柄海鞘（Styela clava）生理活性物质的研究
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摘要
目前，国外对海洋被囊动物海鞘生理活性物质的研究正处于高速发展阶段，但是关于来自柄海鞘鞘囊或鞘体的高效抗肿瘤提取物以及具有其它生理活性的海鞘提取物的仅有零星报道。我国学者对柄海鞘的研究更为少见。我国海洋海鞘资源丰富，尤其是山东沿海海域海鞘资源更是极其丰富，与国外相比，不仅没有得到开发，而且还极大的困扰着养殖业和航海业的发展。因此，应当引起人们极大的关注。本文从以下几个方面对柄海鞘生理活性物质进行了初步的研究。
一、脂溶性物质的研究
首先，将柄海鞘的鞘囊和鞘体进行精细剥离，避免了物质的相互干扰，更好的进行物质的定位。实验中，我们单独对柄海鞘鞘囊中的化学成分进行研究。按照目前比较常用的天然产物的提取分离办法，将样品冷冻干燥后，用100％氯仿浸泡，收集氯仿滤液，减压蒸馏浓缩去除浸提溶剂，得到黑褐色的浸膏状物质。采用硅胶作干层析柱填充物，用氯仿作展开剂和洗脱液，反复硅胶层析，对柄海鞘鞘囊氯仿浸提物进行分离、提取与纯化，最后得到一浅黄绿色的油状液体fraction Ⅰ(以下均称FⅠ)。
其次，利用高效液相色谱，蛋白核酸分析仪，薄层层析，凝胶色谱等检测手段，从各个角度入手，证明该FⅠ为单一组分。
第三，利用核磁共振，红外光谱，紫外分析，质谱等现代分析测试技术对分离提取物FⅠ进行结构鉴定，解析出FⅠ的结构，初步推断该物质为邻苯二甲酸-(3-甲基)壬酯(PDNE)，根据目前所掌握的资料，还没有检索到该物质相关的报道，初步认定为一种新型的天然产物。
第四，利用癌细胞培养等技术进行抗肿瘤活性等生理活性的研究。我们研究了该天然产物对癌细胞的活性的影响，通过MTT法初步检验了其对癌细胞细胞活性的抑制作用。实验表明，PDNE在40μg／ml浓度下即表现出对BEL7402肝癌细胞细胞活性有显著的抑制作用，这种抑制作用具有浓度依赖性。但是对于B37乳腺癌细胞细胞活性的抑制作用不明显。
山东师范大学硕士学位论文 柄海鞘生理活性物质的研究
二、水溶性物质的研究
我们还进行了柄海鞘鞘囊水溶性生理活性的研究。其提取步骤和脂溶性物质
PDNE的提取分离稍有差别。对于水溶性组分，目前还没有进行物质纯度和结构的分
析，仅仅进行了抗癌生理活性的初步检验，实验表明，所提取的绿色HQLF组分能够
抑制B37乳腺癌细胞的活性。
相信，通过进一步的研究，必然会推进我国在此领域天然产物研究与开发的进
度，开创海鞘生理活性物质研究的新局面。
Nowadays, research on the physiological active substances from the marine tunicates is in a high speed progress. But it hardly can be seen on papers that some tunicate extracts which have high effective anticancer potential are obtained from Styela clava'?, tunica and body or other kinds of substances. And the research work carried on Styela Cla\>a in homeland is rarely reported. Our country especially coastal areas in Shandong province abounds tunicates, But comparing with the overseas, the resources greatly disturb the cultivation and sailing, not mention they are utilized. Therefore we should pay great attention to them. In this paper, we made a basic research on the physiological active substance from Styela clava in following aspects stated below. I . Research on liposoluble constituent
First, Styela clava's tunica and its body were separated carefully so that interactions among some substances are prevented. In this paper, we focus mainly on the chemical compound from the tunica of Styela clava. According to the frequently used methods of extraction and isolation of natural products from organic body, the freeze-dried ascidian tunica was exhaustively extracted with 100% chloroform then the combined extracts were filtered and the solvent was separately concentrated under vacuum to yield a dark-brown gum. This material was loaded onto a silica gel column and the column was eluted with chloroform several times until the fraction of interest was collected. Finally we obtained some light yellow green ointment liquid F I .
Second, we used high performance liquid chromatography (HPLC), thin-layer chromatography(TLC), gel chromatography (GC), and protein and nucleic acid analytic apparatus to evaluate F I ' s purification. All data indicated that F I was a pure substance. Third, using modern analytic techniques such as nucleic magnetic resonance(NMR), infrared spectroscopy (IR), ultraviolet visible absorption spectra (UV) and mass spectrometry(MS), we analyzed the basic chemical structure of F I and nominated it phthalic acid di (3-methyl) nanyl ester (PDNE). We estimated primarily it was a novel natural compound.
Fourth, we investigated the PDNE's anticancer activity by culturing cancer cells such as
B37 mammary cancer cell and BEL7402 liver cancer cell through MTT method. From the data of the experiment we found that PDNE was able to inhibit the activity of BEL7402 liver cancer cell significantly under the concentration 40 u g/ml and this effect was concentration-dependent. But the effect of this compound on B37 mammary cancer cell with the same concentration was very mild. We believe that research on physiological substances in this field in our country will lead to a rapid progress through further investigation about this compound. II. Research on water-soluble constituent
We also did some research on the water-soluble physiological active constituent from the tunica of Styela clava. The method we used to extract and purify the water-soluble one was somewhat different from that we used in liposoluble PDNE. As for the water-soluble constituent, analysis about purification and structure on it was still not carried out, and only some basic research on its anticancer activity was finished. Data from the experiment indicate that the green extract HQLF could inhibit the activity of B37 mammary cancer cell.
