- 學位論文
中草藥對於免疫調節與抗病毒功效之研究
In Vivo Cytokine Immunomodulating Effect and In Vitro Antiviral Effect Of Herbal Medicines
摘要
我們的研究是使用中草藥紅景天對於免疫的調節作用與小蘗鹼的抗病毒功效。薔薇紅景天或玫瑰紅景天 (Rhodiola rosea (L.)) 是一種生長在東歐和亞洲高海拔及寒冷環境的植物，同時也是東方醫學常被使用的珍貴和可以用來食用的一種中草藥。到目前紅景天在體內對細胞激素 (Cytokine) 的免疫調節機制仍不清楚。在紅景天植物所含有生物活性的成份中，紅景天苷 (Salidroside) 是紅景天植物所含有的成份中最有生物活性的化合物。我們研究的目的主要是在評估使用紅景天標準溶液 (Rhodiola rosea Standardised Solution，RRSS) 和紅景天苷對生物的毒性和細胞激素的免疫調節作用。研究中使用高效能液相層析儀，定量分析出紅景天苷在紅景天標準溶液的含量百分比是4.39％ (W/V)。 研究實驗是使用經過卵清蛋白 (Ovalbumin，OVA) 致敏 (Primed) 的Balb/c小鼠；小鼠分成六組，每組有四十隻的Balb/c小鼠來進行研究。每天每組的小鼠分別給予餵食不同濃度，分別為50mg/kg，100mg/kg和200mg/kg的紅景天標準溶液或餵食含紅景天苷4.4mg/kg的溶液。實驗的陽性對照組是給予小鼠餵食5mg/kg的咖啡酸苯乙酯（Caffeic acid Phenethyl Ester，CAPE），而陰性對照組為給予小鼠餵食蒸餾水。 對於中草藥的抗病毒功效我們是使用一種從黃連根莖 (Coptidis Rhizoma) 中萃取出來的植物鹼黃連素 (Berberine) 又称為小蘗鹼。黃連也是清胃散 (Ching-Wei-San) 所含有的中藥成份中，具有最強抗菌效果的草藥成份。我們使用高效能液相層析儀 (High-pressure liquid chromatography，HPLC)，從6.25mg/mL (w/v) 的黃連根莖中可萃取出的植物鹼小蘗鹼溶液，或從50.00mg/mL (w/v) 的清胃散溶液中，定量分析出的小蘗鹼含量為0.26mg/mL。 紅景天的急性和亞急性生物毒性試驗結果顯示治療組的小鼠在體重，肝，腎功能及病理的變化在統計上不具顯著性的差異。在評估紅景天在體內對細胞激素的免疫調節作用，實驗的設計是從第一至第四週，每組的小鼠分別給予餵食紅景天標準溶液或紅景天苷。餵食紅景天標準溶液的小鼠，其細胞激素測定的結果發現Th1細胞分泌的細胞激素白細胞介素 (Interleukin，IL) IL-2和干擾素 (Interferon) IFN-γ和Th2細胞分泌的細胞激素IL-4和IL-10都有顯著增加而且這結果在統計上是具顯著性的差異 (P<0.05)。此外研究也發現，餵食紅景天苷的小鼠，其細胞激素的免疫調節作用比餵食紅景天標準溶液的小鼠來得差，而且在體外細胞激素的免疫調節作用也沒有劑量的依賴性。餵食時間的相關性除了細胞激素IL-2不具有顯著性的差異(p =0.114，p >0.05) 外，其他的細胞激素的分泌都與劑量和餵食時間的相關性有顯著性的差異(p =0.000，p <0.05)。 清胃散在對於抗單純皰疹病毒 (Herpes Simplex Virus) 感染時所辨演的角色，實驗是使用繼代細胞培養的非洲綠猴腎細胞 (Vero Cell)，來檢測其被皰疹病毒感染後對細胞所產生的細胞毒性 (Cytotoxicity)，及抗單純皰疹病毒一型 (Herpes Simplex Virus Type 1， HSV-1) 和抗單純皰疹病毒二型 (Herpes Simplex Virus Type 2， HSV-2) 病毒的活性。研究的結果顯示小蘗鹼的選擇性指數 (Selectivity Index) 比從黃連根莖和清胃散中所萃取得的植物鹼小蘗鹼的選擇性指數好1.2-1.5倍，而且黃連根莖的萃取物和清胃散的抗病毒活性是與小蘗鹼在水溶液中的含量成正比。 這研究也發現黃連素及RRSS的免疫的調節作用與抗病毒能力並不受準備過程所影響。研究的結果顯示體內的Th1型和Th2型細胞激素的分泌可以經由餵食RRSS和紅景天苷而增加分泌。黃連素的抗病毒作用機轉可能是在皰疹病毒在病毒穿透細胞壁後及脫氧核糖核酸 (Deoxyribonucleic Acid DNA ) 的合成過程前，其干擾皰疹病毒的複製週期。在所有含有小蘗鹼天然成分的植物中，例如黃連的根莖和清胃散都具有抗單純皰疹病毒的作用。
並列摘要
Although Rhodiola rosea (L.) is widely used and disseminated in oriental medicine, its in vivo effects on cytokine modulation remain unclear. Among the biologically active components of Rhodiola rosea, salidroside was suggested to be the most active compound. The objectives of this study were to assess the toxicity and cytokine modulation effects of Rhodiola rosea-Standardised solution (RRSS) and salidroside. Berberine is an alkaloid extracted from Coptidis rhizome. Among the individual herbal components of a Chinese herb medicine, Ching-Wei-San, Coptidis Rhizoma has the most potent antimicrobial activity. Quantitative HPLC analysis determined the content of salidroside in RRSS to be 4.39% (W/V) and the quantitative analysis of berberine from 6.25 mg/mL (w/v) Coptidis rhizome extract or 50.00 mg/mL (w/v) Ching-Wei-San was determined to be 0.26 mg/mL. Group of Balb/c mice were fed daily with different doses of RRSS or salidroside, with CAPE or distilled water used as positive and negative controls, respectively. To explore the potential use of Ching-Wei-San against herpes simplex virus (HSV) infection, the cytotoxicity, anti-HSV-1 and anti-HSV-2 activity in Vero cells were assayed. The selectivity index of berberine was about 1.2–1.5 times higher than that of Coptidis rhizome extract and Ching-Wei-San. The acute and subacute toxicity tests of RRSS did not reveal weight difference, pathological change, or abnormality in liver or kidney function indices among the treated groups. Ovalbumin (OVA)-primed mouse cytokine assays demonstrated that both Th1 (IL-2 and IFN-γ) and Th2 (IL-4 and IL-10) cytokines, apart for IL-2 secretion which is not associated with time, the orther cytokine secretion were associated with dose and time (p <0.05). Moreover, the cytokine modulation effects of salidroside were less prominent than that of RRSS treatment and not dose-dependent. The antiviral activities correspond to the content of berberine in the aqueous solution. Berberine may interfere with the viral replication cycle after virus penetration and no later than the viral DNA synthesis step. The activities of the RRSS and berberine were not affected by the preparation processes. Berberine, the natural plants that contain this component, including Coptidis rhizome, and Ching-Wei-San have all shown anti-HSV effects and the increased secretion of both Th1- and Th2- pattern cytokines can be achieved with RRSS and salidroside treatment.