TWI448294B - 增強免疫活性之樟芝子實體水萃取物及其製備方法 - Google Patents
增強免疫活性之樟芝子實體水萃取物及其製備方法 Download PDFInfo
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Description
本案係關於樟芝子實體萃取物及其製備方法。尤其，本案係關於一種樟芝子實體水萃取物及其製備方法，對樹突細胞具有免疫刺激的效果。
樟芝(Antrodia camphorata
)又稱樟菇、牛樟菇、牛樟芝等，為台灣特有的真菌菌種，生長於海拔400至2000公尺特有的牛樟樹(Cinnamomum kanehirai
)樹幹腐朽的心材內壁，或枯死倒伏的牛樟木材陰暗潮濕的表面。因此，要尋找到野生的樟芝子實體(fruiting body)或確認此多孔菌目(Aphyllophorales)真菌菌株的外觀並不容易，也由於其生物活性成分具潛在的醫藥價值，因此樟芝的價格居高不下。
由於樟芝子實體不易採集也難以人工方式培養，目前市面上多為樟芝菌絲體(mycelia)產品，其宣稱具有抗癌、減少治療引起的症狀、減少副作用、抗氧化、抗過敏、免疫刺激效果等。
在免疫反應中，樹突細胞(dendritic cell,DC)受到刺激與活化，成為抗原呈現細胞，表現抗原以與未成熟的T淋巴球辨識(3)
，而樹突細胞的成熟及功能受細胞介素(cytokine)及共刺激信號(costimulatory signals)調節(6)
。近來研究發現，以樹突細胞為基礎的免疫療法可應用於治療惡性腫瘤上(1)
。成熟過程(maturation process)為樹突細胞的主要功能，可使細胞依序執行不同的、高度分化的功能。而許多刺激物，包括脂多醣體(lipopolysaccharide,LPS)，具有誘導樹突細胞成熟的功能。
而菇類所產生的多醣體，在現今研究中發現其可成為一種誘使樹突細胞成熟及功能活化的新藥劑。所以，在尋找新的免疫治療藥劑時，於傳統醫學中被應用治療癌症的食藥用真菌被視為極具潛力的候選藥物，其中以樟芝最為國人所知曉。
中華民國專利號I299665揭露了樟芝萃取物及其製備方法，其係以乙醇萃取樟芝菌絲體獲得多醣體，用以抑制基質金屬蛋白酶的活性，但並非以樟芝子實體進行萃取，其產物也未能抑制癌細胞的生長；中華民國專利號I279439揭露了以樟芝菌絲體進行培養，藉由調整培養時的酸鹼值獲得到培養物，並未揭露萃取方法；而中華民國專利號591110揭露了由樟芝菌絲體萃取出γ-胺基丁酸，其係先以冷凍乾燥樟芝菌絲體，再以水或有機溶劑萃取。上述這些發明皆非以樟芝子實體進行有機溶劑或水萃取，而且未確認樟芝子實體水萃取物涉及在樹突細胞免疫反應中的功效。
本案申請人鑑於習知技術中的不足，經過悉心試驗與研究，並一本鍥而不捨之精神，終構思出本案「增強免疫活性之樟芝子實體水萃取物及其製備方法」，能夠克服先前技術的不足，以下為本案之簡要說明。
本發明以水萃取樟芝子實體，獲得樟芝子實體水萃取物，此可有效促進免疫反應及刺激樹突細胞。再分別以乙酸乙酯、乙醇及水進一步萃取樟芝子實體水萃取物，獲得到樟芝子實體乙酸乙酯、乙醇萃取物及水再萃取物，確定其含有的多醣體可促進樹突細胞成熟及活化T細胞。
本發明提供一種樟芝子實體水萃取物的製備方法，包括下列步驟：(a)研磨樟芝子實體成粉狀；及(b)水煮、迴流萃取該粉狀的樟芝子實體，過濾及離心，獲得樟芝子實體水萃取物。
根據上述構想，該製備方法還包括下列步驟：(c)以至少一有機溶劑萃取該樟芝子實體水萃取物，獲得樟芝子實體有機溶劑萃取物。
根據上述構想，該有機溶劑包括乙酸乙酯、乙醇及其組合，該步驟(c)還包括：(c1)以乙酸乙酯萃取該樟芝子實體水萃取物，獲得樟芝子實體乙酸乙酯萃取物及第一樟芝子實體殘留物；及(c2)以乙醇萃取第一樟芝子實體殘留物，獲得樟芝子實體乙醇萃取物及第二樟芝子實體殘留物。
根據上述構想，製備方法還包括下列步驟：(d)以水萃取第二樟芝子實體殘留物，獲得樟芝子實體水再萃取物。
根據上述構想，樟芝子實體水再萃取物的1
H核磁共振圖譜在化學偏移δ3.0-5.5之醣類化合物特徵訊號比例最高。而且在化學偏移4.44、4.46、4.91、5.00及5.15ppm處呈現多醣體結構中異位性碳原子(anomeric carbon)上氫的特徵訊號。
本發明另提出一種樟芝子實體水萃取物，其係由研磨樟芝子實體，並水煮迴流萃取、過濾及離心該樟芝子實體所獲得。
