第一部分：桑葚萃取物降低四氯化碳、STZ、脂多醣體 引起大鼠肝損傷之研究 第二部分：探討N-去甲荷葉鹼致人類乳癌細胞 MCF-7 凋亡之作用

一、Study on the effect of Mulberry (Morus alba L) in CCl4, STZ and lipopolysaccharide-inducing liver injury in rats 二、Study on the effect of N-nornuciferine induced human breast cancer cell apoptosis

彭日新(Jih-Shin Peng)

指導教授 ： 王朝鐘

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2010年)

https://doi.org/10.6834/CSMU.2010.00133

摘要

第一部分:
截至目前為止，許多的研究都已證實桑椹之水萃取物（mulberry water extracts ; MWEs）具有抗氧化與抗發炎的能力，本研究欲驗證一天然植物桑椹之水萃取物是否具有抑制肝發炎的能力。以六週齡體重約150-170克雄性Sprague-Dawley(S.D.)與wistar rats進行動物實驗。利用腹腔注射四氯化碳(化學性)、STZ(高醣)、脂多糖體(細菌性)三種不同的模式去誘導大鼠產生肝損傷，於飼料中混入0.5%、1%、2%MWEs餵食八到十週(LPS模式餵食一週)。結果發現單獨添加MWEs的組別（MWEs組）與單獨腹腔注射CCl4、STZ、LPS(Lipopolysaccharide)組別(誘導組）相較，其動物血清AST(GOT)、ALT(GPT)、鹼性磷酸酶(ALKP)濃度均降低（p<0.05），且血清中中三酸甘油酯（TG）、總膽固醇（TC）、低密度脂蛋白（LDL）的濃度均降低（p<0.05），罹患心血管疾病的風險值（riskratio）也下降（p<0.05）；尿素氮(BUN)與肌酐酸(CRE)的濃度也下降。顯示MWEs具有降低由CCl4、STZ與LPS誘導的肝損傷作用。另外以TBARs assay加以分析脂質過氧化的程度，可觀察到當給予MWEs後可以有效降低三種模式誘導肝損傷產生脂質過氧化的現象。結果證實MWEs可以有效的減少CCl4、STZ與LPS所誘導產生的肝損傷。因此我們推測，MWEs降低肝三種模式肝損傷的機制中，部分是透過脂溶性抗氧化作用而達成。

第二部分:

癌症，近十年來由衛生署所作的台灣十大死亡原因調查，仍舊是高居榜首，為十大死因之第一名，而乳癌是全世界女性最常見的癌病，世界一年約有50萬人死於乳癌。在歐美國家，平均每四個女性癌症患者中就有一個罹患的是乳癌。2007年台灣的醫療統計報告發現乳癌位居女性癌症死亡率的第一名。所以如何預防及延緩癌症形成是目前研究的熱門主題。而目前廣泛研究趨向於天然物質來抑制癌細胞之增生及惡化。
荷葉是睡蓮科多年生草本植物，其化學成分含有葉鹼(nuciferine)、N-去甲荷葉鹼(N-nornuciferine)、O-去甲荷葉鹼(O-nornuciferine)、牛心果鹼(anonaine)、繞袂鹼(roemerine)、亞美帕鹼(armepavine)、N-甲基衡州烏藥鹼(N-methylcoclaurine)、原荷葉鹼(pronuciferine)、鵝掌楸鹼(liriodenine)及去氧繞袂鹼(dehydroroemerine)等多種生物鹼屬於異喹啉類生物鹼中苄基異喹啉和阿朴芬結構 ，有許多研究證實荷葉中的生物鹼具有抗氧化之功效，
但目前荷葉鹼的醫學功能並沒有深入的研究，本研究將探討N-Nornuciferine對於人類乳癌細胞株MCF-7是否具毒殺之作用。MCF-7人類乳癌細胞株處理在不同劑量N-Nornuciferine下，觀察細胞凋亡(apoptosis)的現象。結果顯示，隨著N-Nornuciferine濃度增加對於造成細胞凋亡的現象也越顯著：包括細胞型態的改變。利用流式細胞儀分析在相同劑量下，N-Nornuciferine會對MCF-7造成細胞凋亡和細胞週期停滯在G0/G1 phase的現象。相同的結果也出現在Wsstern Blotting的分析當中，發現促進G0/G1 phase相關蛋白有被抑制的現象、caspase-3及caspase-9活化態蛋白也會被N-Nornuciferine活化。因而造成MCF-7人類乳癌細胞株產生細胞凋亡之現象。

