TWI821584B - 新穎抗發炎化合物,其製法及其應用 - Google Patents
新穎抗發炎化合物,其製法及其應用 Download PDFInfo
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- TWI821584B TWI821584B TW109124130A TW109124130A TWI821584B TW I821584 B TWI821584 B TW I821584B TW 109124130 A TW109124130 A TW 109124130A TW 109124130 A TW109124130 A TW 109124130A TW I821584 B TWI821584 B TW I821584B
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- pineapple
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Abstract
Description
本發明係關於一種具有分離自天然水果之抗發炎化合物,更特別地,係關於一種分離自鳳梨萃取物之PGE2類似物。
環氧合酶(Cyclooxygenase,簡稱COX)是一種參與合成前列腺素家族的酵素。於九○年起,發現在許多細胞含有兩種COX,分別命名為COX-1及COX-2。COX-1可維持胃腸道微血管的完整性而具有胃黏膜保護作用,可合成Thromboxane A2來調節血小板凝集作用,並藉由調整腎血管阻力、血流量、鈉離子的排泄及ADH的拮抗作用,而達到控制腎臟血流的功能。COX-2則是一種受發炎而誘發的酵素,在正常細胞中幾乎無法偵測到其存在,只會在病態時,如受到發炎性刺激或細胞激素(cytokines)刺激時才會被誘導生成,並使花生四烯酸大量轉變為PGE2、PGF2α及血栓素等前列腺素。其中PGE2為一種發炎因子(proinflammatory factor),其利用自分泌(autocrine)及旁分泌(paracrine)的方式作用於各組織,已知PGE2為發炎反應中的重要分子之一,故已有研究指出,降低PGE2的生成或其作用,應可緩和發炎反應中的不適症狀(即,紅、腫、熱、痛)。
當體內巨噬細胞被細菌活化時,會誘發巨噬細胞產生免疫反應以及發炎作用,進而使細胞內的一氧化氮合成酶(iNOS)釋出一氧化氮
(NO)。人體中適量的一氧化氮雖具有消滅外來細菌的作用,但是當NO被過度產生時,便會造成體內組織的急性或慢性發炎反應。已有研究發現,PGE2透過活化其受體蛋白EP2,藉由胞內環單磷酸腺苷/蛋白質激酶A/鈣離子(cAMP/PKA/calcium)訊息調控路徑,提高iNOS的表現量及NO的生成量(Tzeng SF等人,Glia.15;55(2):214-223,2007)。
已知有多種天然蔬果具有抗發炎的功效,包括奇異果、鳳梨、青木瓜、香蕉、葡萄、蔓越莓、番茄與青花菜等。另外,薑黃素含有抗氧化的功用,可以適時幫助肝臟排毒消炎;綠茶則因其中的兒茶素含量很高,能減少細胞受傷與發炎反應。一般認為,鳳梨中具有一種獨特的鳳梨酵素,藉著刺激細胞漿素的製造來阻止發炎現象,可以有效舒解疼痛發炎症狀。
普遍所稱的鳳梨酵素(Bromelain)實際上並非單一種物質,而是綜合多種在鳳梨汁和鳳梨植物莖中發現的蛋白質消化酶,在胃的酸性環境和小腸的鹼性環境中都具有活性,使其在幫助消化上有優異的效果,適合用於改善消化不良的狀況。除了幫助消化外,鳳梨酵素也常用於手術後、運動損傷的恢復,以及改善鼻竇炎和靜脈炎,但目前鳳梨酵素改善術後發炎及腫脹的研究結果不太一致。市面上所稱的鳳梨酵素是鳳梨莖經過打碎、榨汁、離心、超過濾和冷凍乾燥等過程,最後產出的淡黃色粉末,通常以粉包、錠劑或膠囊的方式在市場上銷售,但本質上是一種組成不明確的混合物質。