根據上述構想，樟芝子實體水萃取物經由活化PI3K/Akt及p38/MAPKs訊息傳遞路徑而誘發樹突細胞成熟，促進T細胞增生及干擾素γ的分泌，趨化輔助性T細胞(T helper 1)反應。此外，樟芝子實體水萃取物亦經由抑制細胞凋亡作用促進樹突細胞之活性。
本案所提出之「增強免疫活性之樟芝子實體水萃取物及其製備方法」將可由以下的實施例說明而得到充分瞭解，使得熟習本技藝之人士可以據以完成之，然而本案之實施並非可由下列實施例而被限制其實施型態，熟習本技藝之人士仍可依據除既揭露之實施例的精神推演出其他實施例，該等實施例皆當屬於本發明之範圍。
依照過去文獻製備多醣體的方法(5)
，分別將乾燥的樟芝(Antrodia camphorata
)、靈芝(Ganoderma lucidum
)及姬松茸(Agaricus blazei
)的子實體研磨成細粉，再以1：10比例(重量/體積)水煮、迴流萃取8小時，進一步將上清液過濾、以3,000rpm離心30分鐘以移除沈澱物，再進行冷凍乾燥。所獲得的樟芝水萃取物(ACW)、靈芝水萃取物(GLW)及姬松茸水萃取物(ABW)保存於-70℃。此外，以酚-硫酸法測定其多醣體含量(2)
。
將216.4mg的樟芝子實體水萃取物以極性漸增的溶劑(依序為乙酸乙酯(ethyl acetate)、乙醇及水)萃取，並依序獲得8.9%(19.3mg)樟芝子實體乙酸乙酯萃取物、38.5%(83.3mg)樟芝子實體乙醇萃取物及52.6%(113.8mg)樟芝子實體水再萃取物三種不同的萃取物。百分比表示佔樟芝子實體水萃取物之重量百分比，毫克表示對應的萃取後的乾重。各萃取物以酚-硫酸法測定其多醣體含量(2)
，並以核磁共振圖譜分析其光譜特性。
上述的萃取物溶解於氫的同位素(氘，deuterium)溶劑中，例如D2
O及C5
D5
N等，再以Varian Unity Inova-600MHz NMR測定1
H核磁共振圖譜。化學位移(chemical shift)以ppm(δ)表示。
樹突細胞主要以Lin等人於2006發表的方法製備並加以修飾(4)
。首先，以12位健康捐贈者並以Ficoll-Hypaque密度低度離心法(Amersham Bioscience,Uppsala,Sweden)獲得周邊血液單核球細胞(PBMC)。將周邊血液單核球細胞培養於添加10%胎牛血清、2mM麩胺酸、100μg/ml鏈黴素及100U/ml盤尼西林的RPMI 1640培養基中2小時。移去未附著的細胞，以尼龍絲分離法(nylon wool separation)純化T細胞，做為單核T淋巴球的來源。將T細胞注入管柱，而被洗下的非附著性細胞富含T細胞。以流式細胞儀分析最終獲得的細胞中，80%的T細胞具有高度表現的CD3蛋白。將附著的細胞培養於添加50ng/ml顆粒單核球群落刺激生長因子(GM-CSF)及介白素-4(interleukin-4,IL-4)的RPMI 1640培養基中6天，每隔2至3天以含有生長因子的新鮮培養液替換原先一半體積的PRMI 1640培養液。於第6天收集細胞，並將細胞與抗CD11C+
的微珠混合，並以mini MACs系統(Becton Dickinson,Mountain View,CA)進行。超過90%的CD11C陽性細胞為未成熟的樹突細胞，此未成熟的樹突細胞再分別以PBS、100ng/ml脂多醣體及不同劑量的樟芝水萃取物處理2天，再以流式細胞儀分析。
經過處理的樹突細胞先於37℃、以25ng/ml的絲裂黴素C(mitomycin C)處理30分鐘，再以培養液清洗3次，將樹突細胞與T細胞分別以1：10及1：20的比例混合，再將混合後的細胞培養於96孔盤5天。在第5天的最後4小時，每個孔洞加入20μl WST-1(Roche,Germany)。T細胞增殖的程度下列公式計算：450nm波長的吸光值-650nm波長的吸光值(背景值)。收集細胞培養後的上清液，並以酵素結合免疫吸附分析法(ELISA)分析IL-4及干擾素γ(interferon-γ,IFN-γ)含量。
為了分析樹突細胞的外在表現型，於37℃、以螢光染料(fluorochrome)結合抗體或同型匹配對照抗體(isotype-matched control antibody)標定樹突細胞30分鐘，再以FACScalibur流式細胞儀及Cell Quest軟體(Becton Dickinson,Mountain View,CA)分析細胞。
