Phytochemistry and Pharmacogenomics of Natural Products Derived from Traditional Chinese Medicine and Chinese Materia Medica with Activity Against Tumor Cells
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152 Phytochemistry and pharmacogenomics of natural products derived from traditional chinese medicine and chinese materia medica with activity against tumor cells Thomas Efferth,1 Stefan Kahl,2,3,6 Kerstin Paulus,4 Introduction 2 5 3 Michael Adams, Rolf Rauh, Herbert Boechzelt, Cancer is responsible for 12% of the world’s mortality and Xiaojiang Hao,6 Bernd Kaina,5 and Rudolf Bauer2 the second-leading cause of death in the Western world. 1 Limited chances for cure by chemotherapy are a major German Cancer Research Centre, Pharmaceutical Biology, contributing factor to this situation. Despite much progress Heidelberg, Germany; 2Institute of Pharmaceutical Sciences, University of Graz; 3Joanneum Research, Graz, Austria; 4Institute in recent years,a key problem in tumor therapy with of Pharmaceutical Biology, University of Du¨sseldorf, Du¨sseldorf, established cytostatic compounds is the development of Germany; 5Institute of Toxicology, University of Mainz, Mainz, 6 drug resistance and threatening side effects. Most estab- Germany; and State Key Laboratory of Phytochemistry and Plant lished drugs suffer from insufficient specificity toward Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China tumor cells. Hence,the identification of improved anti- tumor drugs is urgently needed. Several approaches have been delineated to search for Abstract novel antitumor compounds. Combinatorial chemistry,a The cure from cancer is still not a reality for all patients, technology conceived about 20 years ago,was envisaged as which is mainly due to the limitations of chemotherapy a promising strategy to this demand. The expected surge in (e.g., drug resistance and toxicity). Apart from the high- productivity,however,has hardly materialized (1). throughput screening of synthetic chemical libraries, Other sources are natural products. Marine and terres- natural products represent attractive alternatives for drug trial plants and animals are a fertile ground to find novel development. We have done a systematic bioactivity- drugs. Prominent examples for the success of natural based screening of natural products derived from medic- products originally obtained from plants are the Vinca inal plants used in traditional Chinese medicine. Plant alkaloids from Catharanthus roseus G. Don. (Apocynaceae), extracts with growth-inhibitory activity against tumor cells the DNA topoisomerase I inhibitor camptothecin from have been fractionated by chromatographic techniques. Camptotheca acuminata Decne. (Nyssaceae),the terpene We have isolated the bioactive compounds and elucidated paclitaxel from Taxus brevifolia Nutt. (Taxaceae),or the the chemical structures by nuclear magnetic resonance lignan podophyllotoxin isolated from Podophyllum peltatum and mass spectrometry. By this strategy, we identified L. (Berberidaceae; ref. 2). 25-O-acetyl-23,24-dihydro-cucurbitacin F as a cytotoxic Evidently,69% of anticancer drugs approved between constituent of Quisqualis indica. Another promising com- the 1980s and 2002 are either natural products or developed pound identified by this approach was miltirone from Salvia based on knowledge gained from natural products (1). miltiorrhiza. The IC50 values for miltirone of 60 National Arguably,about three quarters of plant-derived drugs in Cancer Institute cell lines were associated with the micro- clinical use today came to the attention of pharmaceutical array-based expression of 9,706 genes. By COMPARE companies because of their use in traditional medicines (3). and hierarchical cluster analyses, candidate genes were The long-lasting experience of traditional folk medicines identified, which significantly predicted sensitivity or resis- may facilitate the identification of novel agents. Although tance of cell lines to miltirone. [Mol Cancer Ther 2008; in industrialized countries medicinal herbs gradually lost 7(1):152–61] importance in the course of chemistry’s progress during the 20th century,bioactive plant constituents are recently experiencing an impressive revival (4). Most of these potentially useful plant products are products of secondary Received 1/31/07; revised 9/21/07; accepted 12/4/07. metabolic pathways and serve their producers either as Grant support: Joanneum Research, Graz, Austria (S. Kahl). protective agents against herbivores and various pathogens The costs of publication of this article were defrayed in part by the or as growth regulators. Due to these physiologic functions, payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to secondary metabolites could be potential anticancer drugs. indicate this fact. It is estimated