當歸補血湯對鐵吸收之影響

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题名:	當歸補血湯對鐵吸收之影響
作者:	陳詩瑜
贡献者:	生藥學研究所
日期:	2010
上传时间:	2010-10-20 12:49:34 (UTC+8)
摘要:	缺鐵性貧血為貧血分類中盛行率最高的疾病，造成缺鐵性貧血的原因有鐵吸收不良、攝鐵不足、失血過多等，在醫療發達的現今缺鐵性貧血仍是急待解決的問題；而當歸補血湯組成為當歸及黃耆，是傳統醫學之補氣生血方劑。因此本研究利用當歸補血湯與鐵併用治療缺鐵性貧血，以探討古籍上所稱「補血」，是否與治療缺鐵性貧血有關，並觀察當歸補血湯與鐵劑，中西藥合併使用之療效。首先利用Caco-2細胞攝鐵模式，檢測當歸補血湯使鐵蛋白(ferritin)生合成之影響，結果顯示：當歸及黃耆單獨使用不會增強鐵蛋白形成，而當歸補血湯則具劑量依存性的促使鐵蛋白形成，且其指標成分阿魏酸(ferulic acid)亦具有促進鐵吸收之作用。繼而探討當歸補血湯與鐵合併使用及間隔使用探討當歸補血湯對鐵吸收之影響，結果顯示：當歸補血湯與鐵併用時，當歸補血湯對於鐵吸收有抑制作用，與鐵間隔使用時則顯著具促進鐵吸收之作用；最後再以大鼠血紅素再生法探討體內之療效，結果顯示當歸補血湯與鐵經口服併用也會抑制鐵之吸收，而當歸補血湯本身即具促使血紅素上升之作用，且由血紅素再生率(HRE)可得知當歸補血湯中的鐵含量為37.6 mg/g，其生體可用率高達1947%。利用植物化學成分分析發現，當歸補血湯中含酚類化合物(6.51 mg/g)、縮合型丹寧化合(2.31 mg/g)及皂苷類化合物(34.48 mg/g)；而由總二價鐵分析結果中，鐵與當歸補血湯反應後之二價鐵含量減少，推測當歸補血湯中成分如酚類、縮合型丹寧與鐵形成不溶性沉澱物而阻礙鐵吸收。另外，以西方點墨法分析蛋白質表現，亦發現當歸補血湯可以活化DMT-1蛋白質表現量，推測其為促使鐵吸收增強的機轉。綜合結果：當歸補血湯單獨使用時，利用在體內、外試驗，皆顯示其具促進鐵儲存利用之作用，顯示當歸補血湯可應用於缺鐵性貧血患者。但當歸補血湯與鐵劑同時服用時，會抑制鐵之吸收，若間隔使用，則可增強鐵之吸收。因此建議當歸補血湯要與鐵劑合併治療缺鐵性貧血時，要先服用當歸補血湯活化DMT-1，再服用鐵劑會使鐵劑吸收之效果增強。 Iron deficiency anemia, the most common type of anemia, is caused by reasons such as iron malabsoption, iron-deficient diet, and blood loss. The traditional Chinese medicine, Danggui Buxue Tang (DBT), containing Radix angelicae sinensis (Danggui) and Radix astragali (Huangqi) has been used to invigorate “Qi” and nourish “Blood”. In this study, DBT was combined with iron to treat iron deficiency anemia. Another objective was to delineate whether the “nourish the blood”, as described in the ancient medicine books, were related to the iron deficient anemia treatment or not. In the first place, Caco-2 cell iron uptake model was used to detect the effect of ferritin biosynthesis. The results showed that Danggui and Huangqi could not enhance ferritin biosynthesis, but DBT could significantly promote ferritin biosynthesis. The principle constituent of DBT, ferulic acid, also could enhance iron absorption. The treatment program of DBT and iron in Caco-2 cells were exchanged to know the interaction of DBT and iron. The results revealed while Caco-2 cells were co-treated with DBT and iron, DBT could inhibit the absorption of iron. When DBT or iron was pre-treated, DBT could significantly enhance the absorption of iron. Therefore, the rat hemoglobin repletion assay was