本實驗是以高敏感性人類細胞介素套組(R＆D Systems,Minneapolis,U.S.A.)測定樹突細胞或T細胞的培養上清液中的介白素-12、介白素-10、干擾素-γ及介白素-4含量。
本實驗是試驗樹突細胞通過transwell chamber的8μm孔徑聚碳酸酯濾膜(Polycarbonate filter)的遷移能力，以了解樹突細胞的化學趨性。首先，分別以脂多醣體、25μg/ml及50μg/ml的樟芝子實體水萃取物處理樹突細胞48小時。在transwell plate的孔洞中加入含(或不含)T淋巴球(2×106
cells/ml)的500μl無血清培養液，而在transwell chamber中加入含樹突細胞(1×105
cells/0.1ml)的無血清培養液。將transwell chamber置於transwell plate的孔洞中，使樹突細胞於37℃遷移3小時。收集遷移至transwell plate孔洞的樹突細胞，並以流式細胞(計數60秒)分析遷移後的樹突細胞數目。
以脂多醣體或特定劑量的樟芝水萃取物刺激樹突細胞，再以RIPA試劑(含140mM NaCl、10mM EDTA、10% glycerol、1% Nonidet P-40、20mM Ths(PH 7.0)、1μM pepstatin、1μg/ml aprotinin、1μg/ml leupeptin及1mM sodium orthovanadate)裂解細胞，並以BCA蛋白試劑組(Pierce,Rockford,IL)測定蛋白濃度。膠體上的每個孔洞注入20μg蛋白，再進行SDS-PAGE分析，之後將解析凝膠轉漬至硝化纖維膜。依序以初級抗體偵測蛋白、再以HRP酵素連結的二級抗體偵測初級抗體、最後以LumiGLo試劑(Pierce)呈色。
為了分析樟芝水萃取物對樹突細胞成熟的訊息傳導路徑，分別以ERK抑制劑PD98059(25μM)、JNK抑制劑SP600125(25μM)、p38 MAPK抑制劑SB203580(25μM)或PI3K/Akt抑制劑LY294002(25μM)處理樹突細胞2小時，再以樟芝子實體水萃取物刺激樹突細胞。2天後，分析樹突細胞的表現特徵及樹突細胞上清液中的介白素-12產量。
本發明的統計分析是以Student’s t test進行，p值小於0.05表示具有顯著差異。
為了比較各食藥用真菌之多醣體對樹突細胞成熟的影響，將未成熟的樹突細胞分別與樟芝、靈芝以及姬松茸子實體水萃取物進行培養，以流式細胞技術分析輔助性刺激分子CD86及HLA-DR的表現。請參閱第1圖，為樟芝、靈芝及姬松茸子實體水萃取物分別刺激未成熟的樹突細胞後的CD86及HLA-DR表現比較圖。各種處理後的平均螢光強度是計算與PBS相較後的倍數而得。由第1圖可知，以靈芝及姬松茸子實體水萃取物(GLW及ABW)並無法如同樟芝子實體水萃取物(ACW)一樣具有可促進樹突細胞表型成熟的能力。進一步以酚-硫酸法分析這三種子實體水萃取物的多醣體含量，發現ACW(82.6%)具有比GLW(52.3%)及ABW(28.3%)含量更高的多醣體(第2圖)。
為了更進一步確認多醣體是樟芝子實體水萃取物中促進樹突細胞成熟的活性成分，請參閱第3圖，為本發明實施例的樟芝子實體水萃取物及其後續的有機溶劑萃取物及水再萃取物的製備方法流程圖。首先，將樟芝子實體(步驟101)以水進行萃取(步驟102)，獲得到樟芝子實體水萃取物(ACW)(步驟103)。接著，將樟芝子實體水萃取物以乙酸乙酯(ethyl acetate)進行萃取，獲得樟芝子實體乙酸乙酯萃取物(ACW-EA)(步驟104)及第一樟芝子實體殘留物(步驟105)；再將第一樟芝子實體殘留物以乙醇(ethanol)進行萃取，獲得樟芝子實體乙醇萃取物(ACW-E)(步驟106)及第二樟芝子實體殘留物(步驟107)；最後將第二樟芝子實體殘留物以水萃取，獲得樟芝子實體水再萃取物(ACW-W)(步驟108)。
樟芝子實體乙酸乙酯萃取物、樟芝子實體乙醇萃取物及樟芝子實體水再萃取物的產量分別佔樟芝子實體水萃取物重量的8.9%、38.5%及52.6%。
接著，將樟芝子實體水萃取物及其分餾的萃取物分別刺激未成熟的樹突細胞，發現與控制組(PBS)相較，以ACW或ACW-W分別刺激的樹突細胞增加1.6至1.9倍的CD86及1.5至1.7倍的HLA-DR表現量，而由ACW-E及ACW-EA分別誘導的樹突細胞的CD86及HLA-DR表現量明顯的比ACW-W或ACW低(第4圖)。
