I.台灣欒木及荷葉抗癌作用之研究 II.枸杞萃取物對於改善乾眼症之研究

I.Anticancer effect of Koelreuteria formosana and Nulumbo Nucifera leaves II.Improvement of dry eyes using a Lycium barbarum extract

洪啟庭(Chi-Ting Horng)

指導教授 ： 謝易修 ； 王朝鐘

中山醫學大學/醫學院/生化微生物免疫研究所/博士(2017年)

摘要

台灣十大死因之首為惡性腫瘤，前三名分別為肝、肺癌及大腸直腸癌。台灣欒樹為台灣原生種，其花、根、葉可製成中藥具消腫及消炎效果，但種子具有毒性，因此可以用來研究與細胞凋亡相關實驗。荷葉萃取物具有抗氧化能力、保護肝臟效果與抗癌的功效。乾眼症盛行率相當廣，全球約有五分之一的人患有乾眼症。枸杞具有抗發炎、抗氧化能力、抗老化的能力與抗癌的能力等。
因此本論文分為兩個部分，第一部分主要探討台灣欒樹酒精萃取物 (Koelreuteria formosana ethanolic extract；KFEE) 及荷葉萃取物 (Nelumbo nucifera leaves phenolic extract；NLPE) 抗癌作用之研究；第二部分主要探討枸杞萃取物對於改善乾眼症之研究。
第一部分主題一，台灣欒樹酒精萃取物 (KFEE) 以台灣欒樹的樹枝以50 %酒精溶液萃取，在MTT實驗發現KFEE對DLD-1和COLO 205人類結腸癌細胞有毒殺作用，隨著處理藥物濃度增加，細胞的毒殺性也隨之增加。KFEE也可有效抑制癌細胞的增生作用，使p21上升、抑制cyclin B、cyclin E與cdc25C、使腸癌細胞停滯在G2/M phase，而其sub-G1-phase並無隨著藥物濃度增加而增加。同時一系列的凋亡蛋白酶 (caspase) 和特異凋亡相關的因子也並無明顯的變化，顯示KFEE並非使細胞凋亡作用。在實驗中我們也觀察到某些自噬小體 (autolysosome)、酸性囊狀胞器、LC3-II、ATG-3及 ATG-16等自噬作用特定標記物增加，而 Bcl-2、PI3K、Akt 和mTOR 這些與細胞自噬與凋亡相關蛋白的表現量，也在 KFEE處理後下降。而本實驗中我們使用自噬作用抑制劑的3-methyladenosine (3-MA) 與bafilomycin-A1 (BAf-A1) 可以抑制自噬細胞，進而抵銷KEFF作用。因此我們推論KEFF對癌細胞的毒殺作用，最主要是透過細胞自噬，而非細胞凋亡。另外我們也以裸鼠當動物實驗，以其右後肢皮下注射穩定表達Pg14. 50 (luc2/CMV/Hygro) 載體的DLD-1細胞株，利用管餵給予KFEE後，我們發現餵食KFEE的老鼠其長在身體的腸腫瘤生長會受到抑制。
第一部分主題二，本動物實驗是以SD老鼠為實驗標本，以荷葉的酚萃取物餵食，來檢視其對抗方式由二甲基亞硝胺 (DEN) 所誘發的氧化壓力與肝癌病變的相關之情形。誘發方式是以DEN餵食SD老鼠共十二周，使其產生肝細胞病變；本實驗Sprague-Dawley (SD)鼠中分為五組，依不同的餵食荷葉萃取物而定。第一組為控制組，只讓其正常飲水。第二組為服用DEN (以誘發產生肝癌)、第三組口服DEN並以0.5%的荷葉酚萃取物來治療。第四組為口服DEN並以1.0%的荷葉酚萃取物治療、第五組為口服DEN同時服用2.0%的荷葉酚萃取物。另外我們也評估各種臨床化學數值、器官重量、發炎指數、蛋白表現、酶與抗氧化因子之變化情形。我們發現給予DEN會使某些代表肝臟受傷指數如GOP、GPT、總膽固醇與三酸甘油脂等數值降低。另外我們發現荷葉酚萃取物也會使glutathione transferase、superoxidase dismutase、catalase、glutathione peroxidase、reduced glutathione等抗氧化因子與還原劑上升；另外也會使過氧化因子如lipid peroxides降低。另外荷葉酚萃取物也會使已形成的肝臟腫瘤尺寸變小，另外肝臟的Rac1、PKCα與GSTπ 數值亦降低。由以上的種種結果，我們認為荷葉酚萃取物可以改善肝癌的機轉：乃是由於在阻礙細胞脂質的過氧化、肝臟的受損與促進其抗氧化能力。
第二部分，全部共四十五隻 Sprague-Dawley (SD) 的公鼠在本實驗中誘發成乾眼模式。實驗一中，我們先注射阿托平液體注射於五隻SD 的公鼠的右眼 (劑量為 1 毫克/公斤)，同時得到其乾眼狀況下的三種檢查的參考數據 (包含淚液試驗、淚液崩裂時間與角、結膜乾眼程度的染色狀況，目的在找出最低數值 (和左眼正常眼比較)。我們發現以阿托平直接注射於老鼠的淚腺上，在第七天可以完全阻斷乙醯膽鹼之釋放、使乾眼的症狀達到最明顯。我們也發現在嚴重SD鼠其淚液試驗僅為5釐米/5分鐘、淚液崩裂時間小於10秒鐘。實驗二中，以相同方法製造出四十隻左眼均為乾眼的SD公鼠、接著再依照枸杞子的餵食與否、和餵食的濃度等，共分為四組 (每一組均十隻)：第一組為控制組 (不餵食)、第二組 (餵食量為250公克/公斤/每天)、第三組 (餵食量為350公克/公斤/每天) 與第四組 (餵食量為500公克/公斤/每天)，若干時間後以上列三種檢查方式來客觀評估乾眼的改善情形，我們持續並觀察了21天。我們發現淚液試驗的數值的恢復、與淚液崩裂的時間延長等。在餵食低濃度的枸杞後的第14天可逐漸恢復，在餵食中、高劑量之枸杞後第7天即可逐漸改善。此外乾眼所誘發的表皮細胞病變之百分比亦在第21天後，亦有明顯的改善。大致而言，劑量越高、其恢復的時間與表徵也會更快。至於枸杞萃取物的內容物分析，以高效液相色譜方法與標準品進行分析，我們發現枸杞含有豐富的多醣體與甜菜鹼兩種主要物質、其可以有效調控乾眼症所引起的氧化壓力與發炎等機轉，而使乾眼的症狀有所改善。我們的研究是文獻上第一次評估枸杞萃取物，對於動物實驗的乾眼症之症狀改善有所助益。然而對於人類使用的口服枸杞的正確劑量，值得更進一步的深入探討。