由於鳳梨酵素是一種蛋白質,在加熱過程容易被破壞而失去活性,本發明首先嘗試從鳳梨水萃物分離、純化出一種具有抗發炎作用的
單一活性化合物,經分子鑑定含有香豆醯基(coumaroyl)與異檸檬酸(isocitrate)的結構組成,其由實驗證明能有效抑制巨噬細胞因LPS誘導所的一氧化氮產生及NF κ B蛋白表現,且在結構上與PGE2類似,具有與PGE2受器(prostaglandin E2 receptor)EP4的結合能力。
其中R1,R2及R3為相同或相異,個別獨立地係選自由H、鹵素、烷基、烯基、炔基、環烷基、雜環烷基、烷氧基、芳基、雜芳基、烷芳基及CF3所組成之組群。。
於本發明之一些具體實施例,所述之化合物中R1、R2及R3至少其中一者為H,或R1、R2及R3皆為H。於本發明之其他具體實施例,所述之化合物中R1、R2及R3至少其中一者為經取代或未經取代之C1-10烷基,或R1、R2及R3皆為經取代或未經取代之C1-10烷基。
較佳地,所述之經取代或未經取代之C1-10烷基為經取代或未經取代之C1-6烷基,更佳地為經取代或未經取代之甲基、乙基、丙基、異丙基或丁基。較佳地,所述之經取代或未經取代之C1-10烷氧基為經取代或未經取代之C1-6烷氧基,更佳地為經取代或未經取代之甲氧基、乙氧基、丙氧基、異丙氧基、丁氧基、戊氧基或己氧基。所述之C2-10烯基較佳地為C2-6烯基,更佳地為經取代或未經取代之乙烯基、烯丙基、丁烯基或戊烯基。
本發明之化合物亦包含其醫藥上可接受之鹽、酯類及水合物等衍生形式。所述之鹽類包含其藥學上可接受的鹼鹽,包括鹼金屬鹽(例如鈉鹽、鉀鹽)、鹼土金屬鹽(例如鈣鹽、鎂鹽)、銨鹽,以及與有機鹼(例如二環己基胺和N-甲基-D-葡糖胺)形成的鹽。
於另一方面,本發明係關於一種具有通式(I)之抗發炎化合物的製備方法,包含將一水果之水萃物,利用二氯甲烷(dichloromethane)及乙酸乙酯(ethyl acetate)進行分層萃取,之後將有機層以Sephadex LH-20管柱進行純化,而分離得到具有抗發炎活性的通式(I)化合物。於本發明之一具體實施例,所述之水果為鳳梨。較佳地,所述之水果之水萃物為鳳梨植株經過榨汁、粗過濾去除殘渣而取得之鳳梨水萃物。
於另一方面,本發明係關於一種抗發炎組合物,特徵在於包含具有通式(I)之化合物或其藥學上可接受鹽、酯類、水合物,及一藥學上可接受載體、賦形劑或稀釋劑。
圖1為本發明所例舉實例一從鳳梨萃取物分離、純化得到具通式(I)化合
物之方法的流程圖。
圖2為鳳梨萃取物使用LC-MS/MS進行分析,使用波長320nm得到之層析光譜圖。
圖3為純化之鳳梨素PL6之320nm之層析光譜圖(a),及於UV-Vis偵測器得到之光譜圖(b)。
圖4為經鑑定之鳳梨素PL6或合物分子結構,分子式為C15H14O9,分子量為338.27。
圖5係顯示鳳梨素PL6(a)及鳳梨水萃物(b)對於RAW264.7巨噬細胞的細胞毒性測試結果。數據係以平均值±SD表示,為三次獨立實驗的平均。***p<0.001表示相對於對照組之細胞具有統計學上的顯著差異。
圖6係顯示鳳梨素EP6於RAW264.7巨噬細胞模式中,在抑制由LPS-誘發之NO產生的功效。數據係以平均值±SD表示,為四次獨立實驗的平均。**p<0.01及***p<0.001表示對於單獨以LPS處理之細胞具有統計學上的顯著差異。
圖7係顯示鳳梨素EP6於RAW264.7巨噬細胞模式中,對於抑制由LPS-誘發之iNOS蛋白表現表現的功效。數據係以平均值±SD表示,為四次獨立實驗的平均。*p<0.05表示對於單獨以LPS處理之細胞具有統計學上的顯著差異。
圖8係顯示鳳梨素EP6於RAW264.7巨噬細胞模式中,在抑制由LPS-誘發之NF κ B蛋白表現的功效。數據係以平均值±SD表示,為四次獨立實驗的平均。*p<0.05表示對於單獨以LPS處理之細胞具有統計學上的顯著差異。
圖9係顯示以分子對接模擬運算,顯示(a)PGE2,及(b)鳳梨素PL6化合物在EP4結合區域中所產生的鍵結作用力以及其鍵結種類。
