TWI819307B - 萜類化合物在治療或預防纖維化疾病中的用途 - Google Patents
萜類化合物在治療或預防纖維化疾病中的用途 Download PDFInfo
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- TWI819307B TWI819307B TW110118145A TW110118145A TWI819307B TW I819307 B TWI819307 B TW I819307B TW 110118145 A TW110118145 A TW 110118145A TW 110118145 A TW110118145 A TW 110118145A TW I819307 B TWI819307 B TW I819307B
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Abstract
本發明係關於一種預防或治療纖維化病症的方法,包括向有需要的受
試者施用有效量的組合物;其中該組合物包含從樟芝或魚針草中萃取的三萜化合物。
Description
本發明係關於源自樟芝(Antrodia camphorata)及魚針草(又名防風草,Anisomeles indica)萃取物的植物性類萜化合物(terpenoids),特別是一種能緩解纖維化疾病的藥用及膳食配方。
對於越來越多的個體而言,纖維增生性疾病是令人困擾的問題,並且是許多持續性炎症疾病的常見病理後遺症,例如肺纖維化(pulmonary fibrosis)、進行性腎病(progressive kidney disease)、肝硬化(liver cirrhosis)、動脈粥樣硬化(atherosclerosis)及良性***增生(benign prostatic hyperplasia)。
急性腎損傷後的腎臟修復會誘發纖維化,最終可能惡化為慢性腎病。首先,腎損傷活化多能先祖細胞(multipotent progenitor cells)以修復組織,然而,隨著損傷持續引發腎纖維化,這些細胞產生功能失調並誘導纖維化修復。腎纖維化的發病機制是一個漸進的過程,最終導致末期腎功能衰竭,這是一種需要透析或腎移植的重大疾病。
非酒精性脂肪性肝病(Non-alcoholic fatty liver disease,NAFLD)是有醫療迫切需求的慢性肝病主要形式。非酒精性脂肪性肝炎(Non-alcoholic
steatohepatitis,NASH)是NAFLD的進行性變異,可導致纖維化、肝硬化及肝細胞癌。NAFLD及NASH成為醫學界普遍關注的主題,尤其是因為世界人口中糖尿病及肥胖症的患病率增加。每個轉氨酶(aminotransferase)水平異常患者的臨床評估都應考慮到非酒精性脂肪肝及其譜系,尤其是肥胖或糖尿病患者。單純NAFLD的預後通常是良性的,但如果存在纖維化、肝細胞空泡(hepatocellular ballooning)、炎症及馬洛里小體(Mallory bodies),則有進展為肝硬化的風險。
自身免疫性肝炎(Autoimmune hepatitis,AIH)是一種慢性肝病,沒有明確的病因,以肝細胞炎症為特徵。嚴重的AIH可能會發展為肝硬化、肝細胞癌,甚至死亡。根據觀察時間的長短,多達40%的自身免疫性肝炎患者會出現肝硬化。因此除了當前的抗炎及免疫抑制療法,還可以再加上新興的抗纖維化療法,這些療法可望將自身免疫性肝炎的治療目標重新定位為預防、穩定及逆轉肝纖維化。
動脈粥狀硬化(atherosclerosis)是心血管疾病發展的主要原因之一,與血管纖維化有關。血管纖維化涉及細胞外基質(extracellular matrix,ECM)蛋白的積累,尤其是血管介質中的膠原蛋白(collagen)及纖連蛋白(fibronectin),並促成重塑結構及形成疤痕。血管壁缺乏彈性蛋白(elastin)或過多的膠原蛋白會導致血管纖維化及硬度增加。
在良性***增生中,***中膠原纖維的沉積是為了替代斷裂的肌纖維,但會導致肌肉組織僵硬無力,使***液沉積在腺管中。***纖維化為老年男性膀胱出口梗阻的主要因素。
藥用真菌樟芝(Antrodia camphorata)是一種著名的民間中藥,已知具有多種生物活性,尤其是在體外癌細胞及體內動物模型中具有抗腫瘤作用。
鑑於其含有多樣化的生物活性化合物,它被認為是一種有效的替代植物治療劑,或癌症治療及免疫相關疾病的佐劑。迄今共分離、鑑定及闡釋結構了225種化合物,包括了大分子(核酸、蛋白質及多醣)、小分子(苯類(benzenoids)、木脂素(lignans)、苯醌(benzoquinones)及馬來酸(maleic acid)/琥珀酸(succinic acid)衍生物)、萜類(羊毛脂烷三萜(lanostane triterpenes)、麥角三萜(ergostane triterpenes)、二萜(diterpenes)、單萜(monoterpenes)及類固醇(steroids))、核苷酸(核鹼基(nucleobase)及核苷(Nucleoside))、脂肪酸及脂肪酸酯。
累積的體外及體內實驗顯示,其具有抗糖尿病、抗高血脂、抗高血壓、抗炎、抗氧化、抗菌、心血管疾病預防、免疫調節、保肝及神經保護等作用。然而,樟芝及其成分於治療纖維化的功效則尚未被評估過。
