, A” Oberste，S” and Pallansch，M” Enterovirus Surveillance - United States, 1970-2005, Surveillance Summaries, 55: 1-20 (2006). 33 1374749 __ 2012年9月11日修正替換頁Khetsuriani, N·, LaMonte-Fowlkes, A” Oberste, S” and Pallansch, M” Enterovirus Surveillance - United States, 1970-2005, Surveillance Summaries, 55: 1-20 (2006). 33 1374749 __ September 11, 2012 Day correction replacement page

Kim, K.S., Hufiiagel, G., Chapman, N.M., and Tracy, S.5 Rev. Med. Virol., 11:355-368 (2001).Kim, K.S., Hufiiagel, G., Chapman, N.M., and Tracy, S.5 Rev. Med. Virol., 11:355-368 (2001).

Lee, H. and Lin, J. Y., Murat Res., 204:229-234 (1988).Lee, H. and Lin, J. Y., Murat Res., 204:229-234 (1988).

Li, Y., Leung, K.T., Yao, F, Ooi, L.S., and Ooi, V.E., J Nat Prod., 69:833-835 (2006).Li, Y., Leung, K.T., Yao, F, Ooi, L.S., and Ooi, V.E., J Nat Prod., 69:833-835 (2006).

Li, Y., Jiang, R., Ooi, L.S., But, P.P., and Ooi, V.E., Phytother Res., 21:466-470(2007).Li, Y., Jiang, R., Ooi, L.S., But, P.P., and Ooi, V.E., Phytother Res., 21:466-470 (2007).

Lin, T. Y., Chu, C. and Chiu, C. H., J. Infect. Dis., 186:1161-1164 (2002).Lin, T. Y., Chu, C. and Chiu, C. H., J. Infect. Dis., 186: 1161-1164 (2002).

Lu, C. Y.} Lee, C. Y., Kao, C. L., Shao, W. Y., Lee, P. I., Twu, S. J., Yeh, C. C., Lin, S. C., Shih, W. Y., Wu, S. I. and Huang, L. M., J： Med. Virol., 67:217-223 (2000).Lu, CY} Lee, CY, Kao, CL, Shao, WY, Lee, PI, Twu, SJ, Yeh, CC, Lin, SC, Shih, WY, Wu, SI and Huang, LM, J: Med. Virol. , 67:217-223 (2000).

Makarov, V. A., Riabova, Ο. B., Granik, V. G., Wutzler, P., and Schmidtke, M., J. Antimicrob Chemother., 55, 483-488 (2005).Makarov, V. A., Riabova, Ο. B., Granik, V. G., Wutzler, P., and Schmidtke, M., J. Antimicrob Chemother., 55, 483-488 (2005).

Melnick J. L.s Enteroviruses: Polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses. In: Fields, B. N., Knipe, D. M., Howley, P. M., Channock, R. M., Melnick, J. L., Monath, T. P., Roizman, B. and Strauss, S. E. (eds.) Fields Virology. Philadelphia, Lippincott-Raven. New York. pp. 655-712 (1996).Melnick JLs Enteroviruses: Polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses. In: Fields, BN, Knipe, DM, Howley, PM, Channock, RM, Melnick, JL, Monath, TP, Roizman, B. and Strauss, SE (eds .) Fields Virology. Philadelphia, Lippincott-Raven. New York. pp. 655-712 (1996).

Morens, D.M. and Pallansch, M.A., In: Rotbart, H.A. (Ed.), Human &quot; Enterovirus Infections, Epidemiology. American Society for Microbiology, . Washington, DC, USA pp. 3-23 (1995).Morens, D.M. and Pallansch, M.A., In: Rotbart, H.A. (Ed.), Human &quot; Enterovirus Infections, Epidemiology. American Society for Microbiology, . Washington, DC, USA pp. 3-23 (1995).