再以酚-硫酸法分析樟芝子實體水萃取物及其分餾的萃取物的多醣體含量，與樟芝子實體水萃取物相較(ACW)，樟芝子實體水再萃取物(ACW-W)具有含量最多的多醣體(88.2%)，其次依序為樟芝子實體乙醇萃取物(ACW-E)的31.8%及樟芝子實體乙酸乙酯萃取物(ACW-EA)的12.9%(第5圖)。
而由1
H核磁共振圖譜分析樟芝子實體水萃取物及其分餾的萃取物可觀察到以下特性。1
H核磁共振圖譜實驗條件為：解析度600MHz的儀器、濃度為10.0mg/0.75ml，水萃取物(ACW)和水再萃取物(ACW-W)溶於D2
O溶液，分餾後極性較低的乙酸乙酯萃取物(ACW-EA)以及乙醇萃取物(ACW-E)則溶於C5
D5
N溶液，以利比較。
請參閱第6圖(A)至第6圖(D)，分別為(A)樟芝子實體水萃取物、(B)樟芝子實體乙酸乙酯萃取物、(C)樟芝子實體乙醇萃取物及(D)樟芝子實體水再萃取物的1
H核磁共振圖譜。若將第6圖(A)至(D)的1
H核磁共振圖譜部分重疊並相互比較，發現在δ3.0-5.5之醣類化合物特徵訊號於水再萃取物(ACW-W)的比例最高。而且在化學偏移4.44、4.46、4.91、5.00及5.15ppm處呈現多醣體結構中異位性碳原子(anomeric carbon)上氫的特徵訊號(第6圖(E))。
此外，更以多粘菌素B(polymyxin B)試驗確定ACW確實能誘發樹突細胞成熟，此活性非來自脂多醣體的污染(LPS contamination)。多粘菌素B是一種抗生素，在測試藥物之免疫活性的實驗中，能與脂多醣體結合、用以去除脂多醣體污染所造成的實驗誤差。因此，以多粘菌素B測試是否會阻斷由樟芝子實體水萃取物誘導的樹突細胞的表型成熟，結果發現，多粘菌素B加速了由樟芝子實體水萃取物誘導的樹突細胞之CD86及HLA-DR的表現量，但是多粘菌素B卻顯著地抑制由脂多醣體誘導的樹突細胞之CD86及HLA-DR的表現量(第7圖)。因此，ACW在無脂多醣體污染情形下能刺激樹突細胞的成熟。
接著，更以1
H核磁共振圖譜針對水再萃取物(ACW-W)以及脂多醣體(LPS)在相同條件下加以分析比較。請參閱第6圖(D)以及第8圖，將兩圖譜重疊相互比較後可發現ACW-W幾乎僅具有醣類化合物的特徵訊號，而無類似脂多醣體之長鏈上的訊號在化學偏移0.70至2.40ppm處，所以更能排除脂多醣體於實驗中的干擾。
由上述的實驗結果可歸納出樟芝子實體水萃取物之促進免疫活性確實來自於多醣體。
樹突細胞受到外界刺激時會產生許多細胞介素(cytokine)，而細胞介素的產生對於活化nave T細胞扮演重要的角色。其中，介白素-12(IL-12)為樹突細胞的引導因子(DC-derived factor)，能指揮輔助性T細胞的生長，產生大量的干擾素-γ(IFN-γ)。請參閱第9圖，為不同濃度的樟芝子實體水萃取物誘導樹突細胞產生介白素-12能力比較圖。在第9圖中，以25μg/ml及50μg/ml的樟芝子實體水萃取物刺激樹突細胞、產生介白素-12(IL-12)的能力皆比脂多醣體強，而測定的介白素-12濃度分別為235.53±2.54(PBS)、698.45±5.46(LPS)、758.14±26.54(ACW-25)及855.7±56.21(ACW-50)pg/ml。
介白素-12能使巨噬細胞進行趨化作用(chemoattractant)，也為樹突細胞遷移所需。請參閱第10圖，為未成熟的樹突細胞受不同濃度的樟芝子實體水萃取物刺激後的遷移能力比較圖。在第10圖中，與PBS及脂多醣體這兩組相較，以不同濃度的樟芝子實體水萃取物(12.5、25及50μg/ml)刺激未成熟的樹突細胞皆能夠有效促進樹突細胞的遷移能力，其中以25μg/ml的樟芝子實體水萃取物刺激樹突細胞能產生最大的遷移效果，而樟芝子實體水萃取物濃度提高至50μg/ml會使遷移能力降低。
樹突細胞可有效地呈現抗原給T細胞，並依據其表現型造成T細胞的分化。請參閱第11圖(A)，為樟芝子實體水萃取物刺激樹突細胞、活化T細胞的曲線圖。在第11圖中，與PBS及100ng/ml脂多醣體這兩組相較，以25及50μg/ml的樟芝子實體水萃取物皆能有效刺激樹突細胞、活化T細胞。請參閱第11圖(B)，為以不同濃度的樟芝子實體水萃取物刺激樹突細胞、活化輔助性T細胞而產生干擾素-γ的比較圖。在第11圖(B)中，與PBS及100ng/ml脂多醣體這兩組相較，以25及50μg/ml的樟芝子實體水萃取物皆能有效刺激樹突細胞、活化輔助性T細胞，並且有效地增強輔助性T細胞產生干擾素-γ的能力。由上述結果可知：樟芝子實體水萃取物可誘導的樹突細胞產生大量的干擾素-γ，而受樟芝子實體水萃取物誘導的樹突細胞可促進T細胞進入輔助性T細胞免疫反應路徑。
