本研究采用活性追踪的方法，逐步从人工培养蝉花虫草分离获得A（50%乙醇回流提取）、B（膜分离）、C（大孔树脂洗脱）和D（Sephadex LH20柱纯化）等4种样品，单一化合物D纯度为98.62%，鉴定为N6‐(2‐羟乙基)腺苷[N6‐(2‐hydroxyethyl)‐adenosine，HEA]。研究各样品对戊四唑（pentylenetetrazol，PTZ）诱导的小鼠惊厥模型的影响，以及选择性腺苷A1受体（AA1R）拮抗剂DPCPX或选择性腺苷A2A受体（AA2AR）拮抗剂ZM241385对HEA作用的影响，并采用苏木精-伊红（hematoxylin-eosin，HE）染色、免疫组化（immunohistochemical，IHC）染色和蛋白质免疫印迹（Western blot，WB）等技术进一步探究HEA抗惊厥作用的机制。结果表明，各样品（i.p.）均有抗惊厥活性，HEA（40–60mg/kg，i.p.）能显著延长惊厥小鼠的存活时间和降低死亡率，DPCPX（2mg/kg，i.p.）能够拮抗HEA的抗惊厥作用，而ZM241385无此作用；HE、IHC和WB的结果进一步揭示DPCPX显著降低HEA的作用。综上所述，蝉花虫草的HEA具有抗惊厥作用，并且可能通过激活腺苷A1受体而起作用。
Abstract
The active components of cultivated Ophiocordyceps sobolifera were separated by bioassay-guided process. By means of 50% ethanol extraction (A), membrane separation (B), macroporous resin elution (C) and Sephadex LH20 purification (D), four samples were obtained, and the sample D was identified as a single compound N6-(2-hydroxyethyl)-adenosine (HEA). Each sample obtained from each extraction step was collected for testing anticonvulsant actions of mice induced by pentylenetetrazol (PTZ). In addition, the effects of adenosine A1 receptor (AA1R) antagonist DPCPX or adenosine A2A receptor (AA2R) antagonist ZM241385 on HEA function was studied. The anti-seizure mechanism of HEA was analyzed and verified by hematoxylin-eosin (HE) staining, immunohistochemical (IHC) staining and western blot (WB). The result showed each extraction significantly increased the alive time and decreased the mortality of convulsionary mice treated with HEA (40mg/kg to 60mg/kg, i.p.). DPCPX, but not ZM241385, could diminish the anticonvulsant activity of HEA, suggesting that the function of HEA was reduced markedly by DPCPX (2mg/kg, i.p.). Our experiments have proved that the potent compound from Ophiocordyceps sobolifera inducing anticonvulsions is HEA which might act as an agonist mediating anticonvulsive effect through activating AA1R.
关键词
金蝉花 /
N6‐(2‐羟乙基)腺苷 /
腺苷受体 /
抗惊厥 /
机理
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Key words
Ophiocordyceps sobolifera /
N6-(2-hydroxyethyl)-adenosine /
adenosine receptor /
anticonvulsant /
mechanism
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脚注
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基金
浙江省重大优先项目（2008C12004-3）；浙江省自然科学基金（LY12C14004）
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