山藥皂苷元抗過敏及益生素之效用

Abstract

傳統中醫藥典籍及科學文獻記載山藥具有改善免疫及消化功能之效用，本研究之目的在於利用小鼠食物過敏模式，探討山藥中主要免疫活性成份之一〝山藥皂苷元〞的抗過敏及免疫調節作用。BALB/c小鼠分為無處理對照組、每天胃內灌服對照溶劑或山藥皂苷元之組別。除無處理對照組，其他組別小鼠皆以腹腔注射免疫卵白蛋白，並反覆胃內灌服卵白蛋白以誘發腸道過敏反應。口服給予山藥皂苷元顯著減緩卵白蛋白所誘發之腸道發炎，包括過敏性下痢的發生、杯狀細胞黏液的分泌及肥大細胞的浸潤和去顆粒。山藥皂苷元對於卵白蛋白所誘發腸道腺窩增生具減緩及保護之效用。給予山藥皂苷元可促進血清中卵白蛋白專一性抗體IgG2a生成，並促進卵白蛋白所刺激之脾臟細胞分泌細胞激素IFN-γ。然而，抗體IgE及細胞激素IL-4之生成則受到抑制。以免疫組織化學染色及逆轉錄聚合酶鏈式反應評估腸道免疫反應。結果顯示，給予山藥皂苷元可抑制IL-4及GATA3，但促進IFN-γ、T-bet、Foxp3及IL-10之表現。免疫組織化學雙染結果顯示，給予山藥皂苷元可有效回復十二指腸中因食物過敏所造成Foxp3+細胞數量之下降，並促進Foxp3+細胞分泌IL-10，但不影響TGF-β生成。以上結果證實，山藥皂苷元抗過敏之效用與促使Th1/Th2免疫平衡朝Th1為主之免疫反應及增強腸道發炎處調節型T細胞免疫反應相關。 基於山藥皂苷元與某些已報告之益生菌及益生素所表現之免疫調節效用相似，本研究分別以離體及活體試驗測試山藥皂苷元促腸內益生菌生長之效用。口服給予山藥皂苷元可有效回復因食物過敏所造成糞便中乳酸菌密度之下降。Lactobacillus murinus及L. reuteri為餵予山藥皂苷元小鼠糞便中所分離之主要乳酸菌。山藥皂苷元及其他結構相似之固醇類化合物對於此兩株乳酸菌之影響則以離體試驗測試。山藥皂苷元的添加可促進L. murinus及L. reuteri生長，但不影響enterococcus。結構活性關係分析顯示，C5-C6雙鍵及完整E和F環之結構可能為固醇類皂苷元之益生素活性所必需。總結可知，固醇類皂苷元為一可能之新型益生素。 本研究確認了山藥皂苷元抗過敏之效用並探討其可能之機制。實驗結果對於山藥改善消化及免疫系統之效用提供了科學證據，亦顯示山藥皂苷元為山藥中主要具免疫活性植物生化素之一。山藥皂苷元及其結構相似皂苷元之益生素活性未來具有應用及開發之潛力。

The beneficial effects of the Chinese yam to improve the function of the immune and gastrointestinal systems have been documented in the literature of traditional Chinese medicine and previous studies. The current study aimed to investigate the anti-allergic and immunomodulatory effects of diosgenin, the major immunoactive sapogenin contained in the Chinese yam, using a murine model of food allergy. BALB/c mice were either left untreated or administered daily with vehicle and/or diosgenin by gavage throughout the experiment. Except for the naive group, the mice were sensitized with ovalbumin (OVA) and repeatedly challenged with intragastric OVA to induce intestinal allergic responses. Oral administration with diosgenin markedly attenuated OVA-induced intestinal inflammation, including allergic diarrhea, mucin secretion by goblet cells, mast cell infiltration and degranulation. A protective effect by diosgenin on reducing the crypt depth of the intestine was also observed in OVA-sensitized and challenged mice. The serum production of OVA-specific IgG2a and the secretion of IFN-γ by splenocytes re-stimulated with OVA were augmented, whereas the production of IgE and IL-4 was attenuated by diosgenin administration. The immune profile in the duodenum was further characterized. The results of immunohistochemical (IHC) staining and reverse transcription-polymerase chain reaction (RT-PCR) showed that diosgenin administration suppressed the expression of IL-4 and GATA3, but enhanced the expression of IFN-γ, T-bet, Foxp3 and IL-10. The results of IHC double staining revealed that diosgenin treatment restored the diminished number of Foxp3+ cells associated with food allergy and elevated IL-10 secretion by Foxp3+ cells in the duodenum, although the production of TGF-β was unaltered. These results clearly demonstrated the anti-allergic effects of diosgenin, which is closely associated with a polarized T helper (Th)1/Th2 immunobalance toward the Th1-dominant direction and an up-regulation of the regulatory T-cell immunity in the intestinal inflammatory sites. As the profile of diosgenin-mediated immunomodulatory effects resembles that of certain prebiotics and probiotics reported in the literature, the effect of diosgenin on the growth of enteric flora was examined both in vitro and in vivo. Oral administration with diosgenin markedly restored the diminished density of fecal lactic acid bacteria (LAB) associated with allergic reactions induced by OVA. Lactobacillus murinus and L. reuteri were the major fecal LAB isolated from diosgenin-administered mice. The direct effect of diosgenin and several structure-related steroidal compounds on the growth of the two identified LAB was investigated in vitro. The presence of diosgenin significantly enhanced the growth of both L. murinus and L. reuteri, but not enterococci. Structure–activity relationship analysis showed that the prebiotic activity of steroidal sapogenins on LAB might require structural elements of the C5–C6 double bond and intact E- and F-rings. These results indicate that steroidal sapogenins may be a novel class of prebiotics. Collectively, the current study characterized the anti-allergic effect of diosgenin and elucidated its possible underlying mechanisms. The results provide scientific evidence to substantiate the beneficial effects of the Chinese yam on the gastrointestinal and immune systems, and indicate diosgenin as one of the major immunoactive phytochemicals contained in the Chinese yam. The prebiotic potential of diosgenin and related sapogenins warrants further investigation and exploitation.