魚針草(Anisomeles indica)以「印度貓薄荷(Indian catmint)」之名為人熟知,是一種藥用活性化合物的來源,具有多種藥理作用。該植物傳統上用作鎮痛劑、抗炎劑及治療皮膚問題。醫學已證明的藥理活性有抗氧化、抗菌、抗人類免疫缺乏病毒、抗幽門螺桿菌及抗癌活性等。進一步的研究揭示了各種植物性化學成分的存在,主要是三萜(triterpenes)、β-谷甾醇(β-sitosterol)、豆甾醇(stigmasterol)、黃酮(flavones)、芹菜素(apigenin)及魚針草內酯(ovatodiolides)等。
本發明提供一種預防或治療纖維化病症的方法,包括向有需要的受試者施用醫療有效量的一組合物;其中該組合物包含從魚針草(Anisomeles indica)萃取的三萜類化合物(triterpenes)。
在一些實施例中,該三萜類化合物係經由將魚針草之乙醇萃取物引入一正相層析管柱(normal phase chromatography column),以己烷/乙酸乙酯/甲醇沖提,自一有機沖提液中獲得。
在一些實施例中,該有機沖提液係包含至少一種選自下式的化合物或其組合:
另一方面,本發明亦提供一種預防或治療纖維化病症的方法,包括向有需要的受試者施用醫療有效量之一組合物,係包含選自下式的化合物或其組合:
在一些實施例中,該纖維化病症包括肝纖維化、腎纖維化、血管纖維化、肺纖維化及良性***增生。
在一些實施例中,該組合物進一步減輕腎功能障礙及腎損傷。
在一些實施例中,該組合物進一步減輕非酒精性脂肪性肝炎(non-alcoholic steatohepatitis,NASH)、非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD),及肝臟之炎症(inflammation)、空泡化(vacuolation)及壞死(necrosis)。
圖1 描述了從樟芝萃取物中分離的antcin K、去氫硫色多孔菌酸/硫色多孔菌酸(dehydrosulphurenic acid/sulphurenic acid)、versisponic acid及去氫齒孔酸(dehydroeburicoic acid)。
圖2 樟芝萃取物及化合物對AKI小鼠順鉑誘導的腎損傷的保護作用。為了分析樟芝萃取物及化合物的作用,從第一劑順鉑後3週開始,每天給予小鼠共7天,並在第4週時犧牲。(A)腎臟的形態變化;(B)血尿素氮(blood urea nitrogen,BUN)水平;(C)血清肌酐(serum creatinine,CRE)水平。數據表示為平均
值±SEM(n=5)。###表示與對照組樣本相比,p<0.001。與順鉑組相比,**表示p<0.01,***表示p<0.001。
圖3 樟芝萃取物及化合物對AKI小鼠順鉑誘導的腎損傷的保護作用。為了分析樟芝萃取物及化合物的作用,從第一劑順鉑後3週開始,每天給予小鼠共7天,並在4週時犧牲。腎臟以H&E染色。順鉑誘導後,準備好每組中的腎臟用於組織學評估。腎臟的代表性組織切片以H&E染色、放大倍數400倍。數據表示為平均值±SEM(n=5)。###表示與對照組樣本相比p<0.001。與順鉑組相比,**表示p<0.01,***表示p<0.001。管狀細胞壞死用箭頭標記;線條比例尺為50μm。
圖4 樟芝萃取物及化合物調節血清中的(A)TNF-α、(B)IL-1β、(C)IL-6(D)TGF-β及(E)白蛋白。TNF-α、IL-1β、IL-6、TGF-β及白蛋白的血清水平由市售ELISA試劑盒測定。數據表示為平均值±S.E.M.(n=5)。###表示與對照組樣本相比p<0.001。與僅使用順鉑組相比,**表示p<0.01,***表示p<0.001。
圖5 ARH005-EA(A)及ARH(B)對順鉑誘導的腎臟TWEAK、α-SMA、P53及P21訊息表達的影響。在順鉑激發後,以西方墨點法分析腎勻漿中TWEAK、α-SMA、P53及P21蛋白表達的水平。
圖6 描繪了建立CCl4誘導纖維化模型的過程
圖7 描繪了(A)重量差,(B)肝臟重量,及(C)肝/體重比。
圖8 描繪了CCl4誘導的肝損傷後大鼠(A)AST,(B)ALT,及(C)AST/ALT的血清水平。
圖9 描繪了肝臟中的(A)炎症,(B)空泡化,(C)壞死,(D)纖維化,(E)總組織學評分。
圖10 肝臟的代表性組織切片以H&E進行染色。
圖11 描繪了建立Con A誘導急性肝炎模型的過程。
圖12 描繪了魚針草內酯(AR100-DS1)對GOT、GPT及體重的影響。Con A誘導後24小時的(A)血清GOT及(B)血清GPT。(C)Con A誘導前後的體重。數據表示為平均值±SEM(n=9)。
圖13 魚針草內酯(AR100-DS1)對肝損傷的影響。(A)Naïve、(B)15mg/kg Con A(Veh)、(C)AR100-DS1及(D)***的肝臟組織病理學,以及(E)壞死的組織病理學評分。
圖14 描繪了建立動脈粥狀硬化兔模型的過程。
圖15 描繪了兔子的初始及最終平均體重。描繪了兔子的初始及最終平均體重。†及*分別表示與對照組及HF組相比P<0.05。
圖16 描繪了每組兔子中W0組之間AST、ALT、BUN的變化,†及*分別表示與對照組及HF組相比P<0.05。
圖17 描繪了各組兔子中W0組之間TG、TC、HDL-C、LDL-C的變化,†及*分別表示與對照組及HF組相比P<0.05。
