Screening And Study Of Active Compounds From Five Traditional Minority Medical Plants Targeted On DNA G-quadruplex

Due to the lack of anti-tumor drugs, malignant tumors seriously threaten the life, health and the socio-economic development of human beings, especially for people in minority areas, and have become a major obstacle to the construction of a well-off society in China. Chinese minority traditional medicine is the crystallization of the traditional culture and knowledge of Chinese minorities over a period of thousands of years. It has made a significant contribution to the survival and flourishing of their own nations in history, and still plays an important role in the health services of native and other communities today. The hope for discovering novel anti-tumor drugs has placed on minority medicines, which have experienced a long period of research and clinical practice, and are pharmacologically validated for the treatment of cancer.The present project is based on the cognition and respect for the knowledge of Chinese minority traditional medicine. We aim to discover novel anti-tumor lead compounds with high specificity, low toxicity and independent intellectual property right from ethno-medicine, and select ethno-medicine which has rich resources and the potential anti-tumor ability via targeted DNA G-quadruplex and interdisciplinary research methods. This project includes the following researches:1. We studied the anti-tumor activity of effective monomer compounds from Chinese minority traditional medicine by testing its capacity to bind targeted G-quadruplex. It has been reported in minority medicine literatures that Tibetan medicine Rheum palmatum L., Uygur medicine Rubia cordifolia L. and Arnebia euchroma (Royle) Johnst, and Pattra medicine Manglietia fordiana Oliv. were used for detoxification by local minority. Some effective monomer compounds in these minority medicines, including Aloe-emodine, alizarin, shikonin, magnolol and honokiol, may have anti-tumor activity at cell level, but it is not clear whether these medicines work via affecting the G-quadruplex. Theoretically small molecule compounds are easier, relative to large molecule compunds, to bind targeted G-quadruplex since they have planar structure in the space. Therefore, the objective of this study was to discover novel anti-tumor lead compounds by testing the activity of selected components, which have planar structure, of minority medicine for binding targeted DNA G-quadruplex. Specific research results are summarized as follows:(1) C-myc 2345 sequence can form parallel-stranded G-quadruplex structures in K+-containing solution, thus, could be used as the molecular target in the study of the interaction between traditional minority medical plant and G-quadruplex. Aloe-emodine, the component of Tibetan medicine Rheum palmatum L., exhibited good capability for stabilizing G-quadruplex by π-π stacking. These results suggest that it might be a new class of G-quadruplex-targeted agents that could become potential antitumor drugs.(2) Alizarin, the component of Uygur medicine Rubia cordifolia L., can bind to the G-quadruplex structure by π-π stacking. Shikonin, the component of Uygur medicine Arnebia euchroma (Royle) Johnst., can stabilize G-quadruplex structure by groove binding and π-π stacking in DNA 3’end. These two compounds exhibit the ability to target the G-quadruplex.(3) Magnolol, the component of Pattra medicine Manglietia fordiana Oliv., cannot bind to G-quadruplex. Its isomer, honokiol, however, can interact with G-quadruplex by groove binding and π-π stacking in DNA 3’ end, thus it could be the new G-quadruplex ligands.(4) UV-visible molecular absorption and fluorescence spectroscopy methods are probably the simplest and most commonly employed instrumental technique for studying the interaction between a small molecule and biological macromolecules. However, if the compound shows an absorption band that can be not clearly distinguished from G-quadruplex in the visible region, we cannot apply this experimental method. NMR can be used to discover a variety of compounds that are bound through weakly binding and the different binding mode.2. We developed a new scientific method to screen the anti-tumor lead compounds quickly and efficiently from ethno-medicine using CD and NMR (CD-NMR method) based on the exploration of experimental materials and laboratory equipment in previous studies. Compounds that have potential anti-tumor activity were obtained from Magnolia, liliiflora Desr. by tracking the G-quadruplex activity part using the CD-NMR method. Specific research results are summarized as follows:(1) The results of CD melting targeted G-quadruplex experiment suggested that 95% alcohol and ethyl acetate layer were the most suitable extraction solvent and the key pole position. According to tracking G-quadruplex test results,5 distillates were extracted in ethyl acetate layer through gradient elution using the petroleum ether-acetone solvent system. Furthermore, NMR spectrum show the field shift of G-quadruplex peaks upon addition of Fr.2(5:1) and Fr.3(3:1) targeted G-quadruplex, indicating that they are the key parts for separation.(2) By using silica gel column chromatography (Silica gel CC), Sephadex LH-20 column chromatography (Sephadex LH-20 CC), and preparing thin-layer chromatography (PTLC),31 compounds were purified from M. liliiflora Desr. Structures of 31 compounds were elucidated by UV, IR, EI-MS,!H-NMR,13C-NMR,2D-NMR (1H-1H COSY, HMQC, HMBC) and other modern spectroscopy techniques, including 4 new lignans [liliflorin B (1), liliflorin C (2), liliflorin D (3), liliflorin E (4)],18 known lignans (compounds 5-22),5 phenyl compounds (compounds 23-27) and 4 sesquiterpene compounds (compounds 28-31). Considering the properties of compounds, eight of these compounds were selected to study targeting G-quadruplex activity by NMR. Compounds 1,2,3,12,22 were found to have the ability to bind targeted G-quadruplex.(3) Due to the good quality and activity, liliflorin A (compound 12), which was extracted from M liliflora, was selected as the research object for the further study of interaction between with small molecule compounds with targeted DNA G-quadruplex. Liliflorin A exhibited good capability for improving stabilization of G-quadruplex by increasing the Tm value of G-quadruplex to 69.93℃. It can bind human telomeric G-quadruplex selectively by groove binding and form a 1:1 stoichiometry, without binding other DNA secondary structures, such as double-stranded DNA ds26, the hairpin loop structure F10T and the other G-quadruplex sequence c-kit. Liliflorin A was the first lignan derivative that could stabilize HTG21 selectively and provided a new candidate for antitumor drug design targeting on human telomeric G-quadruplex.Our results provide the material foundation in finding anti-tumor active monomer compounds that targeting G-quadruplex for scientific development and utilization of minority traditional medicine Chinese minority traditional medicine. Our project also developed a scientific method of CD-NMR to screen the anti-tumor lead compounds quickly and efficiently from ethno-medicine, which offers basic scientific data and technical support for Chinese minority traditional medicine.