Purification and Biological Activity of Ecdysterone from Korean Achyranthes radix

韓國産 牛膝의 Ecdysterone 抽出과 그 生理活性에 관한 硏究

It has been known that the insect molting hormone and its analogues exist also in plant kingdom and their concentration has been found to be about 0.1~2.0% of dry matter, which is equivalent to $10^3{\sim}10^5$ times of those in insects. This study was carried out; 1) to isolate the phytoecdysones from Korean Achyranthes radix and characterize their physico-chemical properties. 2) to investigate the biological activity of this phytoecdysone on Bombyx mori larvae. The resuls were summarized as follows; 1. The extraction method of phytoecdysones was optimized by three consecutive reflux for 1hr using 200g of dried and milled radix per 1l methanol. 2. The purification from the crude extract was made by a series of steps such as precipitation of gum-type polymer with n-Butyl acetate, adsorption on technical grade silica and chromatography with neutral alumina. The conditions of each step were optimized and the resulting crude crystal was about 500mg per kg dry radix. 3. The crude crystal from the cultivated Achyranthes(Achyranthes japonia) contained ecdysterone (20-hydroxyecdysone) and inokosterone in the proportion of one to one. In order to separate these, a series of processes such as acetylation, separation by alumina column chromatography deacetylation by alcoholysis, deionization and crystallization were introduced and optimized 125mg of ecdysterone and 18mg of inokosterone per kg dry radix were thus obtained. 4. The wild Achyranthes (Achyranthes obtusifolia) radix was found to contain the ecdysterone only. A 285mg of ecdysterone was crystallized per kg dry radix. 5. Isolated ecdysterone, inodosterone and acetylated compounds were characterized by IR., UV., NMR spectroscopy, mp, TLC and densitometry. 6. Ligation experiment was undertaken to confirm the biological activity of the purified ecdysterone; the ecdysterone could induce larval-pupal metamorphosis in the ligated abdomen of 4th instar larvae injecting 0.5~1.0${\mu}g$. 7. By ecdysterone feeding experiment using artificial diet, it was elucidated that the critical time of feeding would be the first half of each instar resulting in increased weight of silk layer. 8. The ecdysterone was fed to 5th instar silkworm at the level of 1, 2, 3, 5ppm of dry feed of artificial diet containing 5% mulberry leaves for 72hrs. At 2ppm of the concentration. body weight and silk layer weight were arrived at maximum. But at higher concentrations body weight and silk layer weight decreased than the control group. At 2ppm of the concentration, body weight was increased by 12.5%. 9. Feeding 2ppm of ecdysterone at the later half of 5th instar, the duration of larvae was shortened.

植物界에 昆蟲의 脫皮 호르몬이 存在하고 있는 것은 잘 알려져 있다. 本實驗에서는 韓國에서 栽培또는 自生하고 牛膝(Achyranthes 屬)에서 昆蟲 脫皮 호르몬을 抽出 精製하고 이의 最適抽出條件과 現化學的性狀을 檢討했으며 이 物質의 家蠶에 對한 生理活性을 檢定하였는 바 이 結果를 要約하면 다음과 같다. 1. 乾燥 粉碎한 牛膝뿌리 200g에 對하여 1l의 메탄올로 1時間씩 連續 3回 還流冷却 抽出하는 方法에서 ecdysterone 抽出의 收率이 가장 높았으며 試科의 粉碎 粒度가 重要함을 알았다. 2. 粗結晶의 離分 精製過程은 n-Butyl acetate에 의한 gum 狀의 高分子物質을 沈澱시킨 후 工業用硅素에 吸着을 거쳐 알루미나 chromatography에 의해서 이루어 졌으며 最適狀態에서의 粗結晶의 收率은 牛膝뿌리 kg當 約 500mg程度였다. 3. 원래 野生種이나 現在 栽培되기도 하는 Achyranthes japonica에서는 ecdysterone (20-hydroxyedysone)과 inokosterone이 抽出되었으며 이들의 含量比는 1:1임을 알았다. 이들을 純粹 分離하기 위하여 아세틸化를 한 後, 알루미나 chromatography와 alcohol分解에 의한 脫아세틸化, 이온 交換法을 거쳐 最終結晶을 얻었다. Achyanthes japonica에서는 뿌리 kg當 125mg의 ecdysterone과 小量의 inokosterone이 結晶化 되었고 inokosterone은 分離 精製過程에서 많이 消失됨을 알았다. 4. 野生種으로 알려진 Achyranthes obtusifolia에서는 ecdysterone만이 分離 結晶되었고 收率은 뿌리 kg當 284mg 程度였다. 5. 分離된 ecdysterone과 inokosterone의 理化學的性狀을 얻기 위하여 赤外線 分光分析, 紫外線分光分析, 核子氣共嗚法, 融點測定, 薄層 chromatography를 하여 標準品 또는 文獻値와 一致함을 알았다. 6. 이 物質의 生理活性을 대認하기 위하여 4齡의 家蠶幼蟲을 結紮한 後 ecdysterone을 注射에 의하여 幼蟲脫皮가 誘導되었다. 7. 人工飼科를 利用하여 ecdysterone 給與에 따른 絹物質 增加效果의 臨界期를 알기 위한 試驗結果 ecdysterone 給與는 各齡의 前半部가 效果的임을 알았다. 8. 5齡期 家蠶幼蟲에 5% 뽕잎 분말이 含有된 人工飼科를 利用하여 1, 2, 3, 5ppm의 ecdysterone을 脫皮後72時間동안 給與한 바 2ppm 添加區에서 體重 및 繭層重이 가장 무거웠고 그 以上의 添加濃度에서는 오히려 體重과 繭層重 增加의 沮害效果를 나타내었다. 2ppm 添加區에서 體重은 12%, 繭層重은 12.5% 增加를 보였고 全齡期間中 各齡의 前半部에 ecdysterone 2ppm添加의 人工飼科로 누에를 飼育하여 全繭重 6.6% 繭層重 12.9%의 增加를 보였다. 9. 5齡 後半의 ecdysterone 급여는 5齡 幼蟲期間을 20時間 단축시켰고 結果的으로 繭重 繭層重을 減少시켰다.