圖10係顯示於EP4分子中,(a)PGE2及(b)鳳梨素PL6化合物與關鍵殘基的模擬分子對接態勢。烷基作用力(alkyl interaction)、一般作用力(conventional interaction)、碳原子作用力(carbon interaction)、負-負作用力(negative-negative interaction)及電荷-電荷作用力(charge-charge interaction)各別以虛線及其名稱標示於虛線旁。
其中R1,R2及R3係個別獨立地選自由H、鹵素、烷基、烯基、烷氧基及CF3所組成之組群。
本文中,“鹵素”係指,氟、氯、溴或碘。
本文中,術語“經取代”係指以一或多個取代基(可為相同或不相同)各取代一個被取代基團上的氫原子。取代基的實例包括(但不限定
於)鹵素、氰基、硝基、羥基、胺基、巰基、烷基、烯基、炔基、芳基、雜芳基、環基、雜環基、烷基氧基、芳基氧基、烷磺醯基、芳基磺醯基、烷基胺基、芳基胺基、二烷基胺基、二芳基胺基、烷基羰基、芳基羰基、雜芳基羰基、烷基羧基、芳基羰基、雜芳基羧基、烷基氧基羰基、芳基氧基羰基、雜芳基氧基羰基、烷基羰基、烷基胺甲醯基、芳基胺甲醯基、雜胺甲醯基等,其中每一烷基、烯基、芳基、雜芳基、環基與雜環基視需要可以具有鹵素、氰基、硝基、羥基、胺基、巰基、烷基、芳基、雜芳基、烷基氧基、芳基氧基、烷基羰基、芳基羰基、烷基羰基、芳基羧基、烷基氧基羰基或芳基氧基羰基取代基。
本文中,術語“烷基”或“C1-10烷基”係指,含有1-10個碳原子之經取代或未經取代的直鏈或支鏈飽和烴基基團。較佳地,所述之烷基為經取代或未經取代的C1-6烷基,包括(但不限定於)經取代或未經取代的甲基、乙基、正丙基、異丙基、正丁基、第二丁基、第三丁基、正戊基、異戊基、正己基等。
本文中,“烯基”或“炔基”係指,含有2-10個碳原子及至少一個雙鍵或參鍵之經取代或未經取代的直鏈或支鏈不飽和烴基基團。較佳地,所述之烯基為經取代或未經取代的C2-6烯基,包括(但不限定於)經取代或未經取代的乙烯基、烯丙基、丁烯基、戊烯基、1,4-己二烯基等。較佳地,所述之炔基為經取代或未經取代的C2-6烯基,包括(但不限定於)經取代或未經取代的乙炔基、丙炔基、丁炔基等。
本文中,術語“環烷基”係指,含有4-14個碳原子之部分或完全飽和之單環或雙環體系。較佳地,所述之環烷基為經取代或未經取代的
C4-8環烷基,包括(但不限定於)經取代或未經取代的環丁基、環戊基、環己基等。
本文中,術語“雜環基”係指包含一或多個雜原子(例如,O、N或S)作為環體系之一部分,而其餘為碳的環狀官能基團。雜環基之實例包括(但不限定於)經取代或未經取代的吖丁啶基、六氫吡啶基、四氫吡咯基、四氫呋喃基、氮雜環庚烷基、1,4-oxazepane基等。
本文中,術語“烷氧基”係指,經取代或未經取代的C1-10烷基與氧原子連結後所生成的基團。較佳地,所述之烷基為經取代或未經取代的C1-6烷基,包括(但不限定於)經取代或未經取代的甲氧基(-OCH3)、乙氧基、丙氧基、丁氧基、戊氧基、己氧基等。
本文中,術語“芳基”係指至少具有一個芳香環體系之環狀烴基基團,可為單環或雙環。芳基之實例包括(但不限定於)經取代或未經取代的苯基、萘基、蒽基、及芘基等。
本文中,術語“雜芳基”係指至少具有一個芳香環體系之環狀烴基基團,可為單環或雙環或稠合環系,該芳香環包含至少一為環體系一部分之雜原子(例如,O、N或S),而其餘為碳原子。雜芳基之實例包括(但不限定於)呋喃基、吡咯基、噻吩基、噁唑基、咪唑基、吡唑基、吡啶基、嘧啶基、噻唑基、呋喃基、吲哚基等。
本文中,“藥學上可接受”係指,在合理醫學判斷範疇內,適合與人類或動物組織接觸,而不會產生過度毒性、刺激性、過敏性反應或其他併發症狀者。
本文中,“藥學上可接受鹽、酯類、水合物”係指,本發明之