Ning H. C., Taso, K. C., and Shih, S. R., Laboratory diagnosis of enterovirus infection in Chang Gung memorial Hospital during 1998 summer outbreak. Bulletin of Association of Laboratory Medicine, ROC 13:56 (1998)Ning H. C., Taso, K. C., and Shih, S. R., Laboratory diagnosis of enterovirus infection in Chang Gung memorial Hospital during 1998 summer outbreak. Bulletin of Association of Laboratory Medicine, ROC 13:56 (1998)

Olivetti, G., Abbi, R., Quaini, F., Kajstura, J., Cheng, W., Nitahara, J.A., 34 1374749 ______ 2012年9月11曰修正替換頁Olivetti, G., Abbi, R., Quaini, F., Kajstura, J., Cheng, W., Nitahara, J.A., 34 1374749 ______ September 11, 2012 Revision Replacement Page

Quaini, E., Di Loreto, C., Beltrami, C.A., Krajewski, S., Reed, J.C., and Anversa, P.,N. Engl J. Med., 1131-1141 (1997).Quaini, E., Di Loreto, C., Beltrami, C.A., Krajewski, S., Reed, J.C., and Anversa, P., N. Engl J. Med., 1131-1141 (1997).

Ouellet, A., Sherlock, R., Toye, B., and Fung, K.F., Infect. Dis. Obstet Gynecol., 12:23-26(2004).Ouellet, A., Sherlock, R., Toye, B., and Fung, K.F., Infect. Dis. Obstet Gynecol., 12:23-26 (2004).

Shen, W. C., Tsai, C., Chiu, H., and Chow, K. Neuroradiology, * 42:124-127 (2000).Shen, W. C., Tsai, C., Chiu, H., and Chow, K. Neuroradiology, * 42:124-127 (2000).

Shia, K. S., Li, W. T., Chang, C. M., Hsu, M. C., Chem, J. H., Leong, M. K., Tseng, S. N., Lee, C. C., Lee, Y. C., Chen, S. J., Peng, K. C., Tseng, Η. Y., Chang, Y. L., Tai, C. L., and Shih, S. R., J. Med. Chem., 45:1644-1655 (2002).Shia, KS, Li, WT, Chang, CM, Hsu, MC, Chem, JH, Leong, MK, Tseng, SN, Lee, CC, Lee, YC, Chen, SJ, Peng, KC, Tseng, Η. Y. , Chang, YL, Tai, CL, and Shih, SR, J. Med. Chem., 45:1644-1655 (2002).

Shih, S. R., Tsai, K. N., Li, Y. S., Chueh, C. C., and Chan, E. C., J. Med. F/ro/., 70:119-125 (2003).Shih, S. R., Tsai, K. N., Li, Y. S., Chueh, C. C., and Chan, E. C., J. Med. F/ro/., 70:119-125 (2003).

Shih, S. R, Tsai, M. C., Tseng, S. N., Won, K. F., Shia, K. S., Li, W. T. Chem, J. H., Chen, G. W., Lee, C. C., Lee, Y. C., Peng, K. C., and Chao, Y.S., Antimicroh. Agents Chemother., 48:3523-3529 (2004).Shih, S. R, Tsai, MC, Tseng, SN, Won, KF, Shia, KS, Li, WT Chem, JH, Chen, GW, Lee, CC, Lee, YC, Peng, KC, and Chao, YS, Antimicroh. Agents Chemother., 48:3523-3529 (2004).

Shindarou L. M., Chumakov, Μ. P., and Voroshilova, Μ. K., J. Hyg. Epidemiol. Microbiol. Immunol., 23:284 (1979).Shindarou L. M., Chumakov, Μ. P., and Voroshilova, Μ. K., J. Hyg. Epidemiol. Microbiol. Immunol., 23:284 (1979).

Su, M., Li, Y., Leung, K.T., Cen. Y., Li. T., Chen. R., and Ooi. V.E., Phytother Res., 20:634-639 (2006).Su, M., Li, Y., Leung, K.T., Cen. Y., Li. T., Chen. R., and Ooi. V.E., Phytother Res., 20:634-639 (2006).

Tait, S., Salvati, A.L., Desideri, N., and Fiore, L., Antiviral Res., ^ 72:252-255 (2006). -+ Wu, M. J. Yen, J. H., Wang, L., and Weng, C. Y., Am. J. ChineseTait, S., Salvati, AL, Desideri, N., and Fiore, L., Antiviral Res., ^ 72:252-255 (2006). -+ Wu, MJ Yen, JH, Wang, L., and Weng , CY, Am. J. Chinese

Medicine, 32:681-693 (2004).Medicine, 32:681-693 (2004).

