愛玉子種子水萃物癌症預防活性之體外試驗

Abstract

愛玉子為台灣地區特有的富含果膠之木質藤本植物種子，分布在海拔800至1800公尺之間的山區。本研究將愛玉子種子，以果汁機粉碎，加水萃取的方式，得到含有可溶性固形物的水萃液，再經由冷凍乾燥，獲得愛玉子種子水萃物 (water extract from jelly fig seeds, WEJFS)，為試驗原料。 利用體外 (in vitro) 試驗探討愛玉子種子水萃物的細胞毒性、保護牛胸腺細胞DNA、大鼠肝臟clone 9細胞的DNA和Clone 9細胞的作用，以及對小鼠大腸癌細胞CT-26、人類肝癌細胞HepG2和人類雌激素依賴型乳癌細胞MCF-7生長之影響，並且分析硫酸銨和膜區分 (fraction) 成分與癌細胞生長抑制的關係。研究結果顯示每毫升培養液含有0至200 μg的愛玉子種子水萃物無細胞毒性。愛玉子種子水萃物對DNA和Clone 9細胞具有保護作用，包括降低氫氧自由基誘導的DNA損傷，與抑制過氧化氫造成Clone 9細胞的死亡。在水萃物保護Clone 9細胞之測定結果顯示提高細胞的存活率，降低Clone 9細胞的caspase-3、亞雙倍體比率，並阻止DNA產生片段化。愛玉子種子水萃物對於小鼠大腸癌細胞CT-26、人類肝癌細胞HepG2和人類雌激素依賴型乳癌細胞MCF-7皆有生長抑制的效果，且以抑制CT-26和HepG2細胞的生長能力較佳。使用截留分子量 (molecular weight cut off, MWCO) 為0.5、3.0、10.0及70.0 kDa的過濾膜過濾所獲得的膜區分水萃物，以3.0至10.0 kDa膜區分物對HepG2細胞具有較高的生長抑制能力。此3.0至10.0 kDa膜區分水萃物在三甲基甘胺酸胜肽電泳表現7 kDa的主要色帶，並無洋刀豆血球凝集素A親和式膠體鍵結的能力。利用不同比例的飽和硫酸銨分劃與HepG2細胞培養，結果發現40至80%的分劃對細胞具有生長抑制能力，在此比例的硫酸銨分劃中，是以蛋白質為主要的化學成分。以上各試驗所呈現的結果，顯示愛玉子種子水萃物潛在具有癌症之化學預防活性。

Jelly fig (Ficus awkeotsang Makino) achenes are the seeds of a native woody vine that grows on 800-1800 m high hillsides in Taiwan. In the present study, a water extract from jelly fig seeds (WEJFS) was prepared to investigate the cytotoxicity on rat hepatocyte clone 9 cells, and the protective effect on clone 9 cells and their DNA against oxidative stress damage. The growth inhibition on three cancer cell lines, namely mouse colon cancer CT-26, human hepatoma HepG2 and breast cancer MCF-7 were also studied. Components from ultrafiltration and ammonium sulfate fractionations were analyzed for their activities on the growth inhibition of HepG2 cancer cells. Results showed that WEJFS at 0-200 μg total solids/mL concentration had no cytotoxicity on Clone 9 cells, and that WEJFS inhibited the hydrogen peroxide induced damage in Clone 9 cells as shown by the caspase-3 activity, SubG1 (%), and DNA fragmentation. The anticancer activities on the above-mentioned cancer cell lines were also shown. Among them, CT-26 and HepG2 cells were inhibited more effectively. Filtrates of ultrafiltration within different membrane cut-off ranges were used to test the activity of growth inhibition from WEJFS. The 3.0-10.0 kDa fraction exhibited the highest inhibition ability on HepG2 cells. SDS-PAGE of the fraction found a major band at 7 kDa. The 3.0-10.0 kDa fraction was found without concanavalin A sepharose affinity column binding ability. Among the saturated ammonium sulfate precipitation fractions, the 40-80% fraction showed the highest growth inhibition ability on HepG2 cells. Protein was found to be the major component in the fraction. In conclusion, these results suggested that WEJFS can be a decent potential agent for chemoprevention of cancers.