used to explore the above therapeutic activity in vivo. The result showed that the absorption of iron was reduced after co-treatment with DBT and iron in Wistar rats. However, the hemoglobin level was increased in the DBT group. The bioavailability of the iron was increased up to 1947% in DBT group in the hemoglobin repletion efficiency (HRE) test. The results of phytochemical analysis revealed that there are phenol compounds (6.51mg/g), condensed tannins (2.31mg/g), and saponins compounds (34.48mg/g) in DBT. In addition, the total ferrous content was decreased in the mixture of DBT and iron, indicating of that certain components of DBT like phenols, condensed tannins could chelate the iron. Besides, DBT could activate the DMT-1 expression in Caco-2 cells by Western blotting assay. Therefore, we assumed the iron promoting absorption of DBT was mediated through the activation the DMT-1 in Caco-2 cells. In conclusion, the DBT enhanced the bioavailability of the iron both in vitro and in vivo. The absorption of iron was inhibited by DBT and iron co-treatment, but enhanced by DBT and iron pre-treatment. Based on the above results, we suggested DBT and iron should be administrated separately for iron deficient anemia patients.
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描述:	目錄……………………………………………………………… I 圖目錄…………………………………………………………….. V 表目錄…………………………………………………………….. VII 縮寫表…………………………………………………………... 1 中文摘要………………………………………………………….. 2 英文摘要………………………………………………………... 4 緒論……………………………………………………………….. 6 一、當歸補血湯之文獻回顧……………………………………… 7 (一) 當歸補血湯之出典………………………………………... 7 (二) 當歸補血湯之耆歸比例………………………………….. 8 1. 成分變化…………………………………………….. 10 2. 藥理活性…………………………………………….. 11 (三) 當歸補血湯「補氣生血」與現代藥理活性之關連…….. 11 1. 與「補氣生血」相關之活性……………………….. 12 2. 與「補氣」相關之活性…………………………….. 12 3. 與「生血」相關之活性…………………………….. 13 (四) 當歸補血湯之品質管制………………………………….. 13 1. 黃耆………………………………………………….. 13 2. 當歸………………………………………………….. 15 (五) 當歸補血湯與現代醫學之貧血………………………… 16 二、缺鐵性貧血之簡介…………………………………………… 19 (一) 貧血之定義………………………………………………... 19 (二) 貧血之致病原因…………………………………………... 19 (三) 缺鐵性貧血………………………………………………... 20 1. 缺鐵性貧血之致病原因……………………………... 21 2. 缺鐵性貧血之治療………………………………….. 21 3. 使用鐵劑之注意事項……………………………….. 22 三、鐵的平衡……………………………………………………… 22 (一) 鐵在人體的功用…………………………………………... 22 (二) 人體之鐵流失…………………………………………….. 24 (三) 人體之鐵機收機制……………………………………….. 