通式(I)化合物上的酸性基團與一鹼類或醇類形成之鹽類或酯類,或是其官能基藉由配位與水相連而形成的含水化合物。例如,藥學上可接受的鹽包括(但不限定於),鹼金屬鹽(例如鈉鹽、鉀鹽)、鹼土金屬鹽(例如鈣鹽、鎂鹽)、銨鹽及有機鹼鹽(例如與二環己基胺、N-甲基-D-葡糖胺所形成之鹽)。
本發明亦提供一種抗發炎組合物,包含所述之具通式(I)化合物或其鹽、酯類或水合物及一藥學上可接受之載體、賦形劑或稀釋劑。本文中,“藥學上可接受之載體、賦形劑或稀釋劑”係指,藥學上可接受的材料、基底等載劑和賦形劑或稀釋劑,例如液體、固體填料、穩定劑、分散劑,懸浮劑、增稠劑、溶劑或包封材料,其作用是運輸本發明中的有效成分,使其在患者體能發揮應有的作用。每一個載體必須和組合物中的各個配方成分(包括發明之通式(I)化合物)相容,使其對患者不產生負面影響。藥學上可接受的載體包括:糖類,例如乳糖,葡萄糖和蔗糖;澱粉,例如玉米澱粉和馬鈴薯澱粉;纖維素,例如羧甲基纖維素鈉,乙基纖維素和乙酸纖維素;粉末黃蓍膠;麥芽;明膠;滑石。藥學上可接受之賦形劑或稀釋劑包括可可脂和栓劑蠟;油類,例如花生油、棉籽油、紅花油、芝麻油、橄欖油、玉米油和大豆油;二醇,例如丙二醇;多元醇,例如甘油,山梨醇,甘露醇和聚乙二醇;酯,例如油酸乙酯和月桂酸乙酯;瓊脂;緩衝劑,例如氫氧化鎂和氫氧化鋁;表面活性劑;藻酸;無熱原水;等滲鹽水;林格氏溶液;乙醇;磷酸鹽緩衝溶液;以及其它無毒之藥學上配方相容物質。
本文中,“抗發炎”係指,利用物質或治療抑制或減少發炎反應的症狀與發生。發炎反應是指具有血管系統的活體組織對致炎因子及局部損傷所發生的防禦性為主的反應,其中包括了紅腫、發熱、疼痛等症狀。
發炎反應可分為急性發炎和慢性發炎。急性發炎是生物體應對有害刺激的初步反應,更多的血漿和白血球(特別是粒細胞)從血液移往受損組織。慢性發炎引致發炎部位的細胞類型改變,組織的毀滅與修復同時進行。目前在抗發炎研究方面,主要係以脂多醣(LPS)誘導巨噬細胞的模式,來評估是否能抑制發炎物質如:一氧化氮(NO)、可誘導一氧化氮合成酶(iNOS)、前列腺素E2(PGE2)、環氧合酶-2(COX-2)等的產生,及降低NFκB蛋白的表現量。
本發明之其他特色及優點將於下列實施範例中被進一步舉例與說明,而該實施範例僅作為輔助說明,並非用於限制本發明之範圍。
實施例一、自鳳梨萃取物分層分離抗發炎化合物
本實例係從鳳梨水萃物,利用二氯甲烷(dichloromethane)及乙酸乙酯(ethyl acetate)進行分層萃取,之後將有基層以Sephadex LH-20(30cm*3cm i.d.)管柱進行純化,而分離得到具有抗發炎活性的單一化合物PL6,其流程參見圖1所示。詳細的製備方法及分離條件如下述。
將鳳梨植株經過榨汁、粗過濾去除殘渣後取得鳳梨水萃物,再將所得之鳳梨水萃物進行冷凍乾燥,而得到鳳梨萃取物粉末。利用HPLC分析鳳梨萃取物中主要化合物組成:將萃取物粉末以水回溶使最終濃度為200mg/ml,將欲分析樣品以0.45μm濾膜(13mm syringe filter with 0.45μm PP membrane,PALL)過濾後,取適量體積加入樣品瓶以高效能液相層析儀(high performance liquid chromatography,HPLC)進行分析,分析條件:層析管柱,Mightysil RP-18 GP(4.6mm * 250mm,粒徑:5μm);進樣體積:20μl;流速:0.8ml/min;偵測波長:320nm;100%乙腈(溶液A);1%(v/v)甲酸水溶液
(溶液B);沖提條件,95-70% B(50min),70-50% B(60min),50-95% B(70min)。
結果顯示,萃取物中主要多酚類化合物之吸收峰為280nm,而在320nm有較強吸收訊號,因此選定於320nm波長進行分析。在鳳梨萃取物中,在滯留時間17、20.5、24.5、28、34.8、41及42.8分鐘有較明顯的波峰出現(圖2),初步將其分別命名為PE1、PE2、PE3、PE4、PE5、PL6及PE7。