過去的研究發現癌細胞會藉由誘發細胞凋亡來逃避免疫系統的監視，抑制樹突細胞的凋亡則可提高抗原專一性的免疫反應(antigen-specific immune responses)。因此，需檢測樟芝子實體水萃取物是否會對樹突細胞有細胞毒殺作用。實驗結果發現，以樟芝子實體水萃取物刺激樹突細胞8天，超過90%以上的樹突細胞已成熟，而且經過長時間培養，樟芝子實體水萃取物對樹突細胞並不會產生細胞毒殺作用。此外，以樟芝子實體水萃取物刺激樹突細胞，會顯著降低Bax蛋白表現量，也會增加Bcl-2及NFκB p65的表現量(第12圖(A))。
樹突細胞的成熟、功能化與PI3K/Akt以及MAPKs訊息傳導路徑相關，樹突細胞被樟芝子實體水萃取物刺激後的細胞內訊息傳導路徑也被進一步確定。請參閱第12圖(B)，為樟芝子實體水萃取物刺激樹突細胞後的訊息傳導路徑蛋白表現圖譜。在第12圖(B)中，與PBS及脂多醣體這兩組相較，樟芝子實體水萃取物可活化樹突細胞內的磷酸化p38(p-p38)、磷酸化JNK(p-JNK)及磷酸化AKT(p-Akt)。此外，以12.5μg/ml的樟芝子實體水萃取物可刺激樹突細胞中大量的磷酸化ERK(p-ERK)表現，但是當樟芝子實體水萃取物濃度增加，p-ERK表現量降低，而未磷酸化的ERK表現量在不同濃度的ACW條件下仍相當穩定。
為了分析樹突細胞中PI3K及MAPKs訊息傳導路徑之間的關係，先將訊息傳導路徑蛋白抑制劑(SB203580、PD98059、SP600125及LY294002)分別與樹突細胞作用，以抑制p38、ERK1/2、JNK、PI3K/Akt的活性，再以樟芝子實體水萃取物刺激樹突細胞。結果顯示：p38(SB203580)、JNK(SP600125)、Akt(LY294002)會抑制樹突細胞受樟芝子實體水萃取物所誘導的CD86表現(第13圖(A))。而被p38及AKt抑制劑抑制、再以樟芝子實體水萃取物刺激的樹突細胞，其介白素-12蛋白表現相當低；ERK1/2抑制劑則會促進介白素-12的蛋白表現(第13圖(B))。由以上結果可歸納出樟芝子實體水萃取物是藉由活化p38和Akt而導致樹突細胞的成熟、功能化。
本發明的樟芝子實體水萃取物與靈芝子實體水萃取物及姬松茸子實體水萃取物相較，樟芝子實體水萃取物具有較好的促進樹突細胞成熟的能力。樟芝子實體水萃取物可經由活化PI3K/AKt以及p38/MAPKs訊息傳遞路徑而誘發樹突細胞成熟化，促進T細胞增生及干擾素γ的分泌，趨化輔助性T細胞反應；也可經由抑制細胞凋亡作用促進樹突細胞功能上之活性。因此，樟芝子實體水萃取物可應用於免疫治療上。
由樟芝子實體水萃取物依序以乙酸乙酯、乙醇及水萃取所獲得的萃取物發現，內含的多醣體含量影響了樹突細胞的活化程度，證實了樟芝子實體水萃取物之促進免疫活性源自多醣體。
本發明實屬難能的創新發明，深具產業價值，援依法提出申請。此外，本發明可以由本領域技術人員做任何修改，但不脫離如所附權利要求所要保護的範圍。
10．．．製備方法
101．．．樟芝子實體
102．．．水萃取
103．．．樟芝子實體水萃取物(ACW)
104．．．樟芝子實體乙酸乙酯萃取物(ACW-EA)
105．．．第一樟芝子實體殘留物
106．．．樟芝子實體乙醇萃取物(ACW-E)
107．．．第二樟芝子實體殘留物
108．．．樟芝子實體水再萃取物(ACW-W)
EDTA 2-[2-(Bis(carboxymethyl)amino)ethyl-(carboxymethyl)amino]acetic acid
HRP horseradish peroxidase
NFκB nuclear factor kappa B
PBS phosphate buffered saline
SDS sodium dodecyl sulfate
SDS-PAGE SDS-polyacrylamide gel electrophoresis
WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H
-tetrazolium
PBMC peripheral blood mononuclear cell
GM-CSF granulocyte macrophage colony-stimulating factor
ERK extracellular signal-regulated kinase