We believe that through further research, the work in this field in our country will make a rapid progress and a branded-new prospect will come out.
一、脂溶性物质的研究
首先，将柄海鞘的鞘囊和鞘体进行精细剥离，避免了物质的相互干扰，更好的进行物质的定位。实验中，我们单独对柄海鞘鞘囊中的化学成分进行研究。按照目前比较常用的天然产物的提取分离办法，将样品冷冻干燥后，用100％氯仿浸泡，收集氯仿滤液，减压蒸馏浓缩去除浸提溶剂，得到黑褐色的浸膏状物质。采用硅胶作干层析柱填充物，用氯仿作展开剂和洗脱液，反复硅胶层析，对柄海鞘鞘囊氯仿浸提物进行分离、提取与纯化，最后得到一浅黄绿色的油状液体fraction Ⅰ(以下均称FⅠ)。
其次，利用高效液相色谱，蛋白核酸分析仪，薄层层析，凝胶色谱等检测手段，从各个角度入手，证明该FⅠ为单一组分。
第三，利用核磁共振，红外光谱，紫外分析，质谱等现代分析测试技术对分离提取物FⅠ进行结构鉴定，解析出FⅠ的结构，初步推断该物质为邻苯二甲酸-(3-甲基)壬酯(PDNE)，根据目前所掌握的资料，还没有检索到该物质相关的报道，初步认定为一种新型的天然产物。
第四，利用癌细胞培养等技术进行抗肿瘤活性等生理活性的研究。我们研究了该天然产物对癌细胞的活性的影响，通过MTT法初步检验了其对癌细胞细胞活性的抑制作用。实验表明，PDNE在40μg／ml浓度下即表现出对BEL7402肝癌细胞细胞活性有显著的抑制作用，这种抑制作用具有浓度依赖性。但是对于B37乳腺癌细胞细胞活性的抑制作用不明显。
山东师范大学硕士学位论文 柄海鞘生理活性物质的研究
二、水溶性物质的研究
我们还进行了柄海鞘鞘囊水溶性生理活性的研究。其提取步骤和脂溶性物质
PDNE的提取分离稍有差别。对于水溶性组分，目前还没有进行物质纯度和结构的分
析，仅仅进行了抗癌生理活性的初步检验，实验表明，所提取的绿色HQLF组分能够
抑制B37乳腺癌细胞的活性。
相信，通过进一步的研究，必然会推进我国在此领域天然产物研究与开发的进
度，开创海鞘生理活性物质研究的新局面。
Nowadays, research on the physiological active substances from the marine tunicates is in a high speed progress. But it hardly can be seen on papers that some tunicate extracts which have high effective anticancer potential are obtained from Styela clava'?, tunica and body or other kinds of substances. And the research work carried on Styela Cla\>a in homeland is rarely reported. Our country especially coastal areas in Shandong province abounds tunicates, But comparing with the overseas, the resources greatly disturb the cultivation and sailing, not mention they are utilized. Therefore we should pay great attention to them. In this paper, we made a basic research on the physiological active substance from Styela clava in following aspects stated below. I . Research on liposoluble constituent
First, Styela clava's tunica and its body were separated carefully so that interactions among some substances are prevented. In this paper, we focus mainly on the chemical compound from the tunica of Styela clava. According to the frequently used methods of extraction and isolation of natural products from organic body, the freeze-dried ascidian tunica was exhaustively extracted with 100% chloroform then the combined extracts were filtered and the solvent was separately concentrated under vacuum to yield a dark-brown gum. This material was loaded onto a silica gel column and the column was eluted with chloroform several times until the fraction of interest was collected. Finally we obtained some light yellow green ointment liquid F I .
Second, we used high performance liquid chromatography (HPLC), thin-layer chromatography(TLC), gel chromatography (GC), and protein and nucleic acid analytic apparatus to evaluate F I ' s purification. All data indicated that F I was a pure substance. Third, using modern analytic techniques such as nucleic magnetic resonance(NMR), infrared spectroscopy (IR), ultraviolet visible absorption spectra (UV) and mass spectrometry(MS), we analyzed the basic chemical structure of F I and nominated it phthalic acid di (3-methyl) nanyl ester (PDNE). We estimated primarily it was a novel natural compound.
Fourth, we investigated the PDNE's anticancer activity by culturing cancer cells such as
B37 mammary cancer cell and BEL7402 liver cancer cell through MTT method. From the data of the experiment we found that PDNE was able to inhibit the activity of BEL7402 liver cancer cell significantly under the concentration 40 u g/ml and this effect was concentration-dependent. But the effect of this compound on B37 mammary cancer cell with the same concentration was very mild. We believe that research on physiological substances in this field in our country will lead to a rapid progress through further investigation about this compound. II. Research on water-soluble constituent
We also did some research on the water-soluble physiological active constituent from the tunica of Styela clava. The method we used to extract and purify the water-soluble one was somewhat different from that we used in liposoluble PDNE. As for the water-soluble constituent, analysis about purification and structure on it was still not carried out, and only some basic research on its anticancer activity was finished. Data from the experiment indicate that the green extract HQLF could inhibit the activity of B37 mammary cancer cell.
We believe that through further research, the work in this field in our country will make a rapid progress and a branded-new prospect will come out.
引文
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