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Chemical Analyses and Biological Evaluation of the Endemic Plant Simarouba Tulae Karla Claudio 1 , Pedro González 2 , Elsa Luciano 2 , Janice Santiago 3 , Marianela Pérez, Ph.D. 3 , Augusto Carvajal 1 , Mayra Pagan, Ph.D. 2 and Claudia A. Ospina, Ph.D. 2 , (1)Department of Biology, University of Puerto Rico at Cayey, Cayey, PR, (2)Department of Chemistry, University of Puerto Rico at Cayey, Cayey, PR, (3)Dept. of Pharmaceutical Sciences, UPR - Medical Sciences Campus, San Juan, PR Abstract : Species of the genus Simarouba have been studied because of its antimalarial, antiinflammatory, antileukemic, antifeedant and antiviral activities. A group of highly oxygenated terpenes called the quassinoids have been isolated from species of the Simarouba genus and are thought to be responsible for its therapeutic properties. The objective of this study is to isolate and evaluate the biological activity against cancer cell lines of the secondary metabolites from Simarouba tulae , an endemic plant of Puerto Rico. Extracts prepared in our laboratory were preliminary screened using the brine shrimp lethality test. Most of the Simarouba tulae extracts were cytotoxic with LC 50 values of 160.69 μg/ml or less. These extracts were further biotested against two breast cancer cell lines (MCF-7 and ZR-75-1) inhibiting more than 80% of cell growth. The experimental methodology and the results will be presented. Discussion Future Work Acknowledgements References Figure 1 . Photo of Simarouba tulae from Maricao, P.R. Introduction ,[object Object],[object Object],[object Object],Objectives Methodology 1. Plant Collection 2. Extractions 3. Brine Shrimp Lethality Test Serial dilutions of plant extracts were made in wells of 96-micro-well plates in triplicate at different concentrations. A suspension of 100 µl containing 10-15 brine shrimp larvae were added to each well and incubated at room temperature for 24 hours. After 24 h the dead nauplii were counted with the aid of a microscope and the lethal concentration (LC 50 value) was calculated by probit analysis. Larvae were considered dead if they did not move during the observation. The LC 50 value was obtained by regression analysis of the data of percentage lethality versus concentration. 4. Purification and Isolation C. NMR ( 1 H and 13 C) D. Infrared Spectroscopy A. TLC B. Column Chromatography C. Mass Spectroscopy Results *LC 50 is the lethal concentration needed to kill the 50 percent of the population ** Values lesser than 200 µg/ml are considered toxic. Table 1. Bioassay of Simarouba tulae extractions of leaves Table 2. Anticancer activity of Simarouba extracts TLC Results of Chloroform extract of the leaves of S. tulae 13 C NMR Spectrum (100 MHz) for the cloroform extract of the leaves of S. tulae (SHC2 sample) Figure 2. Basic skeleton of a quassinoid C-20 A great number of plants have been used to treat diseases. Nevertheless, plants endemic to Puerto Rico are still not well studied for this purpose. Simarouba tulae (Figure 1), a member of Simaroubaceae family is an endemic plant of the island which can be found as trees of two through eight meters long in regions such as Maricao and Patillas. This plant is commonly known as “aceitillo falso”. Simaroubaceae family consists of over 150 species that are known for their chemical composition. This group of plants are been used to treat cancer, malaria, viruses and bacteria and even as antifeedant agents. This has brought the attention of some researchers to identify compounds responsible for these activities. Although Simaroubaceae family has been widely studied, there are no literature reports on the chemical composition or biological activities of the S. tulae species . During the 1930’s, Clark’s group isolated from the Simaroubaceae family, specifically from Picrasma excelsa , a group of compounds called quassinoids. The quassinoids, which have a bitter taste, are a group of compounds that are highly oxygenated triterpenes. Over a 150 quassinoids have been isolated and fully characterized. According to their skeleton, quassinoids are categorized in five groups. Among them, C-20 quassinoids (Figure 2) have been subject of intensive investigation due to the discovery by National Cancer Institute of their antitumor activities. As the mechanism of action it has been proposed that quassinoids inhibit the synthesis of proteins needed by cancerous cells. These compounds are responsible of the bioactivities of this family and also have been recognized as chemical markers for this group of plants. This research seeks to isolate, and identify possible quassinoids or other compounds present in Simarouba tulae in order to expand the knowledge of the chemotaxonomy of the endemic plants of Puerto Rico and to investigate their potential use as anti-cancer agents. Maricao Patillas Anderson, M.; Colin, W.; Gupta, M.; Phillipson, D.; Solis, P. “A Microwell Cytotoxicity Assay using Artemia Salina (Brine Shrimp)”. Planta Med , 1993 , 59, 250-252. Batista, J.; Braz, R.; Curcino, I.; Da Silva, M.; Rodrigues E.; Vireira, P. “20(R)- and 20(S)- Simarolide Epimers Isolated from Simaba cuneata Chemical Shifts Assignment of Carbon and Hydrogen Atoms”. J. Braz. Chem. Soc., 1999 , 10, 76-84. Beutler, J.; Clement, J.; Colburn, N.; Kang, M.; Pelletier, J.; Robert, F. Quassinoid Inhibition of AP-1 Function Does Not Correlate with Cytotoxicity or Protein Synthesis Inhibition. J. Nat. Prod . 2009 , 75, 503-506. Guo, Z.; Sindelar, R.D.; Sindelar, R.W.; Vangapandu, S.; Walker, L.A. Biological Actives Quassinoids and Their Chