Safety and immunomodulation evaluations on Chinese herbal novel formulas

Abstract

本論文之目的在探討中草藥複方A與複方B之口服急性毒性及28天亞急性毒性，以評估此兩種複方中草藥之安全性，並利用體外(in vitro)及體內(in vivo)實驗模式，對其免疫調節功能進行研究。 口服急性毒性試驗結果顯示，複方A及複方B之LD50分別為13.27 g/kg BW與10.00 g/kg BW，依照急性毒性分類，兩者均屬於低毒性物質。口服急性毒性試驗期間，餵食複方A與複方B之BALB/c小鼠，其腹腔與脾臟之抗發炎細胞激素IL-10含量大多顯著增加，推測此兩種複方中草藥在急性毒性試驗下，可能具抗發炎之潛力。 28天亞急性毒性試驗的結果顯示，餵食複方A與複方B之組別，體重與控制組比較下並無明顯差異，且皆呈現緩慢增加之趨勢，血清生化指標方面，連續餵食此兩種複方中草藥28天，血清中之GOT、GPT、BUN與creatinine均無異常上升，顯示複方A及複方B在本實驗之使用劑量下無明顯之肝腎毒性，此外，餵食高劑量(1330 mg/kg BW)複方A還可顯著降低BALB/c雌鼠血清中GOT之含量，而餵食中劑量(330 mg/kg BW)複方B，則會使小鼠血清葡萄糖含量明顯下降。 免疫調節功能研究的結果顯示，複方A與複方B在體外實驗之模式下，皆可分別於培養濃度50、250、500 μg/mL時顯著抑制以LPS (10 μg/mL)刺激誘發之腹腔巨噬細胞發炎反應。在動物體內試驗中，中劑量(440 mg/kg BW)複方A可明顯降低小鼠脾臟細胞之促發炎細胞激素TNF-α、IL-6、IL-1β分泌，而高劑量(1330 mg/kg BW)複方A與中劑量(330 mg/kg BW)複方B可顯著增加腹腔巨噬細胞自發性分泌抗發炎細胞激素IL-10之含量。在TH1/TH2免疫傾向之影響方面，餵食高劑量(1330 mg/kg BW)複方A與低劑量(70 mg/kg BW)及中劑量(330 mg/kg BW)複方B，皆可顯著降低小鼠脾臟細胞以phytohemagglutinin刺激誘發之IL-4分泌，使免疫反應傾向TH1型式，而餵食中劑量與高劑量複方B，可顯著增加小鼠血清中之IgA含量。 綜合本論文實驗結果，中草藥複方A與複方B在口服急性毒性評估下均屬於低毒性物質，且於28天亞急性毒性評估中也顯示此兩種複方中草藥對BALB/c雌鼠並無明顯之毒性反應，因此適量食用下應屬安全，此外，複方A與複方B分別具有不同程度之抗發炎潛力與免疫調節功能。

The aim of this study was to evaluate the acute toxicity, subacute toxicity (28 days) and immunomodulatory effects of Chinese herbal novel formulas of A and B. The results from the acute toxicity study of mice administrated with single oral feeding showed that the values of LD50 (medium lethal dose) of formula A and B were 13.27 and 10.00 g/kg BW, respectively, during the 7 days of experimental period. According to the definition of acute toxicity, formulas of A and B were found to be slightly toxic (LD50: 5-15 g/kg BW). During the experimental period, the administration of formula A and B significantly (P < 0.05) increased the secretions of anti-inflammatory cytokine IL-10 by peritoneal macrophage and splenocyte cultures from BALB/c mice. The results suggest that the Chinese herbal novel formula A and B demonstrated a low toxicity and exhibited an anti-inflammatory potential. Subacute toxicity study (28 days) revealed that supplementation with different doses of formula A and B did not significantly affect the body weight of female BALB/c mice compared to the control group. Most of the levels of GOT, GPT, BUN and creatinine in the serum did not significantly increase, whereas high dose (1330 mg/kg BW) supplementation of formula A significantly (P < 0.05) decreased the serum GOT level. Medium dose (330 mg/kg BW) supplementation of formula B significantly (P < 0.05) decreased the serum glucose level. The results indicate that supplementations of formula A and B demonstrated no obvious toxic effects on the liver and kidney of female BALB/c mice. The immunomodulatory effects of Chinese herbal novel formula A and B were evaluated using both the in vitro and in vivo studies. In vitro study showed that the administrations of formula A and B at the concentrations of 50, 250 and 500 μg/mL, inhibited the inflammatory responses of lipopolysaccharide-stimulated peritoneal macrophages. After 28 days of consecutive tube feeding of formula A and B to female BALB/c mice, the medium dose (440 mg/kg BW) supplementation of formula A significantly (P < 0.05) decreased the levels of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1β, secreted by splenocyte cultures. Supplementation with the high dose (1330 mg/kg BW) of formula A and medium dose (330 mg/kg BW) of formula B significantly (P < 0.05) increased the spontaneous secretion of anti-inflammatory cytokine IL-10 by peritoneal macrophages. In addition, supplementation with high dose (1330 mg/kg BW) of formula A as well as low (70 mg/kg BW) and medium (330 mg/kg BW) doses of formula B significantly (P < 0.05) decreased IL-4 production by phytohemagglutinin- stimulated splenocytes. Supplementation with medium (330 mg/kg BW) and high (1000 mg/kg BW) doses of formula B significantly (P < 0.05) increased the serum IgA levels of female BALB/c mice. Based on the studies of acute toxicity and 28 days of subacute toxicity, Chinese herbal novel formulas of A and B were recognized as safe. Furthermore, both Chinese herbal novel formulas of A and B exhibited differential anti-inflammatory effects and an immunomodulatory potential.