Yabe, N. and Marsui, H., J. Ethnopharmacol., 56:67-76 (1997).Yabe, N. and Marsui, H., J. Ethnopharmacol., 56:67-76 (1997).

Yabe, N. Katsuya, K., and Marsui, H., J. Ethnopharmacol., 59:147-159 (1998). 35 1374749 2〇12年9月11日修正贊換頁Yabe, N. Katsuya, K., and Marsui, H., J. Ethnopharmacol., 59: 147-159 (1998). 35 1374749 Revised on September 11, 2002

Yan, J. J., Su, I. J., Chen, P. F., Liu, C. C., Yu, C. K.5 and Wang, J.~R^-Med. Fz&gt;o/., 65:331-339 (2001). 行政院衛生署食品衛生處，y供會忑资用及存看鲞一實表（2〇〇5)。 行政院衛生署疾病管制局，傳染病統計暨監視年報中華民國九十四 (2006)。 呂俊毅、黃立民、李慶雲，会；f夢荸，3:49-51 (1999)。 崩翠龔，霹立成衫犬學誉事#衡肩項:士論文(2004)。 張次郎，尹#秦猶詰，9:221-228(1998)。 葉彦伯、黃凯琳、許守道、吳聰能、趙黛瑜、李秉賴、何美鄉，会摩 夢荸，3(1):57-61 (1999)。 ^ 劉武哲’夢居苈#學及真坪完才法(修訂版），藝軒圖書出版社，台北， 117 頁(2003)。 鄭佩雯，苈廣磬學乂學夢學哥克考廣士款文（2〇〇3)。 36Yan, JJ, Su, IJ, Chen, PF, Liu, CC, Yu, CK5 and Wang, J.~R^-Med. Fz&gt;o/., 65:331-339 (2001). The Food Hygiene Department, y for the use of funds and deposits a real table (2〇〇5). The Disease Control Bureau of the Department of Health of the Executive Yuan, Annual Report on Statistics and Surveillance of Infectious Diseases, Republic of China, 94 (2006). Lu Junyi, Huang Limin, Li Qingyun, Hui; f Nightmare, 3:49-51 (1999). Cui Cui Gong, Yu Licheng, the dog's reputation, #衡肩项:士论文(2004). Zhang Jilang, Yin #秦犹诘, 9:221-228 (1998). Ye Yanbo, Huang Kailin, Xu Shoudao, Wu Congneng, Zhao Yuyu, Li Binglai, He Meixiang, Huimo Nightmare, 3(1): 57-61 (1999). ^ Liu Wuzhe's "Dream" #学和真坪完才法(Revised Edition), Yixuan Book Publishing House, Taipei, 117 (2003). Zheng Peiwen, 苈广磬学学学学学哥克考广士款文 (2〇〇3). 36

Claims (1)

