| 摘要: | 膽鹼假說、類澱粉蛋白假說、濤蛋白假說，包含在阿茲海默症致病病理假說，且彼此互相影響。此外，蛋白質非酵素醣化反應、自由基與氧化壓力及神經生長因子，也對於神經細胞的存活有深遠影響。何首烏，於典籍《神農本草經》提及可「益血氣，黑髭髮，延年不老」；近代藥理研究也深入探討延緩老化的可能機制與臨床運用。因此，本實驗欲探討未經炮製之何首烏，其不同極性劃分的成分是否具有改善阿茲海默症相關因子之潛力。本實驗以乙醯膽鹼酯酶抑制活性與DPPH自由基清除活性篩選萃取物與劃分物。比較何首烏三種不同萃取物A、B、C之乙醯膽鹼酯酶抑制能力與DPPH自由基清除活性，其50%抑制所需濃度(IC50)均為為C > A > B。抽取物篩選結果，萃取物B具有較佳的抑制乙醯膽鹼酯酶活性與清除自由基能力，繼續進行液-液相區分分配(liquid-liquid partition)，得到三劃分物，分別為B1、B2、B3。劃分物之乙醯膽鹼酯酶抑制能力，IC50為B1 > B3 > B2；DPPH自由基清除活性之IC50為B3 > B1 > B2。劃分物篩選結果，B2具有較佳的抑制乙醯膽鹼酯酶活性與清除自由基能力。另，三抽取物與三劃分物皆表現高抑制乙型類澱粉蛋白胜肽聚集活性；在已聚集之乙型類澱粉蛋白纖維，促進再溶解試驗中，抽取物在1 µg/mL濃度下，再溶解效果為A > B > C，促進再溶解活性最高可達27%；而劃分物在1 µg/mL濃度下，再溶解效果為B2 > B1 > B3，促進再溶解活性最高可達近60%。B抽取物與劃分物B1、B2，在牛血清蛋白/半乳糖與牛血清蛋白/甲基乙二醛模式下，皆表現抑制蛋白質非酵素醣化反應。利用製備式高效液相層析儀進行劃分物B2分離，並針對280 nm吸收有明顯波峰處，以分液收集器進行收集，個別合併得B2之管2、4~8，並進行抑制乙醯膽鹼酯酶活性、抑制乙型類澱粉蛋白胜肽聚集與已聚集之乙型類澱粉蛋白纖維再溶解能力。利用製備式高效液相層析儀進行劃分物B1分離，並針對280 nm吸收有明顯波峰處，以分液收集器進行收集，個別合併得B1-a~f。而於PC12細胞模式的細胞突出分枝生長實驗中，低濃度之萃取物B有類似於NGF之促進效果，B2與其分離物則無促進突出分枝生長效果。而B1則具有類似NGF之促進效果，此外B1之分離物B1-a、d也具促進突出分枝生長之能力。於探討神經細胞保護作用於MGO誘導SH-SY5Y細胞毒性之模式實驗中，除了B1-e，萃取物B與其三劃分物B1、B2、B3，及B2之分離物、B1-a~d與f均有細胞保護作用。由目前結果可確定何首烏萃取物B確實具潛力，而其效果可能來自於其不同劃分物、分離物，其分別藉由降低不同阿茲海默症之致病因子，而使何首烏萃取物B具有預防阿茲海默症或減緩失智症狀之潛力。
The hypothesis of Alzheimer's disease, including choline hypothesis, amyloid hypothesis, and tau hypothesis, did not only work alone, but also affected each other. In addition, non-enzymatic glycation, free radicals, oxidative stress, and nerve growth factors also had a profound impact on the survival of nerve cells. He Shou Wu is a Chinese herbal medicine, derived from the Polygonum multiflorum Thunb. Shennong Ben Cao Jing, a Chinese book on traditionally medicinal plants, reported that He Shou Wu could be used to nourishing blood and benefiting qi、darkening mustache and hair and prolonging life. Recently pharmacological research also explores the mechanism and clinical application of Polygonum multiflorum to anti-aging and reduce the risk of Alzheimer’s disease. Therefore, this experiment used the above-mentioned hypotheses and factors related to Alzheimer's disease to evaluate whether the different polarity of ingredients in unprocessed Polygonum multiflorum had potential to improve Alzheimer's disease or not. Acetylcholinesterase (AChE) inhibitory activity and DPPH scavenger ability were used to assay which extracts and fractions of Polygonum multiflorum was most effective firstly. Compare the AChE inhibitory ability and DPPH free radical scavenging activity of 3 extracts, named A,B,C, from Polygonum multiflorum. The half maximal inhibitory concentration (IC50) of 3 kinds of extract in AChE inhibitory activity assay and DPPH scavenger ability assay were C > A > B. The result showed that B had the best overall effect. B was separated by partition, and 3 fractions were collected and named B1, B2, and B3 respectively. 3 fractions of B were assayed the effect. The IC50 of 3 fractions of B in AChE inhibitory activity assay were B1 > B3 > B2. The IC50 of 3 fractions of B in DPPH scavenger ability assay were B3 > B1> B2. The result showed that B2 had the best overall effect. In addition, both 3 kinds of extracts and 3 kinds of partitions exhibited high activity for the aggregation of beta-amyloid peptides. In re-dissolution of aggregation beta-amyloid ability assay, the performance of 3 kinds of extracts with a concentration of 1 μg/mL were A> B> C, and promoted re-dissolution activity up to nearly 27%. In the same analytical method, the performance of 3 fractions of B with a concentration of 1 μg/mL were B2 > B1 > B3, and promoted re-dissolution activity up to nearly 60%. Either B or B2, b3 could inhibit non-enzymatic glycation reaction in BSA/galactose and BSA/MGO model. B2 was separated by HPLC and collected the obvious peaks with absorbance at 280 nm by fraction collector. The collections were individually combined into 6 tubes, and the ability of AChE inhibitory, anti-aggregation of beta-amyloid peptides and re-dissolution of aggregate beta-amyloid were assayed. B1 also was separated by HPLC and collected the obvious peaks with absorbance at 280 nm by fraction collector. The collections were individually combined into 6 fractions, and were named B1-a~f respectively. The assay of neuron outgrown in PC12 cell model, low concentration of B has an effect similar to NGF, but the expected effective B2 and its isolate, which were expected effective, were ineffective, in fact. On the contrary, B1 had an effect similar to NGF. Also, the B1-a and d, isolated from B1 by HPLC, could promote neuron outgrown. Except of B1-e, B, B1, B2, B3, isolates of B2, B1-a~d, and f have protective effect to anti-MGO-induced SH-SY5Y cytotoxicity. Based on the current results, B extract from Polygonum multiflorum was considered to have the potential. The effect of B extract might come from its different partitions and isolates, which respectively reduce the different pathogenic factors of Alzheimer's disease. These effective partitions and isolates made extract B from Polygonum multiflorum had the potential to prevent Alzheimer's disease or alleviate the symptoms of dementia. |
