民族药石上柏作用于喉癌COX-2和EGFR 多靶标的活性成分及作用机制研究

Laryngeal carcinoma is the very common and refractory malignant tumor of head and neck region. “Shi Shang Bai” originates from the dried whole plant of Selaginella doederleinii Hieron, which is widely used for the treatment of laryngeal carcinoma by ethnic people in southern provinces of China. According to our survey, the main source of “Shi Shang Bai” is S. moellendorffii and minor sources are S. doederleinii and S. delicatula in herbal markets. Therefore, it is of great significance to clarify and determine the original material for “Shi Shang Bai” and guarantee the effectiveness of clinical application. In the study, we will systematically evaluate in vitro and in vivo antitumor activities of “Shi Shang Bai” medicinal plants against laryngeal carcinoma with MTT, ELISA and tumor xenograft animal model, perform large scale separation of active extracts of three “Shi Shang Bai” medicinal plants to establish biflavonoid library, screen antitumor activity of the isolated pure compounds based on multi-targets of COX-2 and EGFR, investigate on the structure-activity relationship of in vitro antitumor activities of biflavonoids and their interactions with targeted proteins with computer aided drug design, evaluate in vivo antitumor effects of biflavonoids in tumor xenograft animal model as wells as elucidate antitumor mechanisms of biflavonoids on molecular, cellular and animal levels. Important theoretical basis for the prevention and treatment of laryngeal carcinoma and developing “Shi Shang Bai” medicinal plants will be provided by this study.

喉癌是头颈部常见难治性恶性肿瘤，而石上柏为南方少数民族广泛使用的治疗头颈部癌症药物，传统基源为深绿卷柏。但目前市场上商品主流品种为江南卷柏，少部分为深绿卷柏和薄叶卷柏。因而，亟待澄清及明确石上柏的药材基源，阐明其药效物质基础和作用机制，保障临床应用的有效性。本研究采用MTT方法、ELISA方法及喉癌裸鼠移植瘤模型系统评价石上柏基源植物的体内外抗喉癌活性；对其活性提取物进行放大分离纯化，建立卷柏双黄酮化合物分子库；采用基于COX-2和EGFR多靶点的抗喉癌筛选方法筛选活性纯化合物，以计算机辅助药物设计研究卷柏双黄酮的构效关系及与靶蛋白的相互作用，采用喉癌裸鼠移植瘤模型评价卷柏双黄酮的体内抗喉癌效果，从分子-细胞-动物水平上阐明卷柏双黄酮的抗喉癌作用机制。本研究为喉癌防治和石上柏的应用与开发提供依据。

喉癌是头颈部常见难治性恶性肿瘤。“石上柏”为南方少数民族人民（壮族和瑶族）广泛使用的抗癌及抗喉癌药物，传统基源为深绿卷柏。但目前市场上商品主流品种为江南卷柏，少部分为深绿卷柏和薄叶卷柏。因而，亟待澄清及明确“石上柏”的药材基源，阐明其药效物质基础和作用机制，保障临床应用的有效性。. 本研究利用各种活性筛选技术筛选卷柏属药用植物，综合运用各种柱层析技术，特别是基于HPLC的活性导向和质谱导向分离技术分离纯化100余个单体化合物，其中有18个活性良好的双黄酮化合物。我们以计算机辅助药物设计研究部分双黄酮化合物与COX-2和EGFR蛋白的相互作用关系。我们采用喉癌裸鼠移植瘤动物模型系统评价了深绿卷柏和江南卷柏的体内抗喉癌活性，运用分子生物学技术从分子-细胞-动物水平上初步阐明了2种卷柏属药用植物的抗喉癌作用机制。深绿卷柏抗喉癌的体外活性明显强于江南卷柏，导致两者活性差异如此悬殊的关键在于穗花杉双黄酮。穗花杉双黄酮抗喉癌活性较弱，其在江南卷柏中的含量是其在深绿卷柏中含量的10倍。由于其高含量、低活性和溶解性差明显降低了江南卷柏的抗喉癌活性。本项目明确了石上柏的正确基原为深绿卷柏。基于本研究，我们开发了一个基于穗花杉双黄酮为原料的抗喉癌活性衍生物的制备方法，得到的活性化合物抗喉癌活性比穗花杉双黄酮提升10倍以上。本项目的研究结果将为喉癌的防治和卷柏属药用植物开发提供依据。依托于本基金项目，发表相关科研学术论文7篇，其中SCI论文7篇，申报发明专利1项，授权1项。