1374749 七、申請專利範圍： — I.一種使用大本山葡萄（Ampeh?psis brevipeduncuka (MaXim.) TraUtV·)萃取物以製備用於預防及對抗腸病毒71 型感染之醫藥品的用途’其中該大本山葡萄萃取物係藉由 下列步驟所製得： 3 將經均質化之大本山葡萄與一溶劑相混合，其中經均 質化之該大本山葡萄係為大本山葡萄之根、莖、葉、果^ 一、 種子全株或其等之組合，且該溶劑之極性指m為介於 一氯甲烷之極性指數與水之極性指數之間；以及 利用該溶劑於4。。至95。。萃取經均質化之該大本山葡 萄，藉以獲取大本山葡萄萃取物； 八中該大本山葡萄萃取物具有抗腸病毒71型之活性。 2.如申4專利範圍第1項所述之用途，其中該部分的 大本山葡萄係為大本山葡萄之根、莖、葉、果實、種子、 全株或其等之組合。 3·如申sf專利範圍第1項所述之用途，其中該部分的 大本山葡萄，為大本山葡萄之根、莖或其等之組合。 月專矛J範圍第丨項所述之用途，其中該溶劑係選 自於由下列所構成之群组：水、丙網、95%乙醇、乙酸乙醋、 氰曱烷以及二氣甲烷。 1項所述之用途，其中利用該溶劑 攪拌、震盪、超音波、索氏熱回 5.如申請專利範圍第 萃取之方法包括以浸潰、 流或其等之組合進行。 種用於抑制腸病毒71型活性之外用消毒劑，其包 3 &quot;&quot;有的JL * 、尺表山葡萄（Ampelopsis brevipeduncu!ata 37 1374749 fMaxim ^ T 2012年9月11日修正替換頁 大本山葡vr以及適合的载劍或 葡萄萃取物係藉由下列步驟所製得·· 將經均質化之大本山葡萄與一溶劑相混合，里中該大 係為大本山葡萄之根、莖、葉、果實、種子。:全 合，且該溶劑之極性指數為介於二氣甲烧 之極性指數與水之極性指數之間；以&amp; 乳甲烷 利用該溶劑於4°Γ 5 A , 萄，葬…J 經均質化之該大本山葡 萄藉以獲取大本山葡萄萃取物； 其中該大本山葡萄萃取物具有抗腸病毒71型之活性。 h申凊專利範圍第6項所述之外用消毒劑，1申該 ::的大本山葡萄係為大本山葡萄之根、莖、葉、果: 種子、全株或其等之組合。 A V申明專利辜巳圍第6項所述之外用消毒劑，JL中該 4分的大本山葡萄係為大 '、〇A 糸為大本山勤萄之根、莖或其等之組合。 9 ·如申請專利範圍第6 紀r 固弟6項所述之外用消毒劑，其中該溶 劑係選自於由下列所構成 稱成之群組·水、丙酮、95%乙醇、乙 酸乙知、氰曱貌以及二氯甲烷。 复 如I專利範圍第6項任—項所述之外用消毒劑， 立冰 哪卒取之方法包括以浸潰、授拌、震盈、超 曰波、索氏熱回流或其等之組合進行。 U.一種活體外抑制腸病毒71型活性之方法，|包括. 2種如申請㈣範圍第6至1()項任—項所述之外用消毒 腸病毒71型接觸’藉以抑制腸病毒7i型之活性。 、12.—種料預防及對抗腸病I 71型感染之保健食品 況成物’其包含—有效數量的大本山葡萄（如吻… 38 1374749 2(312年9月日修正替換頁 心―—μ (Maxim.) Trautv )萃取物 __ 或稀釋劑，該大本山葡萄萃取物係藉由下列步驟所製得： 將經均質化之大本山葡萄與一溶劑相混合，其中該大 •本山葡萄係為大本山葡萄之根、莖、葉、果實、種子、全 j株或其等之組合，且該溶劑之極性指數（户,）為介於二氯甲烷 *之極性指數與水之極性指數之間；以及 利用該溶劑⑥4°Ci 95°C萃取經均質化之該大本山葡 萄’藉以獲取大本山葡萄萃取物； 其中該大本山葡萄萃取物具有抗腸病毒71型之活性。 丨3·如申請專利範圍第12項所述之保健食品組成物，其 中該部分的大本山葡萄係為大本山葡萄之根、莖、葉、果 實、種子、全株或其等之組合。 14. 如申請專利範圍第u項所述之保.健食品組成物，其 中該4为的大本山葡萄係為大本山葡萄之根、.莖或其等之 組合。 15. 如申請專利範圍第12項所述之保健食品組成物，其 中利用該溶劑萃取之方法包括以浸潰 '攪拌、震盪、超音 波、索氏熱回流或其等之組合進行。 16. 如申請專利範圍第12項所述之保健食品組成物，其 : 中該溶劑係選自於由下列所構成之群組：水、丙酮、95。/〇乙 ， 醇、乙酸乙酯、氰曱烷以及二氯甲烷。 391374749 VII. Patent application scope: — I. Use of extracts of Ampeh?psis brevipeduncuka (MaXim. TraUtV·) to prepare medicines for preventing and combating enterovirus 71 infections The Benshan grape extract is prepared by the following steps: 3 Mixing the homogenized Dabenshan grape with a solvent, wherein the homogenized Dabenshan grape line is the root, stem, leaf and fruit of the Dabenshan grape ^ A combination of seeds, whole plants or the like, and the polarity of the solvent means that m is between the polarity index of methyl chloride and the polarity index of water; and using the solvent at 4. . To 95. . The homogenized Dabenshan grape is extracted to obtain the Dabenshan grape extract; the Yazhongshan grape extract has the activity against the enterovirus 71 type. 2. The use according to claim 1, wherein the portion of the Dabenshan grape line is a combination of roots, stems, leaves, fruits, seeds, whole plants or the like of the Dabenshan grape. 3. The use of the first item of the sf patent scope, wherein the portion of the Dabenshan grape is a combination of the roots, stems or the like of the Dabenshan grape. The use of the present invention is selected from the group consisting of water, propylene mesh, 95% ethanol, ethyl acetate, cyanurethane, and di-methane. The use of one of the above, wherein the solvent is stirred, oscillated, ultrasonicated, and Soxhlet heat is recovered. 