侧柏总黄酮的分离纯化及对5-脂氧合酶的抑制作用

 目的从侧柏叶中分离纯化活性成分总黄酮并探讨其对5-脂氧合酶的抑制作用。方法石油醚加热回流除去大部分叶绿素等脂溶性杂质,再用体积分数为50%乙醇加热回流提取;提取液浓缩去除乙醇后用乙酸乙酯提取,减压浓缩得粗黄酮;粗黄酮过聚酰胺色谱柱,用体积分数为70%的甲醇洗脱,浓缩得总黄酮;高效液相色谱法测定大鼠中性白细胞5-脂氧合酶的产物5-羟廿碳四烯酸,Fura-2/AM荧光技术测定细胞内游离钙浓度。结果用芦丁作参照品,测得分离的产物总黄酮含量为93%,总黄酮对5-脂氧合酶抑制的IC₅₀为22.2;25～125 mg·L^-1的总黄酮对fMLP诱导的细胞内游离Ca²⁺水平增高抑制率为15.2%～35.9%。结论该方法较简单,分离的总黄酮纯度高,是一种可行的总黄酮提取方法;侧柏总黄酮对5-脂氧合酶的活性有较强的抑制作用,抑制细胞内游离Ca²⁺水平增高可能是抑制5-脂氧合酶的作用机制之一。

Study on Isolation of Total Flavonoids from Platychadus orientalis and Its Inhibition for 5-Lipoxygenase

OBJECTIVE To isolate total flavonoids from the leaf of Platychadus orientalis and investigate its inhibition for 5-lipoxygenase. METHODS After the dried leaf of Platychadus orientalis was refluxed with petroleum ether,the residue was dried absolutely,and subsequently refluxed with 50% ethyl alcohol.After the removal of the ethyl alcohol solvent in vacuo,residue was extracted with ethyl acetate,and the crude flavonoids was obtained after ethyl acetate was removed.The crude flavonoids was loaded onto a polyamide column and subsequently eluted with 70% methanol solvent.The total flavonoids was obtained after the 70% methanol solvent was removed in vacuo. The levels of 5-hydroxyeicosatetraenoic acid(5-HETE) in the cells of rat polymorphonuclear leukocytes were measured by HPLC.The intracellular free calcium was quantitated by Fura-2 fluoresence technique.RESULTS The content of total flavonoids was 93% with rutin as reference.Total Flavonoids was showed to inhibit the activity of 5-lipoxygenase with IC₅₀ value of 22.2 mg·L^-1.The concentration of 25.0～125.0 mg·L^-1 of total Flavonoids decreased the rise of intracellular calcium level induced by N-formyl-methionine-leucinephenglalanine(fMLP) by 15.8%～35.9% CONCLUSION The method provides a simple and convenient way to obtain high parity of total flavonoids.Total flavonoids inhibit the activities of 5-lipoxygenase.The total flavonoids decrease the rise of intracellular calcium level may be one of mechanisms for inhibiting the 5-lipoxygenase activities.