關鍵字

桑葚水萃 ； 肝損傷 ； 乳癌

並列摘要

PARTⅠ:Recently, increasing evidences showed that many mulberry water extracts (MWEs) had the ability to antioxidant and inhibited inflammatory. In this study, we aimed to examine the effect of MWEs on anti-inflammation in liver.First of all, SD and wistar rats induced liver injury by intraperitoneal injection STZ(streptozotocin), CCl4(carbon tetrachloride) and LPS(Lipopolysaccharide),and then we supplemented with 0.5, 1 and 2%MWEs for 8-10 weeks. The results showed that MWEs possessed the abilities to improve liver and kidney injury, including the reduction of AST,ALT,Blood urine nitrogen (BUN),triglyceride(TC) and total cholesterol (TC) in plasma. MWEs was also found to possess the ability to reduce lipid peroxidation in liver. In conclusion, the result showed MWEs can reduce CCl4,STZ and LPS-induced liver damage, and the effects might partially be due to its ability on improving antioxidative condition.

PART Ⅱ:According to the research form the Department of Health of Executive Yuan,cancer has been first reason of death in Taiwan. But the breast cancer is the world female most common cancer, world one year approximately has 500,000 people to die of the breast cancer. In the European and American countries, what in the average every four feminine cancer patient has one to suffer from is the breast cancer. in 2007 Taiwan's medical statistics report discovery breast cancer is situated the feminine cancer mortality rate first.Therefore,it is a popular topic of discussing how to prevent and postpone the formation of cancer.Nowadays,most researches tend to use natural materials to control the growing and aggravation of the cancer cell.
The leaves of Nelumbo nucifera Gaertnero is perennial aquatic crop grown.Its chemical composition contains：Nuciferine、N-nornuciferine、Anonaine、O-nornuciferine、Anonaine、Roemerine、 N-methylcoclaurine、 Liriodenine and Pronuciferine belonging to aporphine and benzylisoquinoline structure of isoquinoline alkaloids.In the biological activity.Many studies demonstrate that alkaloids in Nelumbo nucifera leaves have the effect of antioxidation.
But upon to now,researchers just emphasized the effects of nelumbine in clinical research but not in the mechanisms.Because of that,we designed a series of experiments to study how N-nornuciferine induceded human breast cancer cell MCF-7 apoptosis.The MCF-7 cell was used as the experimental model to observe the apoptosis phenomena under the circumstances of different concentrations of N-nornuciferine.It is discovered that more N-nornuciferine consistency increased,the more conspicuous apoptosis became,including the changes of the morphology.Analyzing with Flow cytometric,we found that same amount, N-nornuciferine caused MCF-7 cell cycle arrest in the G0/G1 phase and also the death of MCF-7.The same result to occurring in the Western Blotting analysis.We found that the promotes G0/G1 phase related protein were inhibition by N-nornuciferine.And N-nornuciferine also activated cleaved-form-caspase3 and cleaved-form-caspase9.which means N-nornuciferine could induced MCF-7 cell apoptosis.

並列關鍵字

MWEs ； liver injury ； breast cancer

參考文獻

120.Yang, D., et al., Antioxidant activities of various extracts of lotus (Nelumbo nuficera Gaertn) rhizome. Asia Pac J Clin Nutr, 2007. 16 Suppl 1: p. 158-63.

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