基于“活性剪切”色谱分离技术探究三种活血化瘀中药抗肿瘤转移物质基础

Malignant tumors can metastasize to and invade neighboring tissues and organs through the blood or lymphatic system, which has seriously harmed human health. Therefore, it is an urgent need for patients to develop the new anti-tumor metastatic drugs. Salvia miltiorrhiza, Rhizoma corydalis and Curcuma zedoaria are representative traditional Chinese medicines (TCMs) with the effect of promoting blood circulation and removing blood stasis. Modern pharmacological studies have shown that these three TCMs and their components have strong anti-tumor metastatic activity from inhibiting the lysine-specific demethylase 1 (LSD1). However, natural LSD1 inhibitors are rarely studied. It is a big challenge to efficiently isolate LSD1 inhibitors from TCMs with complex composition. Most of the activity-oriented separation techniques reported in the literature only locate the active ingredient, which has obvious shortcomings. This project intends to establish an efficient separation technology: bioactivity-cut high-speed countercurrent chromatography (HSCCC) combing with HPLC. This technology organically combines active screening with on-line preparative separation. We will use this technology to separate LSD1 inhibitors from these three TCMs. Then, we will detect the anti-tumor metastatic activity of the separated LSD1 and discuss the structure-activity relationship. The aims of this project are the investigation of remarkable LSD1 inhibitors and the clarification of the material foundation and mechanism of TCMs with anti-tumor metastasis activity. At the same time, it also promotes the development of the activity-oriented separation method.

恶性肿瘤能通过血液或淋巴系统转移、侵犯邻近组织和器官，具有极强的致命性。因此研发新型抗肿瘤转移药物已成为临床用药的迫切需求。丹参、延胡索和莪术是具有活血化瘀功效的代表性中药，现代药理研究表明三种中药及其所含成分可通过抑制组蛋白赖氨酸特异性去甲基化酶1（LSD1）发挥抗肿瘤转移活性。但是天然产物类LSD1抑制剂研究极少。在组成复杂的中药中快速高效的分离LSD1抑制剂是一大难题。文献报道的活性导向分离技术大多仅实现对活性成分定位，不足之处明显。本项目拟建立一种高效的“活性剪切”高速逆流色谱（HSCCC）联合HPLC分离技术，该技术将活性筛选与在线制备性分离有机结合。在三种中药中分离不同结构类型的LSD1抑制剂，并进行基于LSD1靶标的抗肿瘤转移活性筛选和构效关系分析。本项目致力于发现活性显著的抑制LSD1的单体有效成分，阐明三种活血化瘀中药抗肿瘤转移物质基础，同时也推动活性导向分离方法的发展。

中药是中华民族的宝贵财富，阐明其药效物质及作用机制是中药现代化的关键。本项目在前期研究基础上，对三种活血化瘀中药分别建立了三种不同的“活性剪切”逆流色谱技术。针对丹参药材中含有不同极性成分，建立了综合逆流色谱分离技术；针对延胡索药材中含有生物碱类成分，建立了基于分子对接与LC-MS/MS的精准分离逆流色谱技术；针对莪术中含有倍半萜类成分，建立了活性导向逆流色谱技术。利用所建立的分离技术在三种中药中制备性的分离得到天然组蛋白赖氨酸特异性去甲基化酶1（LSD1）的抑制剂。本项目共发现5类天然LSD1抑制剂22个，包括丹酚酸类2个，丹参酮类4个，生物碱类3个，倍半萜类4个以及睡茄内酯类9个。其中具有显著活性的有5个，分别是丹酚酸B、丹参酮IIA、去氢延胡索素、异吉马呋内酯和醉茄内酯A。丹酚酸B、去氢延胡索素、异吉马呋内酯以及醉茄内酯A均能显著性的在酶水平和细胞水平抑制LSD1的活性，并能抑制MDA-MB-231细胞或U2OS细胞的转移和侵袭。丹酚酸B还能抑制荷瘤小鼠的肿瘤生长。此外，活性成分还能通过诱导LSD1/TGF-β1/EMT信号通路，抑制肿瘤细胞的转移。本项目共计发表标注的SCI论文5篇，其中包括色谱分离类国际著名期刊Talanta、J. Chromatogr. A，以及化学类生物学类国际著名期刊Bioorg. Chem.、Chin. J. Nat. Med.、Theranostics等。本项目作为重要内容获得2019年度江苏省科学技术奖一等奖一项。本项目协助培养博士研究生一名，硕士研究生两名。以上研究成果为阐明这三种活血化瘀中药的物质基础及开发其临床抗肿瘤转移药效打下基础。同时也进一步推动了活性导向分离技术的发展以及为基于LSD1靶标的新药研发奠定了基础。本项目完成了既定目标，取得了预期的研究成果，为进一步深入研究打下了坚实的基础。