that only 25,000 plant species have been Requests for reprints: Thomas Efferth, German Cancer Research Centre, studied exhaustively for possible medical applications. This Pharmaceutical Biology (C015), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Phone: 49-6221-423426; Fax: 49-6221-423433. is a minor fraction of the approximate total of 250,000 E-mail: [email protected] to 300,000 higher plant species in the world (3). Copyright C 2008 American Association for Cancer Research. These facts lead us to pursue the search for new bioac- doi:10.1158/1535-7163.MCT-07-0073 tive lead structures with antitumor activity by a strategy of MolCancerTher2008;7(1).January2008 Downloaded from mct.aacrjournals.org on September 29, 2021. © 2008 American Association for Cancer Research. Molecular Cancer Therapeutics 153 bioactivity-guided isolation from plants used in traditional Biosciences) was used to fill glass columns. The fractions folk medicines. Medicinal plants used in many tribes all obtained were evaporated using a rotavapor system worldwide are studied in ethnobotany and ethnopharma- (RE-111; Bu¨ chi). cology (5). It can be expected that the search for bioactive For medium-pressure liquid chromatography,we used plant compounds is more successful in medicinal plants 56-cm columns (ø 1.9 cm; Bu¨ chi),separation material RP-18 than a search across all plant species. Traditional Chinese (14-63 Am; Merck),and a rotavapor (RE-111). medicine commands a unique position among traditional High-pressure liquid chromatography was done using medicines because its enormous variety of drugs of plant an Agilent 1100 Series with autosampler G1313A (Agilent origin is founded on more than 5,000 years of tradition. An Technologies) and photodiode array detector (Agilent Tech- elaborated system has been developed and many written nologies) run with HP Chemstation (Hewlett-Packard). The documents and textbooks have been handed down over the columnsusedwereXTerraPrep7.8Â 150 mm and millennia. This might imply that inactive plants and recipes 19 Â 150 mm (Waters). RP-10 (10 Am) served as separation have vanished over the centuries and that the materia material. The flow rates were 1 mL/min for analytic high- medica of modern traditional Chinese medicine is enriched pressure liquid chromatography and 10 mL/min for semi- with bioactive plants. This may improve prospects for preparative high-pressure liquid chromatography. Detection identifying novel active constituents from traditional was done at wavelength of 210,230,254,280,and Chinese medicine significantly (6). Based on this rationale, 312 nm. The column temperature was 30jC. we started the systematic isolation of chemical compounds Spectrometric Methods from medicinal plants used in traditional Chinese medicine Nuclear magnetic resonance spectra (1H, 13C,COSY, and their biological and pharmacologic characterization. HMQC,HSQC,HMBC,and DEPT) were recorded at 295K with tetramethylsilane as internal standard by means of an AM-400 MHz spectrometer (Bruker Analytical GmbH) Materials and Methods for 1H and 13C spectra and by means of an DRX-500 MHz Collection of Medicinal Plants in the Yunnan Province spectrometer (Bruker Analytical GmbH) for two- Seventy-six medicinal plants were collected in China. dimensional spectra. Solvents were obtained from Cam- Some samples were from Menglun county,prefecture bridge Isotope Laboratories. A Finnigan-4510 mass spec- Xishuangbanna,in Yunnan province located at 600 to trometer was used for the measurement of electron ionizing 700 m above sea level. The botanical identification was and fast atom bombardment mass spectra and an API done by Prof. Chen Yu,Prof. Tao Guo Da,Prof. Tao De QSTAR Pulsar I mass spectrometer for determination of Ding,and Chen Jia Hui. Voucher specimens are deposited time-of-flight mass spectra. Glycerine served as a matrix. at the herbarium of the Institute of Plant Sciences, Crystal Structure Analysis University of Graz,Graz,Austria (Herbar GZU). Other The analysis of crystal structures was done at the Institute samples were purchased at the medicinal plant market in of Chemistry (University of Graz; head: Prof. Ferdinand Kunming,Yunnan province. Belaj). All measurements were done with graphite-mono- The finely ground plant material was successively chromatized MoKa radiation. A total of 3,970 reflexions Q j extracted with solvents of increasing polarity [petroleum were measured ( max =26.0),of which 3,132 were ether (or n-hexane),ethyl acetate,and methanol]. Organic symmetry independent (Rint = 0.0182) with 2,937, which solvent extracts were made in a Soxhlet apparatus,whereas showed I > 2j(I). Structures were elucidated with direct water extracts were made as decoctions according to the methods (SHELXS-97) and refined with full matrix least traditional medicinal preparation.