圖18 描繪了每組兔子中W4組之間AST、ALT、BUN的變化,†及*分別表示與對照組及HF組相比P<0.05。
圖19 描繪了各組兔子中W4組之間TG、TC、HDL-C、LDL-C的變化,†及*分別表示與對照組及HF組相比P<0.05。
圖20 描繪了每組兔子中W8組之間AST、ALT、BUN的變化,†及*分別表示與對照組及HF組相比P<0.05。
圖21 描繪了各組兔子中W8組之間TG、TC、HDL-C、LDL-C的變化,†及*分別表示與對照組及HF組相比P<0.05。
圖22 描繪了每組兔子中W12組之間AST、ALT、BUN的變化,†及*分別表示與對照組及HF組相比P<0.05。
圖23 描繪了各組兔子中W12組之間TG、TC、HDL-C、LDL-C的變化,†及*分別表示與對照組及HF組相比P<0.05。
圖24 描繪了12週研究後高膽固醇血症兔模型中主動脈硬化斑塊病變的組織病理化學檢查。
圖25 描繪了每組兔犧牲後冠狀動脈切片的H&E染色。
圖26 描繪了每組兔犧牲後冠狀動脈切片的H&E染色。N,neointima layer;M,media layer。
圖27 描繪了血管狹窄的表現,表示為neointima layer與media layer面積的比值(N/M比)N,neointima layer;M,media layer。分別與HFD組相比,*表示p<0.05,**表示p<0.01,***表示p<0.001。
圖28 描繪了12週研究後高膽固醇血症兔模型的心臟組織的組織病理化學檢查。
圖29 描繪了12週研究後高膽固醇血症兔模型的肝臟外觀。
圖30 描繪了12週研究後高膽固醇血症兔模型的肝組織病理化學檢查。
圖31 描繪了動物的體重及肺重。
圖32 描繪了小鼠受博來黴素誘導的肺纖維化中肺的組織病理學變化。
圖33 描繪了小鼠受博來黴素誘導的肺纖維化中肺的Masson三色染色。
圖34 描繪了樟芝萃取物及化合物對博來黴素誘導的小鼠肺損傷中羥脯氨酸含量的影響。
圖35 描繪了BALF中樟芝萃取物及化合物調節了(A)TNF-α、(B)IL-1β、(C)IL-6、(D)TGF-β及(E)。
圖36 描繪了樟芝萃取物及化合物對BLM誘導的小鼠肺MPO活性的調節。
為了便於闡述本發明,本發明的上述發明內容所表達的中心思想透過具體的例子表達。實施例中的各個項目是根據適合於說明的比例、尺寸、變形量或位移量來描繪,而不是按照上述實際元件的比例繪製。
術語「萜」(terpenes)是指一大類且多樣的有機化合物,其基本結構遵循一個一般原則:以2-甲基丁烷殘基(2-Methylbutane residues,通常也以異戊二烯單元(isoprene units)或(C5)n稱之)構成萜烯的碳骨架。目前文獻中已知大約有30000種萜烯,根據2-甲基丁烷殘基的數量,可將其區分為半(hemi-,C5)、單(mono-,C10)、sesqui-(C15)、雙(di-,C20)、sester-(C25)、三(tri-,C30)及四(tetra-,C40)萜類化合物。
術語「受試者」、「個體」、「宿主」及「患者」在本說明書中可互換使用以指活的動物,包括人及非人動物。例如,受試者可以是具有能夠響應抗原刺激以及透過細胞表面受體結合進行刺激及抑制信號轉導的免疫細胞
之生物體。受試者可以是哺乳動物。例如人類或非人類哺乳動物,例如狗、貓、豬、牛、綿羊、山羊、馬、大鼠及小鼠。術語「受試者」並未排除在疾病方面完全正常或在所有方面都正常的個體。
術語「治療」,可以施用於患有醫學病症或最終可能獲得該病症的受試者,以預防、治癒、延遲、降低病症或複發病症的一種或多種症狀之嚴重程度,或延長受試者的存活時間。
術語「醫療有效量」是指可引發所需反應之主題化合物的量,該反應包括研究人員、獸醫、醫師或其他臨床人員尋求的組織、系統、動物或人類的生物學或醫學反應。
生化參數的測定 根據製造商的操作說明書,使用比色試劑盒評估血清肌酐(creatinine)及血清尿素(urea)。前者生物標記的試劑盒購自HUMAN Diagnostics Worldwide,Magdeburg,Germany,化學分析儀(Roche Diagnostics,Cobas Mira Plus,Rotkreuz,Switzerland)。
腎臟組織病理學 將每隻小鼠的左外側肝葉前部固定在10%磷酸甲醛緩衝液中,以石蠟包埋,切成5μm切片,以蘇木精-伊紅(hematoxylin and eosin,H&E)染色處理,在光學顯微鏡下進行組織學檢查(Nikon,ECLIPSE,TS100,Tokyo,Japan)。使用數位相機(NIS-Elements D 2.30,SP4,Build 387)以400倍的原始放大率拍攝圖像。
血清中的細胞因子TNF-α、IL-6及IL-1β 血清中促炎細胞因子濃度(TNF-α、IL-6及IL-1β)藉由酵素結合免疫吸附分析法(Enzyme-linked immunosorbent assay,ELISA)試劑盒(Biosource International Inc.,Sunnyvale,CA,USA),依照製造商的操作說明書進行評估。
