201235046 六、發明說明： 【發明所屬之技術領域】 本發明關於一種豆類萃取液（特別是大豆萃取液）在個體 中誘發細胞自禮以治療或預防疾病或病症（如糖尿病、老 化、神經退化疾病、脂肪肝或癌症）之用途。本發明另關 於一種發酵豆類萃取液（特別是大豆萃取液）透過誘發細胞 凋亡與細胞自禮而選擇性地殺死種瘤細胞之用途。 【先前技術】 細胞凋亡（第I型）及細胞自嗟（第11型）為兩種不同類型的 程序性 $田胞死亡{Shimizu et al.，2004，R〇ie 〇f Bci-2 family proteins in a non-cipoptotic programmed cell death dependent on autophagy genes· Nat. Cell Biol. 6, 1221- 722S)。細胞凋亡及細胞自噬為基因調控、演化上保守的 過程以調節細胞的命運。但是，細胞凋亡總是造成癌症細 胞的死亡，而細胞自噬則扮演癌症細胞生存或死亡之雙面 刃的角色。細胞凋亡為一個有組織性且依賴能量的過程， 它使生物體維持組織的動態平衡。若沒有足夠的細胞凋亡 則有助於癌症的發生機制。 細胞自噬也是一個正常的生理過程，其可促進細胞適應 及生存，但在某些條件下，它可致使細胞死亡 and Zakeri, 2004, Int. J. Biochem. Cell Biol. 36, 2405-2419; Maiuri et al., 2007, Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat. Rev. Mol. ☆ //心〇/· & 7W-752)。細胞自噬的一個重要機制在於標靶 154320.doc 201235046 包a蛋白f、蛋白質聚集物及胞器之細胞成分在溶酶體中 ，解’並與許多疾病有所牵連。細胞自㈣過程包含在自 喔溶小體（autolysosomes ’一種自嗤體及溶酶體的融和體） 中降解及回收細胞胞器及蛋白質。這種分解代謝、細胞自 我消化的過程受飢餓或壓力的誘發，引起一種稱為自噬體 之可吞嗤蛋白質及胞器的雙膜小泡㈣。自嗟體接著與溶 酶體融合’而自㈣及所吞嗟之物f被其中被降解。這^ 溶酶體_介導的細胞自我消化過程，在#ι餓下可回收細胞 内養分來維持細胞的新P東代謝，且在壓力下可移除累積之 文知蛋白及胞器。雖然可以透過泛素介導的蛋白酶降解途 板以移除個體蛋白質，但細胞自噬途徑可移除蛋白質聚積 物及胞器。因此’細胞自噬補充且與蛋白酶功能重疊，來 預防在飢餓及壓力下受損細胞成分的堆積。因為這些功 能，細胞自噬為一種重要的細胞應激反應，維持蛋白質及 胞盗的品質控管、保護基因體免於損害並維持細胞及哺乳 動物生存。自噬功能障礙為疾病（包含但不限於老化相關 疾病、神經退化性疾病、脂肪肝、糖尿病及癌症）的主要 因素。許多人類之神經退化性疾病與異常突變及/或泛蛋 白附著（polyubiquitinated)蛋白質的累積及過多神經細胞死 亡相關，小鼠的神經帶有標靶性細胞自噬缺陷者累積泛蛋 白附著及含P62-蛋白質聚積而導致神經退化性疾病。人類 肝臟疾病-脂肪肝及肝細胞癌（HCCs)之主要一次類與含p62 蛋白質聚積物相關，P62為蛋白質聚積疾病中常見的細胞 質包涵體成分。小鼠的肝有細胞自噬缺陷者具有含p62蛋 154320.doc 201235046 白身聚積物、過多的細胞死亡及HCC，最近的研究更顯 厂、 胞自里對於正確的胰島素分泌及-細胞存活至為 重要。 已有報導私出膳食中攝取大豆及以大豆為成分之產品可 : 降低许多癌症之風險。美國專利公開號第2002/01 82274號 ·- 係關於一種發酵之豆類萃取液之用途，其可促進健康、改 善個體的健康、預防及/或治療癌症、預防感染、降低感 染風險、治療感染、治療氣喘、治療發炎、調整免疫系統 及治療免疫疾病。美國專利號第6,685,973號揭示了 一種發 酵大豆萃取液用於抑制15-脂氧化酶（1 5-Up〇〇xygenase)及 預防/治療疾病之用途，其中抑制1 5_脂氧化酶與心血管疾 病、癌症、免疫病症及發炎相關。此外，先前已有文獻報 導SC-1，一種經過濾（〇 22 μηι)之經細菌（枯草桿菌 (Bacillus subtilis)反短芽抱样菌（Bacillus brevis))所發酵的 黃豆發酵液可顯著地抑制HBV-相關之HCC Hep 3B細胞及 小鼠肝炎ML-1細胞之生長及群落發生性（以α/., 2的7, ： Supernatant of bacterial fermented soybean induces apoptosis of human hepatocellular carcinoma Hep 3B cells via activation of caspase 8 and mitochondria. Food Chem. 45, 3(93-374)’該報導亦表明，SC-1對於體外培養 之Hep 3B細胞的細胞毒性是透過誘發卡斯帕斯酶 (caspase)-8及粒線體相關之細胞凋亡所致。然而，沒有任 何先前技術教示及建議豆類萃取液在誘發及甚至增強細胞 自嗟之間的關係。 154320.doc 201235046 【發明内容】 本發明提供了 一種誘發個體中細胞自噬之方法，其包含 施予該個體有效量之發酵大豆（G7_ycz♦⑽所ax (L·))萃取液或 其組合物。 本發明亦提供了一種誘發個體中細胞自噬及細胞凋亡之 方法’其包含施予該個體有效量之發酵大豆g w似 (L·))萃取液或其組合物。 本發明出人意料的發現發酵之豆類萃取物（特別是大豆 萃取液）可有效的誘發及甚至增進個體中之細胞自噬。特 別是，該所述之萃取液在預防及/或治療癌症上有驚人的 功效。 除非本文另有疋義，否則本文中所使用之本發明有所相 關之科學或技術術語應為本發明所屬技術領域中具通常知 識者所理解之意義。這些術語的意義及範圍應為明確，然 而’-旦有任何不明確的地方，本文中之^義優先於任何 字典或外部定義。 作為按照本文揭露之利用 明’應理解為下列之意義： 下方之術語除非有特別說 種有順序之型態及生化 本文術語「細胞凋亡」係指因 改變而使細胞死亡之過程。 本文術語「細胞自嗟」係扣 係柏—種代謝過程，其涉及細胞 本身成份透過溶酶體機制而陁 』旳降解，且對於細胞生長、發育 及幫助維持細胞羞物合成、p备 争解及隨後的回收之體内平衡 扮演正常角色之-種受緊密調節㈣程。 、 154320.doc 201235046 本文術語「大豆（G7ycz>^ w⑴：（L.))」係指一種兩倍化四 倍體（211=40)為蝶型花科、大豆（G7ycz·似WiUd)屬及豆類 β〇7·α)亞屬（Moench)之植物。用於製備發酵大豆萃取液較 佳之豆類可選自由黃豆及黑豆所組成之群，較佳之豆類為 黃豆。 術語「一種（a)」及「一種（an)」代表於本文中之語法對 象有一個或多於一個（即至少一個）。 術語「治療（treat)」、「治療（treatment)」或「治療 (treating)」意指降低病人經歷癌癌症狀之頻率、程度、嚴 重性及/或持續時間。 術語「防止（prevent)」、「預防（preventi〇n)」或「防止 (preventing)」意指抑制或避免癌癌相關之症狀。 術》吾有效量」係指可有效誘發細胞自嗤或細胞自嗟_ 誘發之細胞凋亡之大豆萃取液之量。 在一方面’本發明提供了一種誘發個體中細胞自噬之方 法’其包含施予該個體有效量之發酵大豆所似 (L.))萃取液或其組合物。在另一方面，本發明提供了—種 發酵大丑萃取液用於製備誘發個體中細胞自噬之藥劑之用 途。 在另一方面’本發明提供了 一種誘發個體中細胞自噬及 細胞祠亡之方法’其包含施予該個體有效量之發酵大豆 (Glycine max (L·))萃取液或其組合物。另一方面，本發明 提供種發酵大丑萃取液用於製備誘發個體中細胞自噬_ 誘發細胞凋亡之藥劑之用途。 154320.doc 201235046 根據本發明’該發酵大豆萃取液係透過液體大豆萃取液 與至少一種細菌發酵而製得。根據本發明，用於製備發酵 萃取液之較佳大旦為黃豆或黑豆，較佳者，本發明之發酵 大豆萃取液為發酵黃豆萃取液。 根據本發明’在發酵大豆萃取液中使用了至少—種細 菌較佳地，3玄發酵所用之細菌係選自由短芽_ (cillus brevis) ' # ^ g (Bacillus subtilis) ' 才干菌（^cz//WlS及糞道腸球菌 (^^er〇COCCWlS /aec^w)所組成之群組。更佳地本發明中 所用之細菌為短芽抱桿菌及/或枯草桿菌。此外，本發明 之电酵中’亦可使用乳酸或酵母菌。當使用—種以上的細 菌發酵時，在發酵時細菌可依順序使用或同時使用。 根據本發明’發酵是在適合細菌生長的條件下進行。較 佳的發酵溫度範圍為32〇c至饥。發酵的時間至少5天， 較佳的發酵時間為5至60天，更佳的時間為10至45天。發 酵後，發酵液可視需要的經消毒以獲得無菌的液體，如透 過加熱或‘射，較佳為加熱。較佳地，該無菌液體可經過 據，離心已移除大部分或所有的死亡微生物已獲得發酵大 五萃取物’較佳係利用離心、更佳地，該㈣的步驟接著 去除遽液巾的水份以濃縮發酵液而獲得發酵大豆萃取液。 根據本發明’該發酵大豆萃取液可誘發細胞自嗟。抗癌 療法’如化學治療、放射及高溫可誘發細胞自嗟並導致乳 癌、大腸癌、前列腺癌及腦癌死亡（自噬性細胞死亡）。然 而，細胞自嗤可能會移除因癌症治療所受損之蛋白質及胞 154320.doc 201235046 器’且對於治療有保護性（保護性細胞自噬）。本發明出人 意料的發現’發酵大豆萃取液可誘發甚至增進細胞自噬。 因此’發酵大豆萃取液可用於預防及/或治療癌症、糖尿 病、神經退化性疾病、脂肪肝或老化。 此外，發酵大豆萃取液可誘發細胞自噬及細胞凋亡。較 佳地’該細胞凋亡係受到細胞自噬的誘發（細胞自噬誘發 之細胞凋亡）。誘發細胞凋亡及細胞自噬導致癌症細胞生 長減少’並代表致癌性活性及免疫反應之間的平衡結果。 此外，施予發酵大豆萃取液透過誘發細胞凋亡及細胞自噬 而抑制癌症細胞的生長，而不會顯著的誘發體重及肝臟重 量的改變，意味著發酵大豆萃取液在體内的安全性及有效 性，且暗示了發酵大豆萃取物對於癌症之化學療法潛力。 因此，發酵大豆萃取液可有效的預防及/或治療癌症。較 佳地，該癌症係乳癌、前列腺癌、白血病、大腸癌、子宮 癌、子宮内膜癌、子宮頸癌、大腸癌、睪丸癌、淋巴瘤、 橫紋肌肉瘤、神經母細胞瘤、胰臟癌、肺癌、腦瘤、皮膚 癌、胃癌、肝癌、腎癌或鼻咽更佳地’該癌症係肝 癌，更佳地，該癌症係人類肝細胞癌。 在本發明_，發酵大豆萃取液可單獨或以含有發酵大豆 萃取液及醫藥上可接受之載體、稀釋劑及，或賦形劑之組 合物投予。較佳地，該發酵大豆萃取液為發酵黃豆萃取液 或發酵黑豆萃取液。發酵大豆萃取液可以約1〇_3至1〇 =/kg體重之劑量投予；較佳地，該發酵大豆萃取液的劑 里為0.01至3 ml/kg ’更佳係〇」至】ml/kg體重。這些劑量 154320.doc 201235046 疋根據發酵大且卒取液濃縮形式而計，但適宜之發酵大豆 萃取液為非濃縮形式或乾粉形式也可據此計算之。該劑量 可根據個體的健康狀況或所欲預防或治療的疾病而調整。 發酵大豆萃取液在長期每日攝取並為期2個月的齧齒類 慢性毒性試驗中被證明為非常安全的，小鼠接受劑量為^ ml/小鼠及1，3 ml/小鼠之口服毒性試驗達6〇天，其平均體 重或平均肝重量未展現任何顯著的差異。