24 (四) 影響鐵吸收之物質……………………………………….. 26 1. 抑制鐵之吸收之物質………………………………... 26 2. 促進鐵之吸收之物質……………………………….. 27 3. 中藥與鐵劑之相關交互作用……………………….. 27 四、缺鐵性貧血之實驗模式簡介………………………………… 28 (一) 體外試驗：Caco-2細胞攝鐵法………………………….. 28 (二) 體內試驗：大鼠血紅素再生法………………………….. 29 研究動機與目的………………………………………………... 31 實驗材料與方法………………………………………………... 32 一、實驗材料……………………………………………………… 32 (一) 當歸相關方劑之成分分析………………………………… 32 (二) 體外試驗：Caco-2細胞攝鐵法…………………………… 33 (三) 體內試驗：大鼠血紅素再生法…………………………… 35 二、實驗儀器……………………………………………………… 37 三、實驗方法……………………………………………………… 38 (一) 當歸相關方劑之成分分析………………………………… 38 (二) 體外試驗：Caco-2細胞攝鐵法…………………………… 45 (三) 體內試驗：大鼠血紅素再生法…………………………… 57 四、統計方法……………………………………………………… 59 結果……………………………………………………………... 61 (一) 當歸相關方劑之成分分析………………………………… 61 (二) 體外試驗：Caco-2細胞攝鐵法…………………………… 73 (三) 體內試驗：大鼠血紅素再生法…………………………… 82 討論……………………………………………………………... 86 一、阿魏酸促進鐵吸收之作用…………………………………… 86 二、當歸補血湯與當歸、黃耆分開煎煮之成分與藥理活性變化…………………………………………………………... 87 三、體外試驗中當歸補血湯與鐵併用對鐵吸收之影響………… 88 四、體外試驗中當歸補血湯與鐵間隔使用對鐵吸收之影響…… 90 五、體內實驗中當歸補血湯與鐵併用對鐵吸收之影響………… 91 結論……………………………………………………………... 93 參考資料………………………………………………………... 94 圖目錄圖一、黃耆之飲片……………………………………..………… 15 圖二、當歸之飲片…………………….…………………………. 15 圖三、當歸補血湯及其相關補益湯劑組成間之關係…..……… 17 圖四、鐵在人體之分佈…………..……………………………… 23 圖五、鐵在人體的循環利用………………………………….…. 25 圖六、阿魏酸(ferulic acid)之結構式……………………….. 40 圖七、Vanillin Assay測定縮合型單寧(Condense Tannin )含量之原理……………..………………. ……………………... 43 圖八、MTT被活細胞粒線體內的去氫酶間接還原成Formazan 之原理…………..………………. ……………………... 48 圖九、細胞攝鐵實驗流程………………………………….……. 50 圖十、大鼠血紅素再生法評估鐵可用率之實驗流程圖………… 60 圖十一、阿魏酸之HPLC 圖譜及標準檢量線……………..…… 64 圖十二、當歸補血湯及當歸、黃耆水萃物之HPLC指紋圖譜… 66 圖十三、Gallic acid之標準檢量線圖………………………… 66 圖十四、Catechin之標準檢量線圖…………………………….. 68 圖十五、Diosgenin之標準檢量線圖……………………………. 69 圖十六、FeSO4之標準檢量線圖……………………………….. 70 圖十七、當歸補血湯對FeSO4之總還原鐵(Fe2+)含量影響…… 72 圖十八、不同濃度之FeSO4對Caco-2之鐵蛋白形成影響……… 74 圖十九、阿魏酸與鐵併用對Caco-2之鐵蛋白形成影響……... 75 圖二十、當歸相關方劑單獨使用對鐵蛋白含量之影響……..… 76 圖二十一、當歸補血湯與其組成當歸、黃耆對鐵蛋白含量之影響 77 圖二十二、當歸補血湯與鐵併用對鐵蛋白含量之影響………… 78 圖二十三、當歸補血湯與鐵間隔使用對鐵蛋白含量之影響…… 80 圖二十四、當歸補血湯與鐵對DMT-1蛋白表現量之影響……… 81 圖二十五、飼以缺鐵飼料之大鼠血紅素下降趨勢……………… 82 表目錄表一、當歸補血湯及其相關補益湯劑組成、出典、效能與適應症…………………………………………………………… 18 表二、大鼠血紅素再生法用「基礎飼料」AIN-93G配方的組成 36 表三、血紅素再生法可用的鐵利用率指標……………………. 59 表四、當歸補血湯及其組成當歸、黃耆之製備產率…………… 61 表五、阿魏酸分析方法之同日內差異、異日內差異分析(A)及添加回收率結果(B)…………………………………………… 62 表六、當歸相關方劑之成分分析.........…………………… 71 表七、當歸相關方劑對Caco-2細胞之毒性分析………………… 73 表八、當歸補血湯對缺鐵貧血大鼠之療效.....……………… 85 1. 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