腎組織的西方墨點法分析 裂解緩衝液由0.6% NP-40、150mM NaCl、10mM HEPES(pH 7.9)、1mM EDTA及0.5mM PMSF組成,在4℃下將肝組織均質化。均質化後的樣品在4℃下以3000轉/分鐘(rpm)離心10分鐘以獲得上清液。以牛血清白蛋白(BSA)標定上清液的總細胞蛋白量。蛋白質樣品(50μg)使用標準方法,以變性10%十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)進行解析,並轉印到PVDF膜(Immobilon,Millipore,Bedford,MA,USA),以10%的脫脂牛奶阻隔。將PVDF膜與適當稀釋的特定一抗在4℃下反應,用TBST緩衝液洗滌3次,然後在37℃下與辣根過氧化物酶偶聯(horseradish peroxidase-conjugated)的二抗反應1小時。將PVDF膜洗滌3次,以ECL試劑(Thermo Scientific,Hudson,NH,USA)檢測免疫反應蛋白,藉由Image J軟體(NIH,Bethesda,MD,USA)與對照組進行比較,量化膠片上的條帶亮度並表示為相對強度。
統計分析 從動物實驗中獲得的數據表示為平均值及平均值的標準誤差(±S.E.M.)。t-test用於檢查多個組之間或兩個組之間的差異。統計顯著性表示為*p<0.05、**p<0.01及***p<0.001。
實施例1 樟芝萃取物的製備
100克樟芝子實體用甲醇回流6小時,收集萃取物並乾燥之,共得到樟芝甲醇萃取物15克。
實施例2 活性成分的製備:Antcin K、去氫硫色多孔菌酸(dehydrosulphurenic acid)/硫色多孔菌酸(sulphurenic acid)、Versisponic acid D及去氫齒孔酸(dehydroeburicoic acid)
樟芝甲醇萃取物以矽膠管柱層析法進一步分離,使用正己烷/乙酸乙酯/甲醇作為層析液,得到以下餾分(如圖1所示):
AR101-DS1(RS-Antcin K)
AR101-DS2(去氫硫色多孔菌酸/硫色多孔菌酸)
AR101-DS3(Versisponic acid D)
AR101-DS4(去氫齒孔酸)
實施例3 ARH003萃取物的製備
100克樟芝(皿培)用甲醇回流6小時,收集萃取物並乾燥之,共得到樟芝ARH003萃取物15克。
實施例4 ARH003-E萃取物的製備
200克樟芝(皿培)用乙醇回流6小時,收集萃取物並乾燥之,共得到樟芝ARH003-E萃取物18克。
實施例5 ARH004萃取物的製備
100克樟芝(木培)用甲醇回流6小時,收集萃取物並乾燥之,共得到樟芝ARH004萃取物18克。
實施例6 ARH005-EA萃取物的製備
100克樟芝(固體培養物)用乙酸乙酯回流6小時,收集萃取物並乾燥之,共得到樟芝EA萃取物12克。
實施例7 魚針草萃取物的製備
魚針草萃取物的製備流程如下:(1)取魚針草乙醇萃取物加入矽膠填充管柱,以沖提液「正己烷/乙酸乙酯」、「正己烷/乙酸乙酯/甲醇」及「甲醇」進行梯度沖提,得到一魚針草分離液;(2)用矽膠填充管柱進一步處理上述魚針草分離液,以沖提液「二氯甲烷」「二氯甲烷/甲醇」「甲醇」進行梯度沖提」,得到一分離的濃縮物;(3)將該分離的濃縮物以「正己烷/乙酸乙酯」溶劑共結晶,得到魚針草微晶。
實施例8 活性成分的製備:魚針草內酯(ovatodiolide,AR100-DS1)
將200克魚針草乙醇萃取物加入矽膠填充管柱(10x15cm),以沖提液「正己烷/乙酸乙酯(比例為10:1、5:1、3:1、1:1)」、「正己烷/乙酸乙酯/甲醇(比例為6:4:1、3:2:1)」及「甲醇」進行梯度沖提,得到一初分離液140克。
取140克上述初分離液加入矽膠填充管柱(10x15cm)進一步分離,以沖提液「二氯甲烷」、「二氯甲烷/甲醇(比例為10:1、5:1、7:3)」及「甲醇」進行梯度沖提,得到一分離的濃縮物。該分離的濃縮物進一步以溶劑「正
己烷/乙酸乙酯」再結晶,得到一晶體。該晶體經核磁共振氫譜(H1-NMR)鑑定化學結構為魚針草內酯的雙萜化合物。經高效液相層析儀(HPLC)分析,將該晶體與魚針草內酯標準品進行比對,確認為魚針草內酯化合物。
魚針草內酯(AR100-DS1)的代謝物:
+O,+Cysteine:m/z:466,M2,M3,M4
+Glutathione:m/z:636,M6,M7
+O:m/z:345,M8,M9
實施例9 順鉑誘導的腎損傷小鼠模型
七至八周齡大的雄性C57BL/6小鼠取得自BioLASCO Taiwan Co.,Ltd.(Taipei,Taiwan)。實驗前將動物圈養在壓克力籠中,溫度為22±1℃,相對濕度為55±5%,在12小時的暗光循環中持續至少2週,動物供自由採食及飲水。所有實驗程序均按照機構動物倫理委員會的指導方針進行,經管理及監督動物實驗委員會批准。
腎纖維化經由多次注射低劑量順鉑誘導。在第0、1及3週進行腹膜內注射順鉑(5mg/kg/每次注射;P4394,Sigma-Aldrich,St Louis,MO),共注射3次。小鼠在第一劑順鉑6周後犧牲(n=6)。為了分析樣品的影響,從第一劑順鉑後4週開始,每天對小鼠進行腹腔注射樣品,持續7天,並在4週時犧牲(n=6)。
實施例10 樟芝萃取物及化合物減少順鉑誘導小鼠的腎功能障礙及組織病理學變化