JNK c-Jun N-terminal kinase
MAPK Mitogen-activated kinase
PI3K Phosphoinositide 3-kinase
HLA human leukocyte antigen
第1圖為樟芝、靈芝及姬松茸子實體水萃取物分別刺激未成熟的樹突細胞後的CD86及HLA-DR表現比較圖。
第2圖為樟芝子實體水萃取物、靈芝子實體水萃取物及姬松茸子實體水萃取物的多醣體含量比較圖。
第3圖為本發明實施例的樟芝子實體水萃取物及其後續的有機溶劑萃取物及水再萃取物的製備方法流程圖。
第4圖為樟芝子實體各種萃取物分別刺激未成熟的樹突細胞後的CD86及HLA-DR表現比較圖。
第5圖為樟芝子實體各種萃取物的多醣體含量柱狀圖。
第6圖(A)至第6圖(D)分別為(A)樟芝子實體水萃取物、(B)樟芝子實體乙酸乙酯萃取物、(C)樟芝子實體乙醇萃取物及(D)樟芝子實體水再萃取物的1
H核磁共振圖譜。
第6圖(E)為樟芝子實體水再萃取物1
H核磁共振圖譜的局部放大圖。
第7圖為以多粘菌素B預先處理樹突細胞、再以脂多醣體或樟芝子實體水萃取物誘導的CD86及HLA-DR表現比較圖。
第8圖為脂多醣體的1
H核磁共振圖譜。
第9圖為不同濃度的樟芝子實體水萃取物誘導樹突細胞產生介白素-12能力比較圖。
第10圖為未成熟的樹突細胞受不同濃度的樟芝子實體水萃取物刺激後的遷移能力比較圖。
第11圖(A)為樟芝子實體水萃取物刺激樹突細胞、活化T細胞的曲線圖。
第11圖(B)為以不同濃度的樟芝子實體水萃取物刺激樹突細胞、活化輔助性T細胞而產生干擾素-γ的比較圖。
第12圖(A)及(B)分別為樟芝子實體水萃取物刺激樹突細胞後的訊息傳導路徑蛋白表現圖譜，其中第12圖(A)包括Bax、Bcl-2、p65及Actin，而第12圖(B)包括p-ERK、ERK、p-p38、p-38、p-JNK、JNK、p-AKt及Actin。
第13圖(A)及(B)分別為樹突細胞受訊息傳導路徑蛋白抑制劑抑制後再以樟芝子實體水萃取物處理樹突細胞的(A)CD86及(B)介白素-12表現比較圖。
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10．．．製備方法
101．．．樟芝子實體
102．．．水萃取
103．．．樟芝子實體水萃取物(ACW)
104．．．樟芝子實體乙酸乙酯萃取物(ACW-EA)
105．．．第一樟芝子實體殘留物
106．．．樟芝子實體乙醇萃取物(ACW-E)
107．．．第二樟芝子實體殘留物
108．．．樟芝子實體水再萃取物(ACW-W)
Claims (8)
- 一種樟芝子實體水再取物的備方法，包括下列步驟：(a)研磨一樟芝子實體成一粉狀；(b)水煮、迴流萃取該粉狀的樟芝子實體，過濾及離心，獲得一樟芝子實體水萃取物；(c)以乙酸乙酯萃取該樟芝子實體水萃取物，獲得一樟芝子實體乙酸乙酯萃取物及一第一樟芝子實體殘留物；(d)以乙醇萃取該第一樟芝子實體殘留物，獲得一樟芝子實體乙醇萃取物及一第二樟芝子實體殘留物；以及(e)以水萃取該第二樟芝子實體殘留物，獲得一樟芝子實體水再萃取物。
- 如申請專利範圍第1項所述的製備方法，其中，該樟芝子實體水再萃取物的1 H核磁共振圖譜在化學偏移δ 3.0-5.5之醣類化合物特徵訊號比例最高，而且在化學偏移4.44、4.46、4.91、5.00及5.15ppm處呈現多醣體結構中異位性碳原子(anomeric carbon)上氫的特徵訊號。
- 一種根據申請專利範圍第1項所述的製備方法所製備出的樟芝子實體水再萃取物，其係對一樟芝子實體進行研磨、並水煮迴流萃取、過濾及離心而獲得一樟芝子實體水萃取物，再依序以乙酸乙酯、乙醇及水對該樟芝子實體水萃取物進行萃取，依序獲得一樟芝子實體乙酸乙酯萃取物、一樟芝子實體乙醇萃取物及該樟芝子實體水再萃取物。
- 如申請專利範圍第3項所述的樟芝子實體水再萃取物，其中該樟芝子實體水萃取物經由活化一訊息傳導路徑，而誘發一樹突細胞之成熟。
- 如申請專利範圍第4項所述的樟芝子實體水再萃取物，其中該訊息傳導路徑之蛋白表現係選自PI3K/Akt、p38/MAPKs及其組合。
- 如申請專利範圍第4項所述的樟芝子實體水再萃取物，其中該 樹突細胞之成熟進一步促進T細胞增升級干擾素γ的分泌，並趨化輔助性T細胞反應。
- 如申請專利範圍第3項所述的樟芝子實體水再萃取物，其中該樟芝子實體水萃取物經由抑制細胞凋亡作用促進一樹突細胞之活性。