在實驗動物中皆 未觀察到總體毒性或死亡徵狀。 本發明之發酵大豆萃取液可根據不同的投藥途徑而視需 要以不同的調配物呈現。可選擇性的投藥途徑較佳為，但 不限於局部及口服。口服投藥之組合物可為含有粉末或顆 粒、懸汁液、乳液或在水性或非水性介質中溶液、油或脂 肪、小袋襞、膠囊、錠劑、膠囊錠、食品添加劑及緩釋劑 形之型式。含有發酵大豆萃取液之食品添加劑可選擇性地 作為食品（例如但不限於麵包、烘焙製品、餅乾、脆餅、 礼製品、蛋糕、巧克力及食用脂肪）食用油中之添加劑。 增稠劑、稀釋劑、香料、維生素分散助劑維生素分散助 齊J乳化劑或結合劑皆可添加於其中。較佳之口服投藥調 配物包含明膠膠囊，較佳含有ASU複合物、維他命、抗氧 化剑及稀釋劑。另-種較佳的口服投藥調配物包含ASU複 合物、維他命、抗氧化劑及食用油。 、下貫施例用於提供技藝人士實施本發明，因此，實施 例不應視為過度地限制本發明。本發明所屬技術領域中具 有通*知識者可在不背離本發明之精神或範的情況下對 154320.doc 201235046 本文所討論之實施例進行修改及變化，而仍屬於本發明之 範圍。 【實施方式】 實施例1、黃豆發酵產品之製備 一公斤的乾黃豆（東石鄉，嘉義，台灣）研磨、煮沸並浸 於水中10天，將黃豆的豆渣去除後，上清液以短芽孢桿菌 (1〇5 cells/ml)及枯草桿菌（1〇5 cells/ml)於37°C下發酵1個月 (Su et al·，2007) ° 實施例2、發酵黃豆萃取液之效能試驗 材料及方法 細胞培養 小鼠ML-1細胞由黎煥耀博士提供（微生物及免疫學系， 醫學院，國立成功大學，台南，台灣）在37°C含5% C02潮 濕環境下，培養於含有10%胎牛血清（GIBCO BRL)、2mM 麵胺酸（Sigma，St. Louis, MO)、100 U/ml 盤尼西林 (Sigma)及100青黴素（Sigma)之完全都貝克改良老鷹培養基 (Dulbecco's modified Eagle medium, DMEM, GIBCO BRL, Grand Island, NY) t ° 動物試驗 由國立成功大學動物中心（NCKU，台南，台灣）獲得約 6-7周大’體重介於18_23克之間之BALA/c小鼠，並飼養且 女置於調控溫度且具空調環境並以1〇/14 h光/暗週期之動 物中心。食物及水採自由取食。ML·!細胞在第〇天時以皮 下植入（s.c·; 2·5χ105細胞/小鼠）baLA/c小鼠側邊，於第4天 154320.doc 201235046 時，小鼠隨機分成三組。治療組織小鼠給予經口投藥之發 酵黃豆萃取液（1.0或1.3 ml/小鼠/天），持續56天，而另一 組則以相同時刻表接受空白對照（水）處理。每隔一日監測 小鼠大體上體型之變化，且每隔一日以游標尺監測腫瘤之 生長。腫瘤體積以公式LxW2/2計算，其中L(長度）及W(寬 度）為毫米，且L大於W。所有的動物實驗皆經過NCKU之 動物研究委員會核准，並以台灣國家研究委員會之準則下 進行（IACUC940047)。 免疫組織化學染色 由發酵黃豆萃取液或空白對照處理的小鼠所得到之腫瘤 在液態氮下冷凍，並保存於-80°C直到使用為止。以末端 脫氧核苷酸移轉酶介導的dUTP缺口末端標記法（TUNEL)法 (ApoAlert DNA Fragmentation assay Kit, Clontech, Palo Alto，CA)偵測細胞凋亡。以抗裂解LC3抗體（ABGENT,聖 地牙哥，加州，1:300)檢測細胞自嗔。腫瘤之切片皆進行 細胞凋亡及細胞自噬分析。簡言之，以 Cryotome0620(Thermo Shandon，Waltham，MA)切片以獲得 腫瘤切片（5 μπι)並以3.7%福馬林（Sigma)在室溫下培育1分 鐘，接著以冰乙醇（Merck，Darmstadt，德國）/乙酸 (Wako，大阪，日本；2:1，v/v)在-20°C下培育5分鐘。接 著，該切片以3%過氧化氫（Wako)焊熄5分鐘，接著根據製 造商的流程法進行TUNEL試驗，而嵌入在DNA碎片3'-羥 基末端之螢光-dUTP即可被偵測。在清洗後，切片在阻斷 緩衝液（SuperBlock Blocking Buffer, Thermo Scientific, 154320.doc -12- 201235046201235046 VI. Description of the Invention: [Technical Field] The present invention relates to a bean extract (especially a soybean extract) for inducing self-sacrifice in an individual to treat or prevent a disease or condition (such as diabetes, aging, neurodegenerative diseases) Use of fatty liver or cancer). The invention further relates to the use of a fermented bean extract (especially a soy extract) to selectively kill tumor cells by inducing apoptosis and self-respect. [Prior Art] Apoptosis (type I) and autologous (type 11) are two different types of procedural $cell death {Shimizu et al., 2004, R〇ie 〇f Bci-2 family Proteins in a non-cipoptotic programming cell reaction dependent on autophagy genes· Nat. Cell Biol. 6, 1221- 722S). Apoptosis and autophagy are genes-regulated and evolutionarily conserved processes that regulate cell fate. However, apoptosis always causes the death of cancer cells, while autophagy plays the role of a double-sided blade of cancer cell survival or death. Apoptosis is an organized and energy-dependent process that allows organisms to maintain a dynamic homeostasis. If there is not enough apoptosis, it will help the mechanism of cancer. Autophagy is also a normal physiological process that promotes cell adaptation and survival, but under certain conditions it can cause cell death and Zakeri, 2004, Int. J. Biochem. Cell Biol. 36, 2405-2419; Maiuri et al., 2007, Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat. Rev. Mol. ☆ //Heart/· & 7W-752). An important mechanism of autophagy in cells is the target 154320.doc 201235046 The protein components of protein a, protein aggregates and organelles are in the lysosomes and are involved in many diseases. The cell (4) process involves the degradation and recovery of cellular organelles and proteins from autolysosomes (a fusion of autologous steroids and lysosomes). This process of catabolism and self-digestion by cells is induced by starvation or stress, causing a double-membrane vesicle called an autophagosome that can be swallowed by proteins and organelles (IV). The autologous body is then fused to the lysosomal body' from (4) and the swallowed material f is degraded therein. This lysosome-mediated cell self-digestion process recovers intracellular nutrients under the hungry to maintain the cell's new P-east metabolism, and removes accumulated proteins and organelles under pressure. Although the ubiquitin-mediated protease degradation pathway can be used to remove individual proteins, the cellular autophagy pathway removes protein aggregates and organelles. Therefore, the autophagy is supplemented and overlaps with the protease function to prevent accumulation of damaged cellular components under starvation and stress. Because of these functions, autophagy is an important cellular stress response that maintains the quality control of proteins and cell pirates, protects the genome from damage and maintains