腎臟的形態變化如圖2A所示。CRE及BUN是腎功能的指標,圖2B及2C顯示,相比於對照組,注射順鉑3劑10mg/kg(在第0、1、3週)的小鼠顯著增加了血清CRE及BUN水平(p<0.001),表示順鉑處理的小鼠產生腎毒性。與順鉑刺激組相比,經歸一化的CRE及BUN證明了用1000mg/kg劑量的ARH005-EA及ARH003-E以及化合物AR101-DS4及AR100-DS1治療的小鼠,以劑量依賴性發揮顯著的腎臟保護作用(p<0.001)。
實施例11 樟芝萃取物及化合物減輕經多次順鉑處理而誘導的腎功能障礙及腎損傷
分析組織病理學變化以確定樟芝萃取物及化合物是否影響順鉑刺激的小鼠腎功能衰竭。對照組腎組織完全正常,呈透明管狀及腎小球結構,細胞核清晰正常。在順鉑刺激的小鼠中,腎臟具有嚴重的損傷,導致腎小管上
皮受損、炎性細胞浸潤、腎小管細胞腫脹、腎小管內管型(intratubular cast)形成及腎小管擴張。然而,施用1000mg/kg劑量的樟芝萃取物(AR005-EA)及化合物(AR100-DS1)顯著改善了腎組織中的壞死及炎症浸潤細胞(見圖3)。
實施例12 樟芝萃取物及化合物減輕順鉑誘導的促炎細胞因子及白蛋白的變化
血清中促炎細胞因子TNF-α、IL-1β、IL-6及TGF-β水平的評估以ELISA進行。與對照組相比,施用順鉑的腎損傷小鼠血清中的NO、TNF-α、IL-1β及IL-6水平顯著增加(分別為圖4A-4E)。以1000mg/kg劑量的樟芝萃取物(AR005-EA)及化合物(AR100-DS1)治療顯著改善腎組織中的壞死及炎症浸潤細胞,也降低NO、TNF-α、IL-1β及IL-6被順鉑激發後的產量。
實施例13 抑制順鉑誘導的腎損傷之TWEAK、α-SMA、P53及P21蛋白表達
檢視施用樟芝萃取物(ARH005-EA)及化合物(AR100-DS1)預處理對抑制順鉑誘導的TWEAK、α-SMA、P53及P21蛋白表達的效果。實驗結果顯示,先施用ARH005-EA及ARH,抑制了順鉑激發後腎組織中TWEAK、α-SMA、P53及P21的蛋白質表達(圖5A及5B)。
實施例14 四氯化碳(CCl
4
)誘導的大鼠慢性肝纖維化
如圖6所示,八周大的雄性SD大鼠每週施用0.4毫克/公斤的四氯化碳,持續8週。在第0、2、4、6及8週收集血液樣本,第8週結束時犧牲動物進行組織病理學檢查。圖7A、7B、7C分別描繪了重量變化、肝臟重量及肝臟/體重比。空白組(Naïve)肝臟重量與載體組(Vehicle)無顯著差異,然而空白組的肝臟/體重
比明顯小於載體組。50mg/kg AR100-DS1組的肝臟重量及肝臟/體重比顯著大於載體及空白組。
實施例15 血清肝酵素分析
評估臨床生化水平,例如天冬氨酸氨基轉移酶(aspartate aminotransferase,AST)、丙氨酸氨基轉移酶(alanine aminotransferase,ALT),以確定對照組及實驗組肝臟的酶活性(如圖8A-8C所示)。空白組的AST、ALT及AST/ALT比值在實驗過程中無明顯變化。各試驗組動物血清AST、ALT水平隨著實驗的進行而顯著升高;然而與載體組相比,在第6週及第8週,50mg/kg AR100-DS1組中可以觀察到較少的AST及ALT增加。
實施例16 肝臟組織學評估
四氯化碳誘導8週後,載體組明顯出現AST及ALT升高、AST/ALT比值降低、炎症、纖維化、空泡化及壞死等肝損傷。如圖9A-9E及10所示,50mg/kg AR100-DS1組的肝臟表面光滑,無萎縮及硬化,肝臟重量及肝臟/體重比明顯大於載體及空白組。整體而言,AR100-DS1證明具有部分修復四氯化碳誘導的肝損傷之潛力。
實施例17 魚針草內酯(AR100-DS1)對BALB/c小鼠中Con A蛋白(concanavalin A)誘發的急性肝炎之影響
靜脈注射Con A蛋白是研究T細胞介導肝炎的廣泛使用策略之一。Con A蛋白是一種凝集素(lectin),可以活化CD4+ T細胞,產生細胞因子,並導致肝細胞損傷。***(Dexamethasone,Dex)是一種長效的合成皮質類固醇,用作抗炎及免疫抑制藥物。在BALB/c小鼠中評估了魚針草內酯(AR100-DS1)對血清穀氨酸-丙酮酸轉氨酶(glutamic-pyruvic transaminase,GOT)、谷氨酸-草酰
乙酸轉氨酶(glutamic-oxaloacetic,GPT)、循環細胞因子及肝臟組織病理學對Con A所誘導之急性肝炎的影響。
Con A及Dex購自Sigma Aldrich(美國)。ProcartaPlexTM免疫分析試劑盒購自Corning Inc.(美國)。Fuji Dri-Chem Slide GOP/GPT血清測試套組購自Winning Medical Inc.(台灣)。
雄性BALB/c小鼠(7-9週齡)購自BioLASCO Taiwan Co.,Ltd或國家實驗動物中心(NLAC,台灣)。在整個實驗過程中,每籠飼養五隻動物,供自由採食及飲水。室溫保持在23±2℃,交替進行12小時明暗循環。動物在實驗前適應一周以將壓力的影響降至最低。所有涉及動物及其護理的實驗方案均由ITRI的機構動物護理及使用委員會(IACUC)批准(ITRI-IACUC-2018-041及ITRI-IACUC-2018-050;由AAALAC認可),並根據根據台灣農業委員會的規定進行。
Con A以3mg/mL的濃度溶於無熱原(pyrogen free)食鹽水中,並以15mg/kg或20mg/kg體重的劑量靜脈注射以誘發肝炎。魚針草內酯(AR100-DS1)及Dex在施用Con A的30分鐘前、4小時後及8小時後口服給藥。施用Con A的24小時後收集血液及肝臟組織(圖11)。血清儲存於-80℃直到取出分析。