分層萃取鳳梨化合物:取60g鳳梨凍乾粉末以300ml二次水回溶,加入600ml二氯甲烷進行分層萃取,取水層加入600ml乙酸乙酯進行二次萃取,取有機層並利用減壓濃縮機進行濃縮,並保存於4℃中。
鳳梨萃取物管柱層析:將上述分層萃取中之有機層利用Sephadex LH-20(30cm*3cm i.d.)純化,以兩倍管柱體積沖提,沖提梯度為純水和20%(v/v)甲醇:水,以10ml收集並以HPLC確認目標物。HPLC分析條件:100%乙腈(溶液A);1%(v/v)甲酸水溶液(溶液B);沖提條件,95-70% B(50min),70-50% B(60min),50-95% B(70min)。
鳳梨萃取物液相層析串聯質譜分析:液相層析條件與HPLC分析條件相同,質譜使用ESI負離子方式,離子化溫度為300℃,噴霧電壓為4.5kV。屏蔽氣體(sheath gas)、輔助氣柱(auxiliary gas)和掃氣(sweep gas)之氣體流速分別為50、13及3任意單位(arbitrary units)。採用資料-依賴型擷取(data-dependent acquisition,DDA)進行最適化的篩選條件,掃描100-1500m/z之訊號並利用資料-依賴型方式進行MSn掃描。
鳳梨萃取物經由二氯甲烷(dichloromethane)及乙酸乙酯(ethyl acetate)分層萃取後,可大略將PE1-PE5與PL6及PE7分開,再將含有PL6之
有機層經由Sephadex LH-20層析,最終可純化得到鳳梨素(Pineapplin)PL6,為分析樣品中主要的訊號(圖3a)。根據質譜數據猜測之結構,推論其可能具有抗發炎的活性,因此進一步純化並在HPLC-PDA層析圖中,具有313nm和230nm之吸收峰值(圖3b)。
鳳梨萃取物核磁共振光譜(NMR)分析:使用The Bruker AV-400MHz NMR光譜儀進行13C及2D NMR光譜分析;使用Jeol JNM-ECA 600 NMR光譜儀進行1H NMR光譜分析,以四甲基矽烷(Tetramethylsilane)作為內標,化學位移(Chemical Shift)以δ values(parts per million,ppm)做紀錄。
純化得到的鳳梨素PL6呈現淡黃色的粉末,經由1H-NMR光譜分析,在苯環上氫原子訊號為δ 6.80(2H,d,J=9.0Hz)和δ 7.48(2H,d,J=9.0Hz),烯烴基上的氫原子訊號為δ 6.39(1H,d,J=16.2Hz)和δ 7.67(1H,d,J=16.2Hz),另外其他的氫原子訊號為δ 2.59(1H,dd,J=17.4,5.4Hz)、δ 2.81(1H,dd,J=17.4,9.0Hz)和3.55(1H,m)表示了有三個羧基的結構存在。再以13C-NMR光譜進行碳原子訊號的確認。利用Correlation Spectroscop Y(COSY)、Nuclear Overhauser Effect SpectroscopY(NOESY)及Heteronuclear Multiple Bond Correlation(HMBC),確認氫原子之間的交互作用和碳原子與氫原子間的交互作用,確認所得之鳳梨素(Pineapplin)PL6化合物命名為1,2,3-三羧酸-丙基-3-羟基苯酚基丙烯酸酯,分子式為C15H14O9,分子量為338.27(圖4)。
實施例二、鳳梨素PL6之細胞毒性測試
RAW264.7細胞使用含有10%胎牛血清、0,2%碳酸氫鈉和1% penicillin/streptomycin之RPMI培養基培養,培養於5% CO2、37℃培養
