根據1999年世界衛生組織（WHO）標準，健康男性的精液品質在過去的數十年來有逐漸惡化的現象，其中包括精液量、精子濃度、活動力和形態正常精子比例的下降。精液品質的惡化，有部分因素為長期暴露在過氧化環境所導致的結果，而長期處於高濃度過氧化環境會使精子功能產生障礙，導致胞內反應性氧化物質（Reactive oxygen species; ROS）增加，進而造成精子活動力及精子對卵的穿透能力下降而降低受孕能力。古書中朱震亨提及香椿可治男性精滑夢遺，但目前並無相關研究報告，因此，香椿對男性精子之生理功能需進一步以科學實驗加以探討。
本研究以不同萃取方法，萃取出不同成分椿葉萃取液，探討香椿對人類精子之生理功能，分為以下兩階段來探討：（一）以不同成份（5-2, 5-3, 5-4, 5-4R, 5-5, 5-5R, black）、不同濃度（0.01, 0.05, 0.1, 0.5, 1 mg/ml）之椿葉萃取液處理人類精子，利用流式細胞儀以DCFH-DA測定精子胞內ROS含量、以DiOC6偵測粒線體膜電位、以Acridine orange分析染色質結構完整性、電腦輔助精子分析系統分析精子活動速度、利用冷光儀偵測螢火蟲螢光素酉每催化ATP產生的冷光、利用螢光分光光譜儀以Fura-2 AM測定胞內鈣離子濃度、利用螢光顯微鏡以EtBr/AO做細胞死亡評估。（二）由第一階段的結果，我們得到對精子功能最佳化之萃取液為5-4R。以0.2 mM H2O2誘發精子產生氧化壓力後，探討5-4R是否能改善氧化壓力所誘發之精子功能障礙。
結果顯示，大部分低濃度（0.01或0.05 mg/ml）的椿葉萃取液對精子生理功能不會造成傷害，然而，除了5-4R以外，高濃度（0.5或1 mg/ml）的椿葉萃取液則表現pro-oxidant的作用，使精子ROS上升、活動速度下降、粒線體膜電位及ATP含量降低、細胞凋亡比例增加、染色質完整性受損。以H2O2誘發精子產生氧化壓力後，發現5-4R可使精子胞內ROS顯著降低、活動速度回升、粒線體膜電位及ATP含量增加、胞內鈣離子及細胞死亡數降低，且可保護染色質結構之完整性。相關性分析結果顯示，精子胞內ROS含量與活動力（r = -0.38, p < 0.05）及正常形態百分比（r = -0.48, p < 0.01）呈顯著負相關。而精子活動力與ATP濃度（r = 0.74, p < 0.05）、粒線體膜電位（r = 0.53, p < 0.05）及胞內鈣離子含量（r = 0.60, p < 0.05）皆呈顯著正相關。精子染色質結構分析顯示變性細胞（denatured cells）比例與人工受精之受精率呈顯著負相關（r = -0.50, p < 0.05），而傳統的精液分析中的精子活動力與人工受精之受精率無相關性（r = 0.12, p = 0.6204）。
本研究結論，不同成分及不同濃度的椿葉萃取液對人類精子具有不同的生理作用，高濃度的5-4R不具pro-oxidant作用並可有效改善氧化壓力所導致的男性精子功能障礙，對於健康男性或不孕男性患者都具有提升精子品質的作用。其他椿葉萃取液在高濃度會呈現pro-oxidant作用，誘發精子凋亡，將來可更進一步利用其他細胞模式探討其抗癌之生理功效。

According to the criteria of World Health Organization (WHO) in 1999, the semen quality of health man, such as semen volume, concentration, motility and the percentage of normal morphology was declined gradually in the past several decades. The decline of semen quality may be caused by the exposure to the oxidative stress environment for long period of time. Oxidative stress resulted in the increase of reactive oxygen species (ROS) leading to the decrease of fertility due to a loss of sperm motility and a decreased capacity for sperm to oocyte penetration. The role of Toona sinensis Roem (TS) in the therapy of wet dream was first reported by Zhu C.H. in Chinese ancient book. However, no any study regarding the effects of TS on the sperm functions was conduced. Therefore, the main purpose of this study was to investigate the physiological functions of human sperm incubated with different components (5-2, 5-3, 5-4, 5-4R, 5-5, 5-5R, black) and concentrations (0.01, 0.05, 0.1, 0.5, 1 mg/ml) of TS extracts. The sperm functions evaluated in this study included the intracellular ROS level detected with DCFH-DA, mitochondrial membrane potential (MMP) measured with DiOC6, and sperm chromatin structure assay (SCSA) detected with acridine orange by using flow cytometry, sperm motility and velocity determined by computer-assisted spermatozoa analysis (CASA), ATP levels measured with firefly luciferase by luminometer, intracellular calcium concentration measured with Fura-2 AM by fluorescence spectrophotometer, apoptosis and necrosis detected with EtBr/AO by fluorescence microscope.
Our data showed that 5-4R was beneficial to the optimal sperm functions. We further investigated the protective effects of 5-4R against oxidative stress induced by 0.2 mM H2O2 in human spermatozoa.
The results indicated that sperms treated with low concentrations (0.01 and 0.05 mg/ml) of every TS extracts did not damage sperm functions. However, high concentrations (0.5 and 1 mg/ml) of TS extracts except 5-4R exhibited the pro-oxidant property, which increased ROS levels, reduced motility, MMP, and ATP levels, enhanced the sperm apoptosis and impaired sperm chromatin structure. Sperms under oxidative stress induced by H2O2 treated with 5-4R improved their functions via the decreased ROS levels, increased motility, MMP, and ATP levels, decreased intracellular calcium and the maintenance of the chromatin integrity. Intracellular ROS levels of sperm was negatively correlated to motility (r = -0.38, p < 0.05) and percentage of sperm with normal morphology (r = -0.48, p < 0.01). Sperm motility was positively correlated to ATP levels (r = 0.74, p < 0.05), MMP (r = 0.53, p < 0.05) and intracellular calcium (r = 0.60, p < 0.05). The percentage of denatured sperms from SCSA was negatively correlated to the fertilization rate of in vitro fertilization (IVF) (r = -0.50, p < 0.05). No significant correlation between motility in conventional semen analysis and the fertilization rate of IVF (r = 0.12, p = 0.6204) was found.
In conclusion, the different components and concentrations of TS extracts caused different physiological functions in human sperm. High concentration of 5-4R did not exhibit pro-oxidant property and could improve the impairment of sperm functions resulting from oxidative stress. Protective effects of 5-4R might enhance sperm quality for both healthy or infertility men. Moreover, other TS extracts showed pro-oxidant property at high concentration leading to the apoptosis in sperm may be used to further investigate the anticancer effects of TS by other cell models.

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王珮憓，椿葉水萃取液在Alloxan所誘發的糖尿病鼠中降血糖作用之研究，高雄醫學大學醫學研究所碩士論文，中華民國九十年六月。

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