為評估施用Con A後肝細胞損傷的程度,以Fuji Dri-Chem Slide套組測量血清GPT及GOT水平。同一組的血清匯集後用於細胞因子測定。依照製造商的說明書,以ProcartaPlexTM免疫測定試劑盒測量細胞因子水平。數據表示為平均值±SEM。t檢定用於分析藥物治療組及載體組之間的差異。當p值小於0.05時,差異被認為具有統計學意義。50mg/kg的魚針草內酯(AR100-DS1)顯著降低
了因Con A而增加的GPT水平(109±25對368±107U/L,p<0.05),並稍微改善了GOT的升高量(261±45對比410±56U/L)(圖12)。
將肝組織固定在10%磷酸鹽緩衝的甲醛中,包埋在石蠟中,並用蘇木精及伊紅(H&E)染色,以確認組織病變。由BioLASCO Taiwan Co.,Ltd.的獸醫病理學家通過顯微鏡檢查組織病變。所有微觀病變嚴重程度分級系統的標準從0到4分級如下:0=無;1=單個細胞壞死;2=30%小葉壞死;3=60%小葉壞死;4=>60%小葉壞死。組織病理學分析顯示魚針草內酯(AR100-DS1)改善了肝壞死(評分0.2±0.2 vs 1.4±0.2,p<0.05)(圖13)。綜合以上結果顯示,魚針草內酯(AR100-DS1)可降低血清GOP及GPT,並減輕Con A誘導的肝壞死。
實施例18 樟芝萃取物及AR101-DS2預防動脈粥樣硬化及肝纖維化的功效評價
實驗模型
將2至3公斤的雄性紐西蘭白兔單獨關在籠子裡,飼養在溫度及濕度可控的房間裡,明暗週期各為12小時。經過幾天的適應後,動物被依次分配到六個餵養組:標準兔糧、含有0.5%膽固醇的標準兔糧、含有0.5%膽固醇及10mg/kg洛伐他汀(Lovastatin)的標準兔糧、含有0.5%膽固醇及1% ARH003的標準兔糧、含有0.5%膽固醇及1% ARH004的標準兔糧、含有0.5%膽固醇及10mg/kg AR101-DS2的標準兔糧。除標準兔糧組外,其餘各組給予含0.5%膽固醇的標準兔糧4週(見圖14-15)。每隻兔子的每日餵食量為每天50克/公斤體重。在動物適應新環境後,進行8週的飲食。在12週研究的開始及結束時,通過肌肉注射Zoletil 50(1mL/kg)(Virbac Ltd.,France)麻醉兔子,並採集血液樣本。最後,在犧
牲兔子後收集主動脈(從主動脈弓到髂動脈分叉處)及整個肝臟,用於進一步的組織病理學分析。
2至3公斤雄性紐西蘭白兔(n=30)分為以下各組:
(ND)標準兔糧,n=5;
(HF)含0.5%膽固醇的標準兔糧,n=6;
(L)含0.5%膽固醇及10mg/kg洛伐他汀的標準兔糧,n=4;
(AR003)含0.5%膽固醇及1% ARH003的標準兔糧,n=5;
(AR004)含0.5%膽固醇及1% ARH004的標準兔糧,n=5;
(AR101-DS2)含0.5%膽固醇及10mg/kg AR101-DS2的標準兔糧,n=5;
每隻兔子的每日餵食量為每天50克/公斤體重。
血液化學分析
動物在抽血前禁食過夜,血液從兔子的耳緣靜脈收集到BD Vacutainer EDTA血液收集管中。在4℃下以3000rpm離心10分鐘來分離血漿。圖16-23描繪了血液化學參數變化的測量值,包括低密度脂蛋白(LDL)、膽固醇(Chol)、三酸甘油酯(triglycerides,TG)、谷氨酸草酰乙酸轉氨酶(GOT)及谷氨酸丙酮酸轉氨酶(GPT)的血清水平。
主動脈硬化斑塊染色(Aortic Fatty Streak Staining)
縱向打開主動脈以暴露內膜表面並用生理鹽水輕輕沖洗(見圖24-26)。將主動脈在2%(w/v)Sudan IV中培養,用數種濃度(100%、90%、80%、70%及60%)的乙醇沖洗1分鐘,然後用純水沖洗。圖28中顯示的照片是使用數位相機(Nikon D80,日本)拍攝的,並在Alpha Imager 2200文件系統(Alpha
Innotech,美國)上進行量化。硬化斑塊病變的進展以染色面積佔總面積的百分比表示(圖27)。
方法
1.水合細胞或組織:
i.使用帶有冷凍切片或再水化組織切片的顯微鏡載玻片(參見石蠟包埋組織的切割切片中的步驟12)(Fischer等人,2008年),用酒精或醛基固定劑固定。
ii.用手攪拌將載玻片浸入H2O中30秒。在H2O中沖洗很重要;蘇木精與鹽及緩衝液一起沉澱。染色可以在免疫組織化學或雜交反應後用非熒光檢測系統進行。
2.將載玻片浸入裝有Mayer’s蘇木精的Coplin染色缸中並攪拌30秒。
3.在H2O中沖洗載玻片1分鐘。估計此時的染色強度,如有必要,重複步驟2及3。
4.用1% eosin Y溶液將載玻片染色10-30秒並攪拌。
5.用兩次95%酒精及兩次100%酒精使切片脫水,每次30秒。
6、Extract the alcohol with two changes of xylene。如果在塑膠培養皿中使用塑膠載玻片或染色,請勿使用二甲苯或基於二甲苯的封固劑,因為它們會溶解塑膠。如果在塑料培養皿中使用塑料載玻片或染色,請勿使用二甲苯或基於二甲苯的封固劑,因為它們會溶解塑料。
7.加入一兩滴封固劑並蓋上蓋玻片。如果不能使用酒精,請使用甘油或其他水性封固劑封固蓋玻片。
試劑
顯微鏡載玻片上感興趣的細胞或組織(參見方法1.i)