cell and mammal survival. Autophagy dysfunction is a major factor in diseases including, but not limited to, age-related diseases, neurodegenerative diseases, fatty liver, diabetes, and cancer. Many human neurodegenerative diseases are associated with abnormal mutations and/or accumulation of polyubiquitinated proteins and excessive neuronal cell death. Mouse neurons with targeted autophagy have accumulated ubiquitin attachment and contain P62. - Protein accumulation leads to neurodegenerative diseases. Human liver disease - the primary primary class of fatty liver and hepatocellular carcinoma (HCCs) is associated with accumulation of p62 protein, a common cytoplasmic inclusion component in protein accumulation diseases. The mice with autophagy defects in the liver have p62 eggs 154320.doc 201235046 white body accumulation, excessive cell death and HCC, recent studies have shown that the correct insulin secretion and cell survival are important. Ingestion of soy and soy-based products in private meals has been reported to: Reduce the risk of many cancers. US Patent Publication No. 2002/01 82274-- relates to the use of a fermented bean extract which promotes health, improves the health of an individual, prevents and/or treats cancer, prevents infection, reduces the risk of infection, treats infection, Treatment of asthma, treatment of inflammation, adjustment of the immune system and treatment of immune diseases. U.S. Patent No. 6,685,973 discloses the use of a fermented soybean extract for inhibiting 15-lipoxygenase (1 5-Up〇〇xygenase) and preventing/treating a disease in which 15 5 lipoxygenase and cardiovascular disease are inhibited. , cancer, immune disorders and inflammation. In addition, it has been previously reported in the literature that SC-1, a soy (Bacillus subtilis Bacillus brevis) fermented soybean broth can be significantly inhibited by filtration (〇22 μηι). Growth and community-generating of HBV-related HCC Hep 3B cells and mouse hepatitis ML-1 cells (according to α,., 7, 7,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Caspase 8 and mitochondria. Food Chem. 45, 3 (93-374) 'This report also indicates that the cytotoxicity of SC-1 on Hep 3B cells cultured in vitro is through the induction of caspase-8 and granules. Line-related apoptosis is caused by. However, there is no prior art teaching and suggestion that the relationship between the bean extract in inducing and even enhancing cell self-twisting. 154320.doc 201235046 SUMMARY OF THE INVENTION The present invention provides an induced individual A method of autophagy in a cell comprising administering to the individual an effective amount of fermented soybean (G7_ycz♦ (10) ax (L·)) extract or a composition thereof. The present invention also provides Individual species induce autophagy and method of cell apoptosis' comprising administering to the subject an effective amount of a similar fermented soybean g w (L ·)) extracted liquid or combinations thereof. The present inventors have surprisingly found that fermented legume extracts (especially soy extracts) are effective in inducing and even enhancing autophagy in cells in an individual. In particular, the extract described has surprising effects in preventing and/or treating cancer. Unless otherwise defined herein, the terms of the invention are to be understood as being understood by those of ordinary skill in the art. The meaning and scope of these terms should be clear, but in the event of any ambiguity, the meaning in this document takes precedence over any dictionary or external definition. As used in accordance with the disclosure herein, it should be understood as the following meanings: Unless the term is specifically described in terms of sequence and biochemistry, the term "apoptosis" refers to the process of cell death due to alteration. The term "cell self-sufficiency" is a metabolic process in which the cell itself is involved in the degradation of the cell itself through the lysosomal mechanism, and contributes to cell growth, development, and maintenance of cell-short synthesis. And the subsequent recovery of the homeostasis plays a normal role - the species is closely regulated (four). 