海洋褐藻叉開(kāi)網(wǎng)翼藻中的單萜成分研究

徐秀麗, 印麗媛, 宋福行, 范 曉, 石建功

(1. 中國(guó)科學(xué)院 海洋研究所, 山東 青島 266071; 2. 中國(guó)醫(yī)學(xué)科學(xué)院暨中國(guó)協(xié)和醫(yī)科大學(xué)藥物研究所, 北京100050; 3. 中國(guó)地質(zhì)大學(xué)(北京)海洋學(xué)院, 北京 100083)

海洋褐藻叉開(kāi)網(wǎng)翼藻中的單萜成分研究

徐秀麗1,3, 印麗媛3, 宋福行1, 范 曉1, 石建功2

(1. 中國(guó)科學(xué)院 海洋研究所, 山東 青島 266071; 2. 中國(guó)醫(yī)學(xué)科學(xué)院暨中國(guó)協(xié)和醫(yī)科大學(xué)藥物研究所, 北京100050; 3. 中國(guó)地質(zhì)大學(xué)(北京)海洋學(xué)院, 北京 100083)

為了研究中國(guó)沿海海藻中的活性化學(xué)成分, 利用正相硅膠柱色譜、Sephadex LH-20柱色譜、反相HPLC及重結(jié)晶等手段, 從采自青島的褐藻叉開(kāi)網(wǎng)翼藻(Dictyopteris divaricataOkam.)中首次分離得到了6個(gè)單萜類(lèi)化合物, 經(jīng)紅外波譜(IR), 質(zhì)譜(MS)及核磁共振波譜(1H NMR、13C NMR)等現(xiàn)代波譜技術(shù)鑒定為loliolide (1), isololiolide (2), 3β-hydroxy-5α,6α-epoxy-7-megastigmen-9-one (3), dehydrovomifoliol (4), (3R)-4-[(2R,4S)-2-hydroxy-2,6,6-trimethylcyclohexylidene]-3-buten-2-oneiol (5), (3R)-4-[(2R,4S)-4-acetoxy-2-hydroxy-2,6,6-trimethylcyclohexylidene]-3-buten-2-one (6)。并通過(guò)MTT法對(duì)所得化合物進(jìn)行細(xì)胞毒活性篩選, 通過(guò)比色法進(jìn)行乙酰膽堿酯酶抑制活性篩選。

叉開(kāi)網(wǎng)翼藻(Dictyopteris divaricataOkam.); 化學(xué)成分; 單萜; 結(jié)構(gòu)鑒定; 活性篩選

叉開(kāi)網(wǎng)翼藻(Dictyopteris divaricataOkam.)為網(wǎng)地藻科網(wǎng)翼藻屬海藻, 廣泛分布于我國(guó)黃海和東海海域。文獻(xiàn)報(bào)道主要從中分離得到了吉馬烷骨架倍半萜[1-2]、蓽澄茄倍半萜[3]、杜松烷類(lèi)倍半萜[4]、降碳倍半萜[5]和新骨架的倍半萜[6]。初步藥理活性篩選表明, 叉開(kāi)網(wǎng)翼藻乙醇提取物的乙酸乙酯可溶性部分對(duì)腫瘤細(xì)胞 B16-BL6 (黑色素瘤) 和A2780 (人卵巢癌) 有較強(qiáng)的抑制作用, IC50值分別為7.61 mg/L和8.06 mg/L。為了開(kāi)發(fā)利用我國(guó)豐富的海藻資源, 對(duì)采自青島的叉開(kāi)網(wǎng)翼藻乙醇提取物的乙酸乙酯可溶性部分, 進(jìn)行了化學(xué)成分研究。從中分離得到了6個(gè)單萜類(lèi)成分, 均為首次從該種海藻中發(fā)現(xiàn), 并對(duì)以上化合物進(jìn)行了抗腫瘤和乙酰膽堿酯酶活性篩選,實(shí)驗(yàn)結(jié)果報(bào)道如下。

1 材料和方法

1.1 儀器與試劑

XT-4 顯微熔點(diǎn)測(cè)定儀(溫度計(jì)未校正); Perkin-Elmer 241型旋光儀; Nicolet impact 400型傅里葉變換紅外波譜儀; Inova 500型核磁共振儀; Autospec-Ultima ETOF型質(zhì)譜儀; Waters 600型高效液相色譜儀(Alltech公司Alltima C18 制備柱22 mm × 250 mm, Waters 2487型紫外檢測(cè)器); ZF-C型三用紫外分析儀; 凝膠 Sephadex LH-20為 Amersham Pharmacia Biotech AB公司產(chǎn)品; 硅膠為青島海洋化工有限公司生產(chǎn)(160～200目); 試劑為分析純和色譜純。褐藻叉開(kāi)網(wǎng)翼藻Dictyopteris divaricataOkam.于2003年4月采集于山東青島的匯泉角, 由中國(guó)科學(xué)院海洋研究所邵魁雙博士鑒定, 標(biāo)本保存在中國(guó)科學(xué)院海洋研究所。