Eosin Y(1%水溶液;EM診斷系統)
乙醇(95%,100%)
可以使用甲醇或Flex alcohols(Richard-Allan Scientific)代替乙醇(參見步驟5)。
Mayer的蘇木精是最容易使用的,並且與大多數比色底物兼容。
Mounting medium(Canada Balsam,Sigma C1795)
如果不能使用酒精,請使用甘油或其他水性封固劑(參見步驟7)。
二甲苯(Xylene)
肝組織冷凍切片
兔肝組織(如圖29所示)用生理鹽水灌注並在10%(v/v)福馬林中和溶液(J.T.Baker,Inc.,USA)中固定24小時,接著將組織包埋在Tissue Tek OCT Compound(#4583;Sakura Finetek Inc.,美國)中。將包埋的組織切成10μm厚的切片,並用Sudan IV及蘇木精(Merck,美國)染色。簡言之,將切片用純水洗滌1分鐘以去除OCT化合物,用50%(v/v)乙醇洗滌30秒,然後用2%(w/v)Sudan IV染色1小時。用50%(v/v)乙醇及純水進一步洗滌2分鐘後,切片用蘇木精複染。圖30中顯示的照片是使用配備10倍放大物鏡的顯微鏡獲得的,並在Alpha Imager 2200文件系統(Alpha Innotech,美國)上進行量化。脂肪肝進展的表現以油滴面積佔總肝組織(細胞)的百分比表示。
脂肪肝評分
0:low-to medium-power evaluation of parenchymal involvement<5%
1:5-33%
2:33-66%
3:>66%
位置
0:區域3,小葉中心
1:2區,中區
2:3區,口周
3:panacinar
纖維化評分
0:無
1:輕度竇週或門靜脈周圍
2:竇周及門靜脈/門靜脈周圍
3:橋接纖維化
4:肝硬化
炎症評分
0:無病灶
1:輕度,每200個視野2個病灶
2:中等,每200個視野2-4個病灶
3:嚴重,每200個視野4個病灶
實施例19 樟芝萃取物及化合物對博來黴素(bleomycin)誘導小鼠肺纖維化的保護作用
動物及處置
無特定病原體的雄性ICR小鼠(體重18-22g)購自BioLASCO Taiwan Co.,Ltd.(台北,台灣)。實驗前將動物圈養在樹脂玻璃籠中,恆溫22±1℃,相對濕度55±5%,12小時暗光循環至少2週,動物供自由採食及飲水。所有實驗程序均按照機構動物倫理委員會的指導方針進行,該協議經委員會批准,用於控制及監督動物實驗。
博來黴素(bleomycin,BLM)誘導的小鼠肺纖維化
小鼠按體重分為以下各組,每組5只:對照組、BLM組、BLM+DEX組(7.5mg/kg)、BLM+ACH劑量組(ARH003 ext.1.0g/kg)、BLM+ACM劑量組(ARH003 ext.0.5g/kg)、BLM+AH劑量組(AR101-DS1 50mg/kg)、BLM+AM劑量組(AR101-DS1 25mg/kg)、BLM+BH劑量組(AR101-DS2 50mg/kg)及BLM+BM劑量組(AR101-DS2 25mg/kg)BLM+CH劑量組(AR101-DS4 50mg/kg)及BLM+CM劑量組(AR101-DS4 25mg/kg)BLM+DH劑量組(AR100-DS1 50mg/kg)及BLM+DM劑量組(AR100-DS1 25mg/kg)BLM+EH劑量組(ARH013-RA1 50mg/kg)及BLM+EM劑量組(ARH013-RA1 25mg/kg)BLM。以單次7.5mg/kg體重氣管內施用BLM在小鼠中建立肺纖維化(PF)。BLM損傷
後21天每天灌胃不同劑量的樣品,以DEX作為陽性對照。對照組及實驗組使用相同的時間表及給藥途徑接受等體積的載體(0.9% NaCl)。
每天記錄小鼠體重,在第21天使用過量的水合氯醛麻醉劑犧牲小鼠,採集血液用於ELISA分析,取出整個肺並稱重。右肺用10%福馬林固定、脫水、石蠟包埋。左肺用於測定羥脯胺酸(hydroxyproline)。肺比重計算公式如下:肺重/體重×100%
實驗設計
雄性C57BL/6小鼠隨機分為以下8組:
(n=6):
1.第一組:對照組;
2.第二組:小鼠接受單次腹腔注射BLM(7.5mg/kg)
3.第三組:單劑量(ACH,ARH003 ext.1.0g/kg)
4.第四組:單劑量(ACM,ARH003 ext.0.5g/kg)
5.第五組:純化的AR101-DS1(50mg/kg)
6.第六組:純化的AR101-DS1(25mg/kg)
7.第七組:純化的AR101-DS2(50mg/kg)
8.第八組:純化的AR101-DS2(25mg/kg)
7.第七組:純化的AR101-DS4(50mg/kg)
8.第八組:純化的AR101-DS4(25mg/kg)
7.第七組:純化的AR100-DS1(50mg/kg)
8.第八組:純化的AR100-DS1(25mg/kg)
7.第七組:純化的ARH013-RA1(50mg/kg)
8.第八組:純化的ARH013-RA1(25mg/kg)
BALF取樣
在麻醉下,通過氣管插管用0.7mL生理鹽水進行四次BALF。在檢查的每隻小鼠中,回收了約2.5mL(90%)的BAL液(BALF)。BALF的上清液保存在-80℃備用。
肺組織病理學