箱。當細胞至七至八分滿將細胞進行繼代。將RAW264.7細胞以每孔4x104cells/well密度培養於96孔盤中,放置一個晚上待細胞貼附後,加入不同濃度的鳳梨素PL6(25、50、100、200、400及800μM)或鳳梨萃取物(3、6、12mg/ml),而空白對照組則不加入任何藥物。培養24小時後,使用Alamar blue來測試藥物是否具有細胞毒性。去除培養基,以PBS清洗2次,並使用不含FBS之培養基稀釋成10倍Alamar Blue試劑,在避光環境下作用6小時,之後使用ELISA reader測量於波長570nm下的吸收光值變化。
結果顯示,鳳梨素PL6在給予上述濃度下,對於RAW264.7細胞並無明顯的細胞毒性(圖5a)。鳳梨素PL6在鳳梨萃取物中的含量約占0.1%,並將濃度擴大進行鳳梨萃取物之細胞毒性,鳳梨萃取物以水回溶後,濃度增加至3、6及12mg/ml,並以Alamar blue進行細胞毒性測試。結果發現,鳳梨萃取物濃度提升至12mg/ml時,即呈現顯著的細胞毒性(圖5b)。
實施例三、鳳梨素PL6抑制由LPS誘導細胞之發炎反應
抑制一氧化氮產生
將RAW264.7細胞以每孔4*105cells/well密度培養於6孔盤中,放置一個晚上待細胞貼附後,加入200ng/ml LPS,空白對照組則不加入LPS,在5% CO2及37℃條件下培養24小時。第二天給予不同濃度(50、100、200和400μM)之鳳梨素PL6處理,空白對照組給予不含LPS培養基,負對照組給予不含藥物之培養基,在5% CO2及37℃條件下培養24小時後,收集每組各150μl的培養基。
配置1%對胺基苯磺酸溶於5%磷酸之溶液,並將其與0.1% N-(1-萘基)伸乙二胺二鹽酸鹽以1比1混合均勻,配置成Griess reagent。配置濃
度為1.5625μM到100μM的亞硝酸鈉當作標準品,製作標準曲線。將150μl收集的培養基與50μl Griess reagent混合,在避光環境下反應30分鐘,使用ELISA reader測量在波長555nm下的吸收光值變化。
結果顯示,鳳梨素PL6在200μM及400μM濃度下,可顯著性的降低LPS誘導RAW264.7巨噬細胞所產生的一氧化氮產量(圖6),表示鳳梨素PL6具有抑制因外物刺激所產生之免疫反應的功效。
抑制iNOS蛋白之表現
測試鳳梨素PL6對於抑制LPS所誘導RAW264.7巨噬細胞中iNOS蛋白表現之功效。iNOS/β-actin為iNOS與β-actin之蛋白相對表現量,表現量越高表示有發炎反應(發炎指標)。將RAW264.7細胞以LPS(200ng/ml)處理24小時後,再與不同濃度(50、100、200和400μM)之鳳梨素PL6,在5% CO2及37℃條件下培養24小時,(-)對照組給予不含LPS培養基,(+)對照組給予不含藥物之LPS培養基。收取不同組別之細胞培養上清液,計算各組別之蛋白濃度後,取30μg進行蛋白質膠體電泳。於膠體轉漬完成後,使用一次抗體Anti iNOS(以1:1000稀釋),與二次抗體Anti rabbit IgG(1:5000稀釋)進行西方點墨法(Western blotting),以PBST清洗後加入呈色劑(Western Chemiluminescent HRP Substrate),利用冷光螢光數位分析系統(ImageQuant LAS 400mini,GE Healthcare Life Sciences)進行冷光呈色。
圖7所列示之結果,係冷光呈色膠片利用掃描光密度法,並相對於β-肌動蛋白標準化,經電腦計算所得之iNOS蛋白與β-肌動蛋白的相對表現量,數值越高表示發炎反應程度越高。由抑制iNOS蛋白表現之結果亦顯示,鳳梨素PL6確實具有抑制發炎反應的功效。
抑制NF κ B蛋白之表現