1.2 提取與分離

新鮮的海藻樣品采集后風(fēng)干粉碎, 用 95%的乙醇提取3次, 每次72 h, 提取液合并后減壓濃縮(溫度低于 40°C), 濃縮物懸浮于蒸餾水中, 依次用乙酸乙酯和正丁醇萃取。乙酸乙酯萃取液經(jīng)減壓濃縮后得到棕黑色浸膏 120.3 g, 將其進(jìn)行正相硅膠(160～200目)柱色譜分離, 以石油醚(60～90°C)-丙酮梯度洗脫, 通過(guò)薄層色譜檢測(cè), 合并成分相似部分,最終得到24個(gè)組分。石油醚-丙酮(10 : 1), (5 : 1)和(3 : 1)洗脫部分。分別經(jīng)Sephadex LH-20柱色譜(石油醚-氯仿-甲醇, 5 : 5 : 1)和反相HPLC柱色譜(流動(dòng)相為75%水性甲醇)純化, 得到化合物1～6(圖1)。

圖1 化合物1～6的結(jié)構(gòu)式Fig. 1 Structures of compounds 1～6

2 化合物結(jié)構(gòu)鑒定

-36°(c0.15, MeOH)。EIMS給出其分子離子峰m/z266 [M]+以及失去甲基的碎片離子峰209。IR (KBr)波譜顯示分子中存在羥基(3 454 cm-1), 累積雙鍵(1 938 cm-1), 酯基(1 738 cm-1)和 α,β-不飽和羰基(1 672 cm-1)。1H NMR (Me2CO-d6, 500 MHz)δH5.73 (1H, s, H-8), 5.38 (1H, dddd,J=12.5,12.5,4.5,4.5 Hz, H-3), 2.23 (1H, ddd,J=12.5,4.5,2.5 Hz, H-4a), 2.15 (3H, s, H-10), 1.98 (3H, s, H-15), 1.97 (1H, ddd,J=12.5,4.5,2.5 Hz, H-2a), 1.54 (1H, dd,J=12.5,12.5 Hz, H-4b), 1.45 (1H, dd,J=12.5,12.5 Hz, H-2b), 1.42 (3H, s, H-11), 1.41 (3H, s, H-13), 1.15 (3H, s, H-12); 表明結(jié)構(gòu)中存在乙?；碗p鍵。13C NMR (Me2CO-d6, 125 MHz)δC210.0 (s, C-7), 197.4 (s, C-9), 170.2 (s, C-14), 119.0 (s, C-6), 100.8 (d, C-8), 71.8 (s, C-5), 68.0 (d, C-3), 46.0 (t, C-4), 45.7 (t, C-2), 36.4 (s, C-1), 31.8 (q, C-12), 30.6 (q, C-13), 29.2 (q, C-11), 26.4 (q, C-10), 21.1 (q, C-15);表明結(jié)構(gòu)中存在連氧次甲基碳、連氧季碳和累積雙鍵以及2個(gè)乙酰基。以上波譜數(shù)據(jù)與文獻(xiàn)[14]報(bào)道的化 合 物 (3R)-4-[(2R,4S)-4-acetoxy-2-hydroxy-2,6,6-trimethylcyclohexylidene]-3-buten-2-one的數(shù)據(jù)基本一致。

3 化合物的活性分析

用MTT法[15], 對(duì)上述6個(gè)化合物在人腫瘤細(xì)胞HCT-8, Bel-7402, A549, BGC-823和A2780模型上進(jìn)行了細(xì)胞毒活性測(cè)試,結(jié)果表明所有化合物對(duì)以上細(xì)胞株均無(wú)明顯毒性( IC50＞ 10 mg/L ) 。

利用比色法測(cè)定了上述 6個(gè)化合物的抗乙酰膽堿酯酶活性, 結(jié)果表明在濃度為 25nmol/L 時(shí), 化合物6表現(xiàn)了一定的酶抑制活性, 抑制率為12.43%。

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(本文編輯:張培新)

Monoterpene constituents of the brown algaDictyopteris divaricataOkam.

XU Xiu-li1,3, YIN Li-yuan3, SONG Fu-hang1, FAN Xiao1, SHI Jian-gong2
(1. Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; 2. Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China; 3.School of Marine Sciences, China University of Geosciences, Beijing 100083, China)

Dec., 12, 2010

Dictyopteris divaricata Okam.; chemical constituents; monoterpene; structural elucidation; bioactive screening

Compounds 1-6 were isolated by recrystallization, normal phase silica gel, Sephadex LH-20 chromatography, and reverse phase HPLC techniques from the brown algaDictyopteris divaricataOkam, collected from Qingdao. Through spectroscopic methods including IR, MS and NMR, their structures were elucidated as loliolide (1), isololiolide (2), 3β-hydroxy-5α,6α-epoxy-7-megastigmen- 9-one (3), dehydrovomifoliol (4), (3R)-4-[(2R,4S)-2-hydroxy-2,6,6-trimethylcyclohexylidene]-3-buten-2-oneiol (5), (3R)-4-[(2R,4S)-4-acetoxy-2-hydroxy-2,6,6-trimethylcyclohexylidene]-3-buten-2-one (6). All compounds were obtained from the Dictyotaceae algae for the first time. Compounds 1-6 were tested for antitumor activity by the MTT method and AchE inhibitory activity.

Q952, Q253

A

1000-3096(2012)10-0081-04

2010-12-12;

2011-02-20

國(guó)家自然科學(xué)重點(diǎn)基金資助項(xiàng)目(20432030); 國(guó)家高技術(shù)發(fā)展計(jì)劃(863計(jì)劃)專(zhuān)項(xiàng)(2001AA620403)

徐秀麗(1976-), 女, 河北省盧龍縣人, 副教授, 主要從事海洋天然產(chǎn)物及活性物質(zhì)的研究。E-mail: xiulixu@163.com