葉下珠分化層及純化合物抗單純疱疹病毒活性及作用機轉探討

只有少數的藥物可供用來治療病毒之感染及抗藥性病毒出現等種種因素，促使尋找更多新抗病毒藥物的需求性。因此，本研究最主要的目的為利用溶斑減少分析法來探討大戟科（Euphorbiaceae）植物，葉下珠（Phyllanthus urinaria L.）抗單純疱疹病毒第一型（HSV-1）及第二型（HSV-2）的活性。實驗結果顯示葉下珠抗HSV-2活性比抗HSV-1來的佳；在各分化層中，以丙酮分化層，乙醇分化層和甲醇分化層的抗HSV-2活性比較好，濃度為100 μg/ml時的抑制能力均在92.7%以上，且抑制能力皆呈現劑量依存之現象。在時間加入效應中發現，於0小時加入上述三種分化層皆能抑制超過85%的HSV-2感染。另外，不同的濃度的丙酮分化層，乙醇分化層和甲醇分化層，在37℃，6小時的條件下，也會完全的將病毒去活化；上述的結果並不會因溫度條件的改變而受影響。在純化合物方面，共探討了葉下珠所含相關成分chebulagic acid，corilagin，euphorbin D，excoecarianin，gemin D，geraniin，hippomanin A，3, 5-di-galloyl-(-)shikimic acid和1, 3, 4, 6-tetra-O-galloyl-β-D- glucose等九種成分；其中以euphorbin D和excoecarianin抗HSV-1及HSV-2的活性最佳。由於euphorbin D和excoecarianin 有較佳的抗病毒活性及較高的選擇係數值，因此以這兩種成分來探討其抗病毒的機制。 Excoecarianin於其抑制50%病毒活性之濃度（IC50）時，就能有效的將病毒去活化；反之，euphorbin D需於高過其IC50濃度時，才會將病毒去活化。此外， ii euphorbin D也會干擾病毒吸附至細胞膜及抑制病毒穿透細胞膜；不過並不會將已附著在細胞膜上的病毒分開。在探討將euphorbin D與ACV合併之抗病毒效果，可發現其併用效果在低濃度時為協同作用；高濃度時為副協同作用。由上述的研究結果可發現，葉下珠的分化層及純化合物各具不同程度的抗HSV-1及HSV-2活性，而其作用機制亦有所差異。本次研究最特別的部份為葉下珠的活性分化層及excoecarianin如何能在低濃度下將病毒去活化，這值得日後更深入的探討。另外，研究也發現euphorbin D呈現多樣化的抗病毒作用機轉。對於euphorbin D與ACV併用後而呈現的副協同和協同作用之抗病毒效果，也是值得深入研究的題材。

關鍵字

天然物；葉下珠；抗單純疱疹病毒活性；作用機轉；協同作用；純化合物

並列摘要

The fewer applicable of antiviral medicine, the emergence of resistant viruses, etc make a desire of searching new effective agent to manage viral infection. In this study, some extracts and pure compounds of Phyllanthus urinaria L. (Euphorbiaceae) were investigated for in vitro anti-HSV-1 and HSV-2 activities by plaque reduction assay. Results showed that the acetone, ethanol and methanol extracts inhibited HSV-2 infection more significance than that of HSV-1. All the three extracts showed more than 92.7% inhibiton against HSV-2 infection at concentration of 100 μg/ml. The time of addition study demonstrated that these three extracts only effective when added during the HSV-2 infection which suggested that they might disturb the initial stage of HSV-2 infection. Furthermore, they can diminish virus infectivity without significantly affecting by incubation time and temperature. Nine pure compounds isolated from P. urinaria were tested for antiviral activity and were found to inhibit HSV replication at different magnitudes of activity. Among them, euphorbin D and excoecarianin had good antiviral activity. Besides, both compounds also had high selectivity index, which served them as the candidates for the following mechanism studies. The results showed that excoecarianin could significantly diminish virus iv infectivity at concentration of IC50, while euphorbin D needed high concentration to achieve the same effect. Euphorbin D was also been found to inhibit HSV-2 from attaching to cell membrane, and prevented HSV-2 from penetrating into cells. However, it can not detach those viruses which had been bound to cells. Euphorbin D, dependent with the used concentrations, acted either synergistically or subsynergistically with ACV in suppressing HSV-2 multiplication. In summary, the extracts and pure compounds of P. urinaria were found to inhibit HSV infection at different magnitudes of activity through different modes of action. The inactivation of virus infectivity at low concentration, and the synergistical or subsynergistical action between euphorbin D with ACV on inhibiting HSV infection, were merits for further investigation.