5. The method of extracting according to the scope of the patent application comprises a combination of impregnation, flow or the like. A disinfectant for inhibiting the activity of enterovirus type 71, which contains 3 &quot;&quot; JL*, and amphibious mountain grapes (Ampelopsis brevipeduncu!ata 37 1374749 fMaxim ^ T September 11, 2012 revised replacement page Dabenshan Portuguese vr and suitable sword or grape extract are prepared by the following steps: Mixing the homogenized Dabenshan grape with a solvent, which is the root, stem and leaf of the Dabenshan grape , fruit, seed.: all-in, and the polarity index of the solvent is between the polarity index of the gas and the polarity index of water; using &amp; milk methane using the solvent at 4 ° Γ 5 A, The burial...J is homogenized by the Dabenshan grape to obtain the Dabenshan grape extract; wherein the Dabenshan grape extract has the activity against the enterovirus 71. The application of the disinfectant described in item 6 of the patent scope of the application 1 Application: The Dabenshan grape line is the root, stem, leaf and fruit of the Dabenshan grape: a combination of seeds, whole plants or the like. AV Declared patents, except for the disinfectant mentioned in item 6, JL The 4 points of the Dabenshan grape line are ', 〇A 糸 is the combination of roots, stems or the like of Dabenshan. 9 · For the application of the scope of the patent, No. 6 ru, the use of disinfectant, wherein the solvent is selected from the following The group consisting of water, acetone, 95% ethanol, acetic acid, cyanide, and methylene chloride. Reuse of the disinfectant as described in item 6 of the I patent scope, The method comprises the following steps: impregnation, mixing, shock, super-chopper, Soxhlet heat reflux or the like. U. A method for inhibiting enterovirus 71 activity in vitro, including: 2. (4) Scope of the sixth to the first (1) of the scope of the use of disinfected enterovirus type 71 contact 'to inhibit the activity of enterovirus type 7i., 12. - seed material prevention and anti-intestinal disease type I 71 infection of health food The condition of 'there' contains an effective amount of Dabenshan grapes (such as kisses... 38 1374749 2 (September 312, revised replacement page heart - μ (Maxim.) Trautv) extract __ or thinner, the big Mountain grape extract is obtained by the following steps: homogenized Dabenshan grape with one The agent is mixed, wherein the big Benshan grape line is a combination of roots, stems, leaves, fruits, seeds, whole j strains of Dabenshan grapes, or the like, and the polarity index (household) of the solvent is inter-dichloro Between the polarity index of methane* and the polarity index of water; and extracting the homogenized Dabenshan grape by using the solvent at 95 ° C i 95 ° C to obtain the Dabenshan grape extract; wherein the Dabenshan grape extract has anti- The activity of the enterovirus type 71. The health food composition according to claim 12, wherein the Dabenshan grape line is the root, stem, leaf, fruit, seed, whole plant of the Dabenshan grape. Or a combination thereof. 14. The composition of the health-care food as described in the scope of claim 5, wherein the 4-year-old Dabenshan grape line is a combination of the root of the Dabenshan grape, the stem, or the like. 15. The health food composition of claim 12, wherein the method of extracting with the solvent comprises performing a dipping, agitation, shaking, ultrasonic, Soxhlet heat reflux, or the like. 16. The health food composition according to claim 12, wherein: the solvent is selected from the group consisting of water, acetone, and 95. / 〇 B, alcohol, ethyl acetate, cyanonane and dichloromethane. 39
TW98111599A 2009-04-08 2009-04-08 Medical products,food composition and external disinfectant preparation containing ampelopsis brevipedunculata (maxim.) trautv. extract and use thereof in prevention and treatment of enterovirus 71 infection TWI374749B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
TW98111599A TWI374749B (en) 2009-04-08 2009-04-08 Medical products,food composition and external disinfectant preparation containing ampelopsis brevipedunculata (maxim.) trautv. extract and use thereof in prevention and treatment of enterovirus 71 infection