154320.doc 201235046 The term "soybean (G7ycz>^ w(1):(L.))") refers to a doubled tetraploid (211=40) of the genus Butterfly, soybean (G7ycz·WiUd) and A plant of the genus Moench, a legume of the genus β〇7·α. The beans suitable for preparing the fermented soybean extract may be selected from the group consisting of free soybeans and black beans. The preferred beans are soybeans. The terms "a" and "an" mean one or more than one (i.e., at least one) of the grammatical objects herein. The term "treat", "treatment" or "treating" means reducing the frequency, extent, severity and/or duration of a patient's progression to a cancerous condition. The terms "prevent", "preventi", or "preventing" mean inhibiting or avoiding symptoms associated with cancer. The "effective amount" refers to the amount of soy extract that can effectively induce cell autologous or cell-induced apoptosis. In one aspect, the invention provides a method of inducing autophagy in an individual' which comprises administering to the individual an effective amount of a fermented soybean-like (L.) extract or composition thereof. In another aspect, the present invention provides a use of a fermented ugly extract for the preparation of a medicament for inducing autophagy in an individual. In another aspect, the invention provides a method of inducing autophagy and cell death in an individual' comprising administering to the individual an effective amount of a fermented soybean (Glycine max (L.)) extract or composition thereof. In another aspect, the present invention provides the use of a fermented ugly extract for the preparation of a medicament for inducing autophagy-induced apoptosis in an individual. 154320.doc 201235046 According to the present invention, the fermented soybean extract is obtained by fermenting a liquid soybean extract with at least one of the bacteria. According to the present invention, the preferred large-sized denier for use in the preparation of the fermented extract is soybean or black bean. Preferably, the fermented soybean extract of the present invention is a fermented soybean extract. According to the present invention, at least one type of bacteria is used in the fermented soybean extract. Preferably, the bacteria used in the 3x fermentation are selected from the group consisting of cillus brevis ' # ^ g (Bacillus subtilis) ' //Group of WlS and Enterococcus faecalis (^^er〇COCCWlS / aec^w). More preferably, the bacteria used in the present invention are Bacillus brevis and/or Bacillus subtilis. Further, the present invention In the electro-fermentation, lactic acid or yeast can also be used. When more than one type of bacteria is used for fermentation, bacteria can be used sequentially or simultaneously during fermentation. According to the present invention, 'fermentation is carried out under conditions suitable for bacterial growth. The fermentation temperature ranges from 32〇c to hunger. The fermentation time is at least 5 days, the preferred fermentation time is 5 to 60 days, and the better time is 10 to 45 days. After fermentation, the fermentation broth can be disinfected as needed. To obtain a sterile liquid, such as by heating or 'shooting, preferably heating. Preferably, the sterile liquid can be centrifuged to remove most or all of the dead microorganisms and the fermented Big Five extract has been obtained. Using centrifugation, more preferably, The step then removes the moisture of the liquid wipe to concentrate the fermentation liquid to obtain a fermented soybean extract. According to the present invention, the fermented soybean extract can induce cell autologous. Anticancer therapy such as chemotherapy, radiation and high temperature can induce cells. Self-deprecating and leading to breast cancer, colorectal cancer, prostate cancer, and brain cancer death (autophagic cell death). However, cell autopsy may remove proteins and cells damaged by cancer treatment. The treatment is protective (protective cell autophagy). The present invention surprisingly found that 'fermented soybean extract can induce or even enhance autophagy. Therefore, 'fermented soybean extract can be used for prevention and / or treatment of cancer, diabetes, neurodegenerative Disease, fatty liver or aging. In addition, the fermented soybean extract can induce autophagy and apoptosis. Preferably, the apoptosis is induced by autophagy (cell apoptosis induced by autophagy). Apoptosis and autophagy lead to a decrease in cancer cell growth' and represent a balance between carcinogenic activity and immune response. In addition, the fermented soybean extract inhibits the growth of cancer cells by inducing apoptosis and autophagy, without significantly inducing changes in body weight and liver weight, meaning that the fermented soybean extract is safe in vivo and Effectiveness, and implies the potential of chemotherapy for fermented soybean extracts for cancer. Therefore, fermented soybean extract can effectively prevent and/or treat cancer. Preferably, the cancer is breast cancer, prostate cancer, leukemia, colorectal cancer, Uterine cancer, endometrial cancer, cervical cancer, colorectal cancer, testicular cancer, lymphoma, rhabdomyosarcoma, neuroblastoma, pancreatic cancer, lung cancer, brain tumor, skin cancer, stomach cancer, liver cancer, kidney cancer or nasopharynx More preferably, the cancer is liver