於本實例亦測試鳳梨素PL6對於抑制LPS所誘導RAW264.7巨噬細胞中NF κ B蛋白表現之功效。p-p65/p65為NF κ B之蛋白相對表現量,表現量越高表示有發炎反應(發炎指標)。將RAW264.7細胞以LPS(200ng/ml)處理24小時後,再與不同濃度(50、100、200和400μM)之鳳梨素PL6,在5% CO2及37℃條件下培養24小時,(-)對照組給予不含LPS培養基,(+)對照組給予不含藥物之LPS培養基。收取不同組別之細胞培養上清液,計算各組別之蛋白濃度後,取30μg進行蛋白質膠體電泳。於膠體轉漬完成後,使用一次抗體Anti phospho-NFkB p65及NFkB p65(以1:1000稀釋),與二次抗體Anti rabbit IgG(1:5000稀釋)進行西方點墨法(Western blotting),以PBST清洗後加入呈色劑(Western Chemiluminescent HRP Substrate),利用冷光螢光數位分析系統(ImageQuant LAS 400mini,GE Healthcare Life Sciences)進行冷光呈色。
圖8所列示之結果係冷光呈色膠片利用掃描光密度法,並相對於β-肌動蛋白標準化,經電腦計算所得之NF κ B蛋白p-p65/p65的相對表現量,數值越高表示發炎反應程度越高。由抑制NF κ B蛋白表現之結果亦顯示,鳳梨素PL6具有抑制發炎反應的功效。
實施例四、鳳梨素PL6之分子對接模擬運算
由於PL6在結構上與PGE2有類似,PGE2類似物在先前文獻表明是具有抗發炎活性的潛力,透過分子模擬軟體GEMDOCK運算,使用前列腺素E2(PGE 2)受體EP4作為PGE2的活性中心標的,比較實施例1中所述之鳳梨素PL6及PGE2,與EP4的結合能力以及與受器的鍵結能量。
於下表1所列之結果顯示,PGE2所需之化學能為-104.7kJmol-1(凡得瓦力-83.3kJmol-1、氫鍵-20.9kJmol-1、靜電力-0.6kJmol-1);PL6所需之化學能為-108.1kJmol-1(凡得瓦力-82.5kJmol-1、氫鍵-21.9kJmol-1、靜電力-3.0kJmol-1)。
以分子模擬軟體Discovery Studio來比較,PGE2與PL6兩化合物與EP4結合區域之間所產生的鍵結作用力,以及其鍵結種類與強度。依結果顯示,PL6與PGE2皆可以進入EP4的結合區域(圖9)。
PGE2與EP4之間主要產生三種分子間作用力,為烷基作用力(alkyl interaction)、氫鍵(hydrogen bond,conventional bond)、非典型氫鍵(non-classical hydrogen bond)與電荷作用力(charge-charge interaction)。由圖10a之分子對接模型顯示,PGE2與EP4具有一處的烷基作用力,在PGE2之碳鏈和EP4上的MET27形成烷基作用力。EP4上的ARG316與PGE2上的羧酸基形成三處氫鍵以及電荷作用力。此外此處羧酸基亦與EP4上的TYR80形成氫鍵。PGE2環上的羥基EP4上的CYS170形成氫鍵;而PGE2另一端的碳鏈則會
與EP4的SER319形成非典型氫鍵。
而在PL6則與EP4之間主要有π-烷基作用力(π-alkyl interaction)、氫鍵、非典型氫鍵與負電荷作用力(negative-negative interaction)五種作用力。PL6的苯環與EP4的MET27和VAL72兩個胺基酸形成π-烷基作用力。在PL6苯環上的羥基與EP4的THR69形成氫鍵。PL6在碳鏈上的羧基與EP4上的THR76、THR79及CYS170形成四處氫鍵且與SER95產生非典型氫鍵(圖10b)。
雖然以有限數量的實施例說明本發明的實施方式,本領域具通常知識的技術人員仍有可能加以修改或變化,因此本發明的保護範圍應只受專利範圍限制,而非受限於上述實施例。
Claims (3)
- 如請求項1所述之用途,其中該通式(I)化合物或其藥學上可接受鹽類、水合物具有與前列腺素E2(PGE2)受體結合的能力。
- 如請求項1所述之用途,其中該通式(I)化合物或其藥學上可接受鹽類、水合物係用於抑制發炎反應。
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