- 一種萃取自一樟芝子實體的樟芝子實體水再萃取物，其係對該樟芝子實體進行研磨、並水煮迴流萃取、過濾及離心而獲得一樟芝子實體水萃取物，再依序以乙酸乙酯、乙醇及水對該樟芝子實體水萃取物進行萃取，依序獲得一樟芝子實體乙酸乙酯萃取物、一樟芝子實體乙醇萃取物及該樟芝子實體水再萃取物，該樟芝子實體水再萃取物具有下列1 H核磁共振圖譜特徵：化學偏移4.44、4.46、4.91、5.00及5.15ppm處呈現多醣體結構中異位性碳原子(anomeric carbon)上氫的特徵訊號。
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| TW98107047A TWI448294B (zh) | 2009-03-04 | 2009-03-04 | 增強免疫活性之樟芝子實體水萃取物及其製備方法 |
| US12/552,115 US20100227404A1 (en) | 2009-03-04 | 2009-09-01 | Water extract of antrodia camphorata for immunostimulatory effect and preparation method thereof |
| US14/577,605 US9241962B2 (en) | 2009-03-04 | 2014-12-19 | Water extract of Antrodia camphorata for immunostimulatory effect and preparation method thereof |
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| TWI448294B (zh) | 2009-03-04 | 2014-08-11 | Univ Kaohsiung Medical | 增強免疫活性之樟芝子實體水萃取物及其製備方法 |
| TW201231474A (en) * | 2011-01-26 | 2012-08-01 | Univ Kaohsiung Medical | Triterpenoid composition of Antrodia cinnamomea, preparation and analysis method thereof |
| US9603882B2 (en) | 2013-08-13 | 2017-03-28 | Industrial Technology Research Institute | Method for modulating Th17 cells and method for treating a disease related to modulation of Th17 cells |
| TWI549614B (zh) * | 2013-08-15 | 2016-09-21 | zong-ming Ye | Extraction of Antrodia extraction method and apparatus |
| TW201521752A (zh) * | 2013-12-05 | 2015-06-16 | Univ Nat Taiwan Normal | 牛樟芝於抗肥胖的應用及其方法 |
| CN103733991B (zh) * | 2013-12-18 | 2016-05-25 | 柳州市天姿园艺有限公司 | 牛樟芝扦插枝的培育方法 |
| TWI574693B (zh) | 2015-03-06 | 2017-03-21 | Separation and Purification of Functional Components in Antrodia camphora by Supercritical Fluid Technology |
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| CN1386545A (zh) * | 2001-05-18 | 2002-12-25 | 葡萄王企业股份有限公司 | 樟芝保肝制剂 |
| TW591110B (en) | 2001-12-03 | 2004-06-11 | Grape King Entpr Inc | A method for producing gamma-aminobutyric acid with Antrodia camphorate mycelium and composition thereof |