cancer, and more preferably, the cancer is human hepatocellular carcinoma. In the present invention, the fermented soybean extract may be administered alone or in a composition containing a fermented soybean extract and a pharmaceutically acceptable carrier, diluent and or excipient. Preferably, the fermented soybean extract is a fermented soybean extract or a fermented black bean extract. The fermented soybean extract may be administered at a dose of about 1 〇 3 to 1 〇 = / kg body weight; preferably, the fermented soybean extract is in the range of 0.01 to 3 ml / kg 'better 〇 至 to 】 ml /kg body weight. These doses 154320.doc 201235046 疋 are based on the large fermentation form and the concentrated form of the draw liquid, but the suitable fermented soybean extract may be calculated in a non-concentrated form or a dry powder form. The dosage can be adjusted depending on the health of the individual or the condition to be prevented or treated. The fermented soybean extract proved to be very safe in long-term daily intake and a 2-month rodent chronic toxicity test. The mice received an oral toxicity test at a dose of 2 ml/mouse and 1,3 ml/mouse. Up to 6 days, the average body weight or mean liver weight did not show any significant difference. No overall toxicity or signs of death were observed in the experimental animals. The fermented soybean extract of the present invention can be presented in different formulations as needed depending on the route of administration. The alternative route of administration is preferably, but not limited to, topical and oral. Orally administered compositions may be in the form of powders or granules, suspensions, emulsions or solutions in aqueous or non-aqueous media, oils or fats, sachets, capsules, lozenges, capsules, food additives and sustained release formulations. . The food additive containing the fermented soybean extract may optionally be used as an additive in food oils such as, but not limited to, bread, baked goods, biscuits, shortbread, gift products, cakes, chocolate, and edible fats. Thickeners, thinners, perfumes, vitamin dispersing aids, vitamin dispersing aids, J emulsifiers or binders can be added to them. Preferred oral administration formulations comprise gelatin capsules, preferably containing an ASU complex, a vitamin, an antioxidant sword, and a diluent. Another preferred oral administration formulation comprises an ASU complex, a vitamin, an antioxidant, and an edible oil. The following examples are provided to provide a person skilled in the art to practice the invention, and thus, the embodiments should not be construed as limiting the invention. Modifications and variations of the embodiments discussed herein may be made without departing from the spirit and scope of the invention, and still fall within the scope of the invention. [Examples] Example 1. Preparation of Soybean Fermented Products One kilogram of dried soybeans (Dongshi Township, Chiayi, Taiwan) was ground, boiled and immersed in water for 10 days. After removing the bean dregs, the supernatant was Bacillus brevis. (1〇5 cells/ml) and Bacillus subtilis (1〇5 cells/ml) were fermented at 37 °C for 1 month (Su et al., 2007) ° Example 2, efficacy test materials for fermented soybean extract and Methods Cell culture mouse ML-1 cells were provided by Dr. Li Huanyao (Department of Microbiology and Immunology, Medical College, National Cheng Kung University, Tainan, Taiwan) cultured in a humidified environment containing 5% CO 2 at 37 ° C. Dulbecco's modified Eagle medium (DMEM, GIBCO) with serum (GIBCO BRL), 2 mM face acid (Sigma, St. Louis, MO), 100 U/ml penicillin (Sigma) and 100 penicillin (Sigma) BRL, Grand Island, NY) t ° Animal Trial Received approximately 6-7 weeks old BALA/c mice weighing between 18 and 23 grams from the National Center for Successful Animals (NCKU, Tainan, Taiwan), and raised and placed Control temperature and air-conditioned environment with 1 / 14 h light / dark cycle of the animal center. Food and water are free to eat. ML·! cells were implanted subcutaneously (s.c.; 2.5 χ 105 cells/mouse) on the side of baLA/c mice on day ,, and on day 4 154320.doc 201235046, the mice were randomly divided into three groups. Treated tissue mice were given oral fermented soy extract (1.0 or 1.3 ml/mouse/day) for 56 days, while the other group received a blank control (water) treatment at the same timetable. Mice were monitored for changes in body size every other day, and tumor growth was monitored every other day with a vernier scale. Tumor volume is calculated by the formula LxW2/2, where L (length) and W (width) are millimeters and L is greater than W. All animal experiments were approved by the NCKU Animal Research Committee and conducted under the guidelines of the