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
TW98111599A TWI374749B (en) 2009-04-08 2009-04-08 Medical products,food composition and external disinfectant preparation containing ampelopsis brevipedunculata (maxim.) trautv. extract and use thereof in prevention and treatment of enterovirus 71 infection

Publications (2)

Publication Number Publication Date
TW201036622A TW201036622A (en) 2010-10-16
TWI374749B true TWI374749B (en) 2012-10-21

Family

ID=44856332

Family Applications (1)

Application Number Title Priority Date Filing Date
TW98111599A TWI374749B (en) 2009-04-08 2009-04-08 Medical products,food composition and external disinfectant preparation containing ampelopsis brevipedunculata (maxim.) trautv. extract and use thereof in prevention and treatment of enterovirus 71 infection

Country Status (1)

Country Link
TW (1) TWI374749B (en)

Also Published As

Publication number Publication date
TW201036622A (en) 2010-10-16

Similar Documents

Publication Publication Date Title
JP7008368B2 (en) Antiviral Chinese herbal medicine composition and its preparation method and use
Lin et al. Anti-enterovirus 71 activity screening of Chinese herbs with anti-infection and inflammation activities
Wu et al. Antiviral effects of Salvia miltiorrhiza (Danshen) against enterovirus 71
Zhang et al. Antiviral activity of shikonin ester derivative PMM-034 against enterovirus 71 in vitro
CN106580979B (en) Application of the pyridine heterocycle ester type compound in the drug for preparing anti-Coxsackie virus type B3
CN112261946B (en) Broad-spectrum enterovirus-resistant polypeptide and application thereof
Song et al. Raw Rehmannia radix polysaccharide can effectively release peroxidative injury induced by duck hepatitis a virus
JP2010222344A (en) Influenza virus suppressing composition and application thereof
KR20220156604A (en) Application of Traditional Chinese Medicine Compositions in Preparation of Drugs for Treatment or Prevention of Coronavirus Infection
CN107753823B (en) Traditional Chinese medicine composition for treating or preventing hand-foot-and-mouth disease
TWI374749B (en) Medical products,food composition and external disinfectant preparation containing ampelopsis brevipedunculata (maxim.) trautv. extract and use thereof in prevention and treatment of enterovirus 71 infection
CN111671846A (en) Application of golden shell preparation in resisting coronavirus
KR101653884B1 (en) A Coxsackie virus proliferation inhibitory composition extracted from Isodon excisus
CN108186866B (en) Application of golden scallop oral liquid in antivirus
JP4102750B2 (en) Herbal extract having antiviral activity and preparation thereof
CN106668015B (en) A kind of fat-based ester type compound WY124 is preparing the application in anti-enterovirus medicines
CN102274234A (en) Application of ganoderic acid Y to preparation of medicament for treating or preventing enterovirus 71 infection
KR101665015B1 (en) Composition for the prevention and treatment of antiviral comprising extracts of crude drug complex
ES2400495T3 (en) Activity against dengue extracts of Cissampelos pareira
CN102440988B (en) Application of arbidol hydrochloride in preparation of anti-enterovirus medicines
JP2012062247A (en) Virus treating agent
TWI304342B (en) An herbal extract having anti- enterovirus activity and preparation of same
CN103655755A (en) Anti-influenza drug and preparation method thereof
CN110201068A (en) The application of a kind of gram of sense relieving sore-throat composition or preparation in preparation prevention and treatment dengue fever medicine
KR100295395B1 (en) Antiviral pharmaceutical composition useful for prevention and treatment of a cold caused by influenza a virus and a functional food