每隻小鼠的右肺前部固定在10%磷酸甲醛緩衝液中,石蠟包埋,切成5μm切片,然後用蘇木精及伊紅(H&E)染色處理,在光學顯微鏡下進行組織學檢查(Nikon,ECLIPSE,TS100,日本東京)。使用數位相機(NIS-Elements D 2.30,SP4,Build 387)以400倍的原始放大率拍攝圖像。
羥脯氨酸(hydroxyproline)的測定
按照羥脯氨酸測定試劑盒(Biosource International Inc.,Sunnyvale,CA,USA)的說明在肺組織中分析羥脯氨酸的含量。將小鼠肺組織磨碎,用1ml 6mol/L氯化鉀溶液勻漿,95℃水解5小時,調pH值至6.0-6.8。根據說明書,將相應的試劑加入反應體系中並充分混合,然後在60℃下培養15分鐘。冷卻後,以3500rpm離心10分鐘後收集上清液。用分光光度計在550nm處測量樣品上清液的吸光度值,並計算各組羥脯氨酸的含量。
血清中的TNF-α、IL-6及IL-1β細胞因子
依照製造商的說明書,使用酵素結合免疫吸附分析法(ELISA)試劑盒(Biosource International Inc.,Sunnyvale,CA,USA)評估血清中促炎細胞因子(TNF-α、IL-6及IL-1β)的血清濃度。
髓過氧化物酶(Myeloperoxidase,MPO)檢測
肺部的MPO活性是評估肺部炎症細胞浸潤的可靠指標。將肺組織勻漿,並根據製造商的說明書用試劑盒檢測MPO水平。
肺組織病理學分析
將右肺用石蠟包埋,10%福馬林固定,加上成切片。切片用蘇木精及伊紅(H&E)染色或進行Masson’s三色染色。
統計分析
從動物實驗中獲得的數據表示為平均值及平均值的標準誤差(±S.E.M.),t檢驗用於檢查多個組之間或兩個組之間的差異,統計顯著性表示為*p<0.05、**p<0.01及***p<0.001。
在整個實驗結束時,記錄動物的體重及肺重。與對照動物相比,施用博來黴素(BLM)的動物的體重變化顯著降低。與其他實驗組相比,肺指數[(肺重/體重)×100]顯示施用博來黴素的動物顯著增加(下表及圖31)。ACH、BH及DH的肺指數顯著降低。
樟芝萃取物及化合物對博來黴素誘導肺纖維化的肺指數
實施例20 樟芝萃取物及化合物減少BLM誘導的小鼠的肺功能障礙及組織病理學變化
評估小鼠的組織病理學肺變化,以探索樟芝萃取物及化合物的治療效果。通過H&E染色觀察到組織結構的炎症浸潤及完整性(圖32);肺組織的纖維化程度通過Masson染色進行(圖33)。對照組表現出一些組織學現象,如肺泡壁薄,肺泡結構完整,肺泡間隔正常,肺間充質炎症細胞浸潤較少BLM給藥21
天後,觀察到肺泡水腫、隔膜寬度顯著增加及炎症細胞浸潤增加。與BLM組相比,樟芝萃取物及化合物的給藥改善了肺組織中的炎症浸潤及受損結構。
在BLM給藥後21天後,Masson染色在肺組織及隔膜中廣泛染成藍色,顯示BLM組的肺纖維化程度比正常組嚴重。樟芝萃取物及化合物治療後,藍色區域減少,纖維化程度減輕。在BLM誘導後21天,樟芝萃取物及化合物治療後肺泡炎及纖維化的評分顯著降低。上述結果表明,樟芝萃取物及化合物減輕了肺纖維化小鼠肺部的炎症及纖維化程度。
實施例21 肺纖維化標記
羥脯氨酸(HP)含量是肺組織膠原沉積的重要指標。為了量化肺纖維化的程度,在每組中測量肺組織中的羥脯氨酸含量並顯示在圖34中。與對照組相比,BLM明顯增加了HP含量(p<0.001)。樟芝萃取物(1.0g/kg)及AH、BH及DH顯著降低肺的HP回升(p<0.001)。
實施例22 樟芝萃取物及化合物提醒博來黴素誘導的促炎細胞因子的變化
以ELISA進行血清中促炎細胞因子TNF-α、IL-1β、IL-6及TGF-β水平的評估。與對照組相比,BLM治療的腎損傷小鼠血清中的NO、TNF-α、IL-1β及IL-6水平顯著增加(分別為圖35A-34E)。以1.0g/kg的樟芝萃取物及化合物(BH及DH)治療顯著改善肺組織中的壞死及炎性浸潤細胞,並改善了BLM誘導後TNF-α、IL-1β、IL-6及TGF-β的生成(p<0.001)。
實施例23 樟芝萃取物及化合物對肺部MPO活性的影響
如圖36所示,與對照組相比,響應BLM誘導的MPO水平顯著增加(p<0.01)。相反地,樟芝萃取物AH、BH、DH及Dex與BLM組相比明顯抑制MPO活性(p<0.001),其作用強於樟芝萃取物及化合物組(p<0.05)(圖36)。
Claims (5)
- 一種魚針草(Anisomeles indica)之萃取物用於製備預防或治療纖維化病症之醫藥組合物的用途,包括向有需要的受試者施用醫療有效量的該醫藥組合物;其中該纖維化病症為腎纖維化、血管纖維化、肺纖維化及良性***增生。
- 如請求項1所述之用途,其中該萃取物係利用乙醇萃取獲得。
- 如請求項2所述之用途,其中該萃取物經乙醇萃取後進一步引入一正相層析管柱(normal phase chromatography column),以己烷/乙酸乙酯/甲醇沖提獲得。
- 一種組合物之用途,其係用於製備預防或治療纖維化病症之藥物,包括向有需要的受試者施用醫療有效量之該組合物;其中該組合物包含魚針草內酯(ovatodiolide),該纖維化病症為腎纖維化、血管纖維化、肺纖維化及良性***增生。
- 如請求項1或4所述之用途,其中該組合物進一步減輕腎功能障礙及腎損傷。
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