National Research Council of Taiwan (IACUC 940047). Immunohistochemical staining Tumors obtained from fermented soybean extract or blank control mice were frozen under liquid nitrogen and stored at -80 °C until use. Apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method (ApoAlert DNA Fragmentation assay Kit, Clontech, Palo Alto, CA). Cell autotrophy was measured with an anti-lytic LC3 antibody (ABGENT, San Diego, CA, 1:300). The sections of the tumor were analyzed for apoptosis and autophagy. Briefly, sections were cut with Cryotome 0620 (Thermo Shandon, Waltham, MA) to obtain tumor sections (5 μm) and incubated with 3.7% fumarine (Sigma) for 1 minute at room temperature followed by ice ethanol (Merck, Darmstadt, Germany) / acetic acid (Wako, Osaka, Japan; 2:1, v/v) was incubated at -20 ° C for 5 minutes. Subsequently, the section was soldered out with 3% hydrogen peroxide (Wako) for 5 minutes, and then subjected to the TUNEL test according to the manufacturer's protocol, and the fluorescent-dUTP embedded in the 3'-hydroxyl end of the DNA fragment was detected. After washing, the sections are in blocking buffer (SuperBlock Blocking Buffer, Thermo Scientific, 154320.doc -12- 201235046
Rockford，IL)中於室溫下培育30分鐘。將腫瘤切片在4°C下 以兔多株抗-裂解LC3抗體（ABGENT，San Diego，CA; 1:300)培育過夜以進行免疫染色，接著在阻斷緩衝液中以 羊抗兔 Alexa Fluor568 共輕二級抗體（Molecular Probes， Inc.，Eugene，OR; 1:450)於室溫下培育2小時。在清洗後， 螢光素-dUTP及/或抗-裂解LC3抗體染色之切片以赫希斯特 (Hoechst) 33258(Sigma-Aldrich，St. Louis, MO; 0.05 pg/ml 於PBS中）於室溫下培育10分鐘，以螢光顯微鏡（OLYMPUS BX51)檢測信號。 以流式細胞儀分析細胞凋亡之細胞 細胞（2xl05)生長於6孔盤中，並在加入SC-1(265 pg/ml) 之前以或不以10 nM之細胞自噬抑制劑3-甲基腺嘌呤（3-MA; Sigma, St· Louis, MO)預處理2小時。收集細胞，並以 800g在4°C下離心10分鐘，且在37°C下黑暗中以含有40 pg/ml破化丙。定及100 mg/ml RNase A之HBS中再懸浮30分 鐘。以 FACScan 流式細胞儀（Becton Dickison，Mountain View，力口州，美國）量測細胞凋亡之細胞。 統計分析 結果以平均值±標準誤（SEM)呈現。腫瘤體積之差異以學 生 t 檢驗分析（Minitab software, version 10.2)。若 ρ<0·05 時 認為有差異，體重、腫瘤重量及肝臟重量皆以單方 ANOVA分析，不同群組之差異以鄧肯（Duncan’s)多重範圍 試驗（SPSS軟體，14.0版），若ρ<0·05時認為有差異。 發酵黃豆萃取液對於ML-1細胞在體内生長之效能 154320.doc -13- 201235046 如上述，小鼠ML-i細胞以皮下植入s.c.近交系bala/c小 鼠的側面，接著連續56天經口投予發酵黃豆萃取液（1〇或 1.3 mi/鼠/天）或空白對照（水）。ml·!細胞之生長每隔天即 偵測直至第60天。如圖1A所示，在第30天時，接受空白對 照之小鼠其ML-1細胞的生長很明顯，然而，小鼠接受發 酵黃豆萃取液（1.3 ml/鼠/天）者則很不顯眼。在第6〇天時， 相較於第30天，控制組小鼠的腫瘤大小變大許多，然而接 受發酵黃豆萃取液（1·3 ml/鼠/天）之群組小鼠腫瘤大小沒有 明顯的變化。在此試驗中，腫瘤的生長以游標尺每隔曰測 里’並分析腫瘤體積的差異。如圖1B所示，投予發酵黃豆 萃取液（1.3 ml/鼠/天）者其腫瘤的大小在整個實驗期間中顯 著地受抑制（p<0.05)。值得注意的是，所有的小鼠一直到 實驗最後皆存活，小鼠接受發酵黃豆萃取液者皆未發現明 顯的疾病，以發酵黃豆萃取液處理者其體重及肝臟重量皆 沒有明顯的改變（Ρ>0·05)(表1)。 表1發酵黃豆萃取液對體重、腫瘤重量及肝臟重量之影響 處理 體重 腫瘤 肝臟 --重量（g)-------------- 控制組 23.3±0.7a 7.3±1.3a 1.69±0.16a SCB (1 .〇 ml/小鼠） 22.9 士 0_7a 3.6 ± 0.7b 1.77±0.04a SCB (U ml/小鼠） 24.6 ± 0.6a 2.1±0.2b 1.59±0.05a P值 0.146 0.005 0.474 154320.doc -14· 201235046 結果以平均值±標準誤（SEM)呈現。SCB代表含有活菌之 未經過濾之發酵黃豆萃取液，平均值在每列中具有不同的 字母代表有顯著的差異，P<〇.〇5。 發酵黃豆萃取液於體内誘發細胞凋亡之效能 為了確認體内誘發之細胞)周亡，腫瘤切片在螢光染色前 先進行TUNEL試驗以確認細胞凋亡之現象，核DNA雙股斷 裂。如圖2A所示，以1 〇〇χ之螢光顯微鏡觀測，與空白對照 控制組相比’發酵黃豆萃取液處理者有增進的陽性TUNEL 染色’圖2Β為以400χ觀測’進一步顯示了經空白對照處理 的小鼠所得到的腫瘤切片中，細胞核中並沒有明顯的綠色 細胞凋亡螢光，意味著細胞凋亡現象在控制組中沒有產 生。相反的，接受發酵黃豆萃取液（1.3 ml/鼠/天）之小鼠之 腫瘤切片中’細胞核展現強烈的綠色螢光，代表核DnA雙 股斷裂，其為細胞凋亡之標諸。 發酵黃豆萃取液於體内誘發細胞自噬之效能 測定腫瘤中之細胞自噬現象。如圖3A所示，在ιοοχ了觀 測裂解LC3分佈之型態分析，經發酵黃豆萃取液處理者與 控制組相比’有較多之裂解LC3染色。使用4〇〇乂之螢光顯 微鏡觀測’進一步發現在控制組中，細胞質内沒有裂解 LC3圍繞細胞核（圖3B)。相反的，在發酵黃豆萃取液處理 的群組中’裂解LC3之表現增加並顯示出點狀染色，代表 自噬體中自噬標記LC3的位置《這些結果顯示發酵黃豆萃 取液在體内ML-1細胞中可誘發細胞凋亡及細胞自噬兩 者。此外，重豐赫希斯特（Hoechst) 33258、TUNEL及裂解 154320.doc -15- 201235046 LC3染色之影像顯示發酵黃豆萃取液可單獨誘發細胞自 噬，而不誘發細胞凋亡，其特徵在於藍色的細胞核圍繞著 紅色的點狀物（圖4A及4B)。有趣的事，發酵黃豆萃取液不 可僅誘發細胞凋亡而未誘發細胞自噬，因為幾乎所有的帶 有綠色染色的核皆圍繞著紅色的裂解LC32點狀。 發酵黃豆萃取液誘發細胞凋亡及細胞自噬之效能 為了確認細胞凋亡前之細胞自噬，將細胞自噬抑制劑3_ MA加入細胞培養，且以流式細胞儀分析凋亡細胞之相對 量。結果顯示3 -Ma以時間依賴方式抑制了無論是發酵黃豆 萃取液誘發之小鼠肝臟ML-1及人類HCC Hep 3B細胞的細 胞凋亡。如圖5A顯示，投予3-MA在48小時，抑制了發酵 黃豆萃取液誘發之ML-1細胞凋亡約45%，且在72小時抑制 了 52%。圖5B中，在第48小時抑制了發酵黃豆萃取液誘發 之Hep 3B細胞凋亡約42%，且在第72小時抑制了 60%。因 此，對於接受發酵黃豆萃取液之小鼠腫瘤在相對長的一段 時間後，所觀察到誘發之細胞凋亡只與細胞自噬結合並不 令人意外。 【圖式簡單說明】 圖1顯示ML-1細胞在BALB/c小鼠中的生長。（a)帶有 紙心細胞之小鼠經口投予空白對照（水，對照組）或發酵黃 旦萃取物（1.3 ml/小氣/天）之代表照片。SCB代表含有活菌 之未經過遽發酵黃豆萃取液β (B)帶有ML]細胞之小鼠腫 瘤體積’數據以平均值±標準誤呈現（每個群組n=4)， 空白對照控制組相對值有顯著差異。 / 154320.doc 201235046 圖2顯示發酵黃豆萃取液誘發植入於BALB/c小鼠中之 ML-1細胞凋亡’ SCB代表含有活菌之未經過濾發酵黃豆萃 取液。（A)在lOOx下觀察之腫瘤切片代表圖。（^)在4〇〇乂下 觀察之腫瘤切片代表圖。腫瘤切片以赫希斯特（H〇echst) 33258(藍色）及螢光d-UTP(TUNEL試驗，綠色）染色以分別 觀察細胞中之細胞核及細胞凋亡之核。結果代表三個獨立 實驗。 圖3顯示發酵黃豆萃取液誘發植入於BALB/c小鼠中之 ML-1細胞自嗤，SCB代表含有活菌之未經過渡發酵黃豆萃 取液。（A)在ΙΟΟχ下觀察之腫瘤切片代表圖。在4〇〇χΤ 觀察之腫瘤切片代表圖。腫瘤切片以赫希斯特（Hoechst) 33258(藍色）及抗-裂解LC3抗體（紅色）染色以分別觀察細胞 中之細胞核及細胞自噬LC3-II的點狀圖樣。結果代表三個 獨立實驗。 圖4顯示了在腫瘤中誘導了細胞凋亡及細胞自嗤兩者， SCB代表含有活菌之未經過濾發酵黃豆萃取液。（A)由經口 投予水（控制組）之小鼠中獲得之腫瘤。（B)由經口投予發酵 黃豆萃取液（1 ·3 ml/小鼠/天）之小鼠中獲得之腫瘤。腫瘤切 片以赫希斯特（Hoechst)33258(藍色）、螢光d-UTP(TUNEL 試驗’綠色）及抗-裂解LC3抗體（紅色）染色以分別觀察細胞 核、細胞凋亡之核及細胞自噬LC3-II的點狀圖樣。結果代 表三個獨立實驗。