關鍵字

台灣欒木 ； 荷葉 ； 大腸癌 ； 肝癌 ； 枸杞 ； 乾眼症

並列摘要

Malignant tumor remained the first rank of ten major death caused resons in Taiwan. The top three of malignant tumor were liver cancer, lung cancer and colorectal cancer. Koelreuteria formosana, which is obtained from natural plants endemic to Taiwan, has inhibited cell metastasis in renal carcinoma cells. Lotus (Nelumbo nucifera Gaertn) possesses antioxidant, hepatoprotective, and anticancer potential. Dry eye prevalence is quite wide, about one fifth people around the world suffering from dry eye. Goji possesses anti-inflammatory, antioxidant capacity, anti-aging ability and anticancer potential.
There are two parts in this study, first is about anticancer effect of Koelreuteria formosana ethanolic extract (KFEE) and Nelumbo nucifera leaves phenolic extract (NLPE), second is about improvement of dry eyes using a Lycium barbarum extract
In part I, theme I, KFEE exerted a strong cytotoxic effect on DLD-1 and COLO 205 human colorectal cancer cell lines. KFEE effectively inhibited cancer cell proliferation, induced G2/M phase arrest associated with down-regulation of cyclin E, cyclin B and cdc25C and up-regulation of p21, and induced cell death by activating autophagy but did not cause apoptotic cell death. Exposed KFEE cells showed increased levels of acridine orange, autophagic vacuoles, and LC3-II proteins, which are specific autophagic markers. Bcl-2, p-Akt, and p-mTOR levels, which have been implicated in autophagic down-regulation, were significantly decreased after KFEE treatment. Autophagy inhibitor 3-methyladenosine and bafilomycin-A1 and genetic silencing of LC3 attenuated KFEE-induced growth inhibition. These findings suggested that KFEE causes cytostatic effect through autophagy. In xenograft studies, oral administration of KFEE had significantly inhibited the tumor growth in nude mice that had received subcutaneous injection of DLD-1 cells.
In part I, theme II, this study determined the protective role of aqueous extract from Nelumbo nucifera leaves (NLPE) against N-diethylnitrosamine (DEN)-induced oxidative and hepatocellular carcinogenesis in a sample of Sprague-Dawley (SD) rats. NLPE was fed orally to rats in which hepatic carcinoma was induced with DEN for 12 weeks. Five groups of 12 groups each were used for the study： Group 1 (control group) rats received distilled water; Group 2 rats were induced with DEN; Group 3 rats were induced with DEN and co-treated with 0.5% NLPE; and Group 4 rats were induced with DEN and co-treated with 1.0% NLPE; Group 5 rats were induced with DEN and co-treated with 2.0% NLPE. Clinical chemistry, organ weight, inflammatory makers, protein expression, enzyme, and antioxidant analyses were conducted. The oral DEN administration to SD rats resulted in significantly decreased levels of serum alanine aminotransferase, aspartate aminotransferase, total cholesterol and triglyceride, which is indicative of hepatocellular damage, compared with the control group. DEN-induced oxidative stress was inhibited by NLPE and the inhibition was paralleled by decreased lipid peroxidases and increased glutathione transferase, superoxide dismutase, catalase, and glutathione peroxidase activity in liver tissues. The status of non-enzymatic antioxidant, such as reduced glutathione, was also found to be increased in NLPE-administration rats. Furthermore, NLPE decreased tumor size, hepatic Rac1, PKCα, GSTπ expressions compared with the DEN-only group. This supplementation of NLPE reduced the adverse changes that occur because of liver cancer. These results prove that NLPE protects against liver carcinogenesis induced because of treatment with DEN through protecting the lipid peroxidation, decreasing hepatic damage, and enhancing the antioxidant ability.
In part II, total 45 male Sprague-Dawley (SD) rats were enrolled in our studies. In experiment 1, all the right eyes of 5 rats were induced into the dry eye model by injecting the atropine solution (1mg/kg). Therefore, we could gain the ranges of three parameters (schirmer’s test, tear break-up time and kerato-conjunctival staining). We found that the maximal level of dry eye should reach after 7 days of atropine injection by blocking the Ach transport which could be proven by the changes of signs and symptoms in 5 male SD rats. Besides, it revealed that the schirmer’s test is only 5mm/5min and the tear break-up time is less than 10 sec. In experiment 2, 40 SD rats with dry eye of all left eyes were created as the above method. Then, they were divided into 4 groups by various concentration of goji extract including group 1 (control group), group 2 (250 mg/kg/day), group 3 (350 mg/kg/day) and group 4 (500 mg/kg/day). We followed the changes of schirmer’s test, tear break-up time and kerato-conjunctival staining for 21 days and recorded. We would observe the changes of symptoms and signs after Goji feeding. In middle and high doses of goji extract group, the dry eye condition should become better; however, the low dose group may improve after 14 days. Furthermore, the degrees of kerato-conjunctival changes of dry eye may also improve significantly after 21 days. Besides, it showed the dose-dependent manner. At the same time, we found the exact amount of polysaccharides and betaine in the goji extract by HPLC method. The two components of the Goji were considered to own the abilities of decrease the inflammation and oxidase stress of ocular surface which may be benefit to the dry eye. This is the first study to evaluate the changes of dry eye by intake of goji under animal model. The exact oral doses in human should be further intervention in the future.

並列關鍵字

Koelreuteria formosana ； Nulumbo Nucifera ； Colorectal cancer ； liver carcinoma ； Lycium barbarum ； dry eyes

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