| TWI279439B (en) | 2002-03-29 | 2007-04-21 | Council Of Agriculture | Processes for producing an antrodia camphorata culture having pharmacological activity, processes for obtaining a pharmacologically active composition from a culture of a. camphorata, products produced thereby and pharmaceutical compositions for the... |
| EP1634877B1 (en) * | 2004-08-17 | 2011-06-22 | Simpson Biotech Co., Ltd. | Mixture and compounds from mycelia of antrodia camphorata and use thereof |
| TWI299665B (en) | 2006-05-09 | 2008-08-11 | Univ Chia Nan Pharm & Sciency | A method for preparing the extracts from taiwanofungus camphoratus with a capacity for inhibiting the activity of matrix metalloproteinases |
| TWI394572B (zh) * | 2007-06-12 | 2013-05-01 | Golden Biotechnology Corp | Application of Cynanchum aurantii Cyclohexenone Compounds in the Preparation of Drugs for the Treatment of Autoimmune Diseases |
| TWI448294B (zh) | 2009-03-04 | 2014-08-11 | Univ Kaohsiung Medical | 增強免疫活性之樟芝子實體水萃取物及其製備方法 |
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Non-Patent Citations (1)
| Title |
|---|
| 徐銘豊，樟芝(Antrodia camphorata) 液態培養生產(1→3)-β-D-glucan多醣體之研究探討，2006/11/7揭示牛樟芝多醣體 (子實體或菌絲體)可萃取出抗腫瘤與免疫調節物質屬習知技術 Toxicology and Applied Pharmacology, vol.201(2004), p186-193，2006/11/7揭示牛樟芝多醣體萃取方法包括先經熱水萃取後再經乙醇萃取之多醣體萃取物具抗腫瘤功效 Nutrition and Cancer, vol.57(1), 2007, p111-121揭示使用乙醇萃取樟芝子實體粉末之含多醣體萃取物與PI3K/Akt蛋白表現有關 Agricultural and Food Chemistry, vol.56 ,2008, p865-874揭示經熱水及乙醇萃取牛樟芝菌絲萃取物與p38/MAPKs蛋白表現有關 複方中草藥調節免疫及樟芝子實體抑制腫瘤之研究，2007揭示經熱水萃取樟芝子實體之萃取物具有活化免疫調節活性，抑制腫瘤 * |
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| Publication number | Publication date |
|---|---|
| US9241962B2 (en) | 2016-01-26 |
| US20150104481A1 (en) | 2015-04-16 |
| US20100227404A1 (en) | 2010-09-09 |
| TW201032816A (en) | 2010-09-16 |
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