Incubate for 30 minutes at room temperature in Rockford, IL). Tumor sections were incubated overnight at 4 °C with rabbit polyclonal anti-lysed LC3 antibody (ABGENT, San Diego, CA; 1:300) for immunostaining followed by goat anti-rabbit Alexa Fluor 568 in blocking buffer. Light secondary antibody (Molecular Probes, Inc., Eugene, OR; 1:450) was incubated for 2 hours at room temperature. After washing, sections stained with luciferin-dUTP and/or anti-lysed LC3 antibody were plated in Hoechst 33258 (Sigma-Aldrich, St. Louis, MO; 0.05 pg/ml in PBS). Incubate for 10 minutes at a temperature and detect the signal with a fluorescence microscope (OLYMPUS BX51). Cellular cells (2xl05) analyzed by flow cytometry were grown in 6-well plates and autophagy inhibitors 3- or 10 nM were added before SC-1 (265 pg/ml). The adenine (3-MA; Sigma, St. Louis, MO) was pretreated for 2 hours. The cells were collected and centrifuged at 800 g for 10 minutes at 4 ° C and 40 μg/ml of c-dissolved C at 37 ° C in the dark. Resuspend in HBS at 100 mg/ml RNase A for 30 minutes. Apoptotic cells were measured by FACScan flow cytometry (Becton Dickison, Mountain View, Likou, USA). Statistical analysis Results are presented as mean ± standard error (SEM). Differences in tumor volume were analyzed by Student's t-test (Minitab software, version 10.2). If ρ<0·05 is considered to be different, body weight, tumor weight and liver weight are analyzed by unilateral ANOVA. The difference between different groups is Duncan's multiple range test (SPSS software, version 14.0), if ρ<0· At 05, I think there is a difference. The efficacy of fermented soy extract for growth of ML-1 cells in vivo 154320.doc -13- 201235046 As described above, mouse ML-i cells were implanted subcutaneously into the side of sc inbred bala/c mice, followed by 56 consecutive The fermented soybean extract (1〇 or 1.3 mi/mouse/day) or the blank control (water) was administered by the celestial mouth. The growth of ml·! cells was detected every other day until the 60th day. As shown in Fig. 1A, on day 30, the growth of ML-1 cells was evident in the mice receiving the blank control, however, the mice receiving the fermented soybean extract (1.3 ml/mouse/day) were inconspicuous. . On the 6th day, compared with the 30th day, the tumor size of the control group mice became much larger, but the tumor size of the mice receiving the fermented soybean extract (1.3 ml/mouse/day) was not obvious. The change. In this test, tumor growth was measured by vernier scale every ’ and the difference in tumor volume was analyzed. As shown in Fig. 1B, the size of the tumor of the fermented soybean extract (1.3 ml/mouse/day) was significantly inhibited throughout the experiment (p < 0.05). It is worth noting that all the mice survived until the end of the experiment. No obvious disease was found in the mice receiving the fermented soybean extract. The body weight and liver weight of the fermented soybean extract were not significantly changed (Ρ> ;0·05) (Table 1). Table 1 Effect of fermented soybean extract on body weight, tumor weight and liver weight Treatment of body weight tumor liver - weight (g) -------------- Control group 23.3 ± 0.7a 7.3 ± 1.3a 1.69±0.16a SCB (1.〇ml/mouse) 22.9 ±0_7a 3.6 ± 0.7b 1.77±0.04a SCB (U ml/mouse) 24.6 ± 0.6a 2.1±0.2b 1.59±0.05a P value 0.146 0.005 0.474 154320.doc -14· 201235046 Results are presented as mean ± standard error (SEM). SCB represents an unfiltered fermented soy extract containing live bacteria, and the average value has a significant difference in each column, representing a significant difference, P<〇.〇5. The effect of fermented soybean extract on apoptosis in vivo In order to confirm the death of cells induced in vivo, tumor sections were subjected to TUNEL test before fluorescence staining to confirm the phenomenon of apoptosis, and nuclear DNA double-strand break. As shown in Fig. 2A, the fluorescence of the 1 〇〇χ microscope showed that the fermented soybean extract treatment had improved positive TUNEL staining compared with the blank control group. Figure 2 shows the observation with 400χ, which further shows the blank. In the tumor sections obtained from the control mice, there was no obvious green cell apoptosis fluorescence in the nucleus, which means that the apoptosis phenomenon did not occur in the control group. In contrast, in the tumor sections of mice receiving fermented soybean extract (1.3 ml/mouse/day), the nuclei exhibited intense green fluorescence, representing a nuclear DnA double-strand break, which is the target of apoptosis. The efficiency of fermented soybean extract in inducing autophagy in vivo Determines autophagy in cells in tumors. As shown in Fig. 3A, the type analysis of the cracked LC3 distribution was observed at ιοο, and the fermented soybean extract treatment had more cracked LC3 staining than the control group. Using a 4 fluoroscopy microscope observation, it was further found that in the control group, no cleavage of LC3 around the nucleus in the cytoplasm (Fig. 3B). In contrast, in the group treated with the fermented soybean extract, the performance of 'cracking LC3 increased and showed dot-like staining, representing the location of the autophagy-labeled LC3 in the autophagosome. These results show that the fermented soybean extract is in vivo ML- Both cells can induce apoptosis and autophagy. In addition, Hoechst 33258, TUNEL and cleavage 154320.doc -15- 201235046 LC3 staining images show that fermented soybean extract can induce autophagy alone without inducing apoptosis, which is characterized by blue The colored nuclei surround the red dots (Figures 4A and 4B). Interestingly, the fermented soy extract did not induce apoptosis but did not induce autophagy, as almost all of the green-stained nuclei were dotted around the red-cleaved LC32. Efficacy of fermented soybean extract to induce apoptosis and autophagy In order to confirm autophagy before apoptosis, the autophagy inhibitor 3_MA was added to the cell culture, and the relative amount of apoptotic cells was analyzed by flow cytometry. . The results showed that 3 -Ma inhibited apoptosis of mouse liver ML-1 and human HCC Hep 3B cells induced by fermented soybean extract in a time-dependent manner. As shown in Fig. 5A, administration of 3-MA inhibited apoptosis of ML-1 cells induced by fermented soybean extract by about 45% at 48 hours, and inhibited 52% at 72 hours. In Fig. 5B, the apoptosis of Hep 3B cells induced by the fermented soybean extract was inhibited by about 42% at the 48th hour, and was inhibited by 60% at the 72nd hour. Therefore, it has not been surprising that the induced apoptosis was observed to bind only to autophagy in mice after receiving the fermented soybean extract for a relatively long period of time. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the growth of ML-1 cells in BALB/c mice. (a) A photograph of a mouse with paper heart cells orally administered to a blank control (water, control group) or fermented yellow extract (1.3 ml/small air/day). SCB represents the tumor volume of mice with live bacteria that have not been fermented soy extract β (B) with ML] cells. Data are presented as mean ± standard error (n=4 per group), blank control group The relative values are significantly different. / 154320.doc 201235046 Figure 2 shows that fermented soybean extract induces apoptosis of ML-1 cells implanted in BALB/c mice. SCB represents unfiltered fermented soybean extract containing live bacteria. (A) Representative representation of tumor sections observed at 100x. (^) A representative view of the tumor slice observed at 4〇〇乂. Tumor sections were stained with H〇echst 33258 (blue) and fluorescent d-UTP (TUNEL assay, green) to observe the nuclei and apoptotic nuclei in the cells, respectively. The results represent three independent experiments. Fig. 3 shows that the fermented soybean extract induces self-sufficiency of ML-1 cells implanted in BALB/c mice, and SCB represents an untransition-fermented soybean extract containing viable bacteria. (A) Representative map of tumor sections observed under the armpits. The tumor slice representative map was observed at 4〇〇χΤ. Tumor sections were stained with Hoechst 33258 (blue) and anti-lysed LC3 antibody (red) to observe the nucleus of the cells and the dot pattern of autophagy LC3-II, respectively. The results represent three independent experiments. Figure 4 shows both induction of apoptosis and autophagy in tumors, and SCB represents unfiltered fermented soy extract containing live bacteria. (A) Tumors obtained from mice orally administered with water (control group). (B) Tumors obtained from mice which were orally administered with fermented soybean extract (1·3 ml/mouse/day). Tumor sections were stained with Hoechst 33258 (blue), fluorescent d-UTP (TUNEL test 'green') and anti-lytic LC3 antibody (red) to observe nuclei, apoptotic nuclei and cells, respectively. A dot pattern of LC3-II. The results represent three independent experiments.
圖5顯示細胞自噬抑制劑對發酵黃豆萃取物誘發之細胞 凋亡之影響。（A)3-MA對ML-1細胞之抑制影響。（B)3-MA 154320.doc 17- 201235046 對Hep 3B細胞之抑制影響。在處理後，細胞以碘化丙啶染 色進行流式細胞儀檢驗。圖中的百分比代表細胞凋亡之細 胞停止於次-G1期之比例。控制組細胞（C)培養於DMEM 中。結果代表三個獨立實驗。 【主要元件符號說明】 無 154320.doc -18-Figure 5 shows the effect of autophagy inhibitors on apoptosis induced by fermented soybean extract. (A) Inhibition effect of 3-MA on ML-1 cells. (B) 3-MA 154320.doc 17- 201235046 Inhibition effect on Hep 3B cells. After treatment, the cells were stained with propidium iodide for flow cytometry. The percentages in the graph represent the proportion of apoptotic cells that stopped in the sub-G1 phase. Control group cells (C) were cultured in DMEM. The results represent three independent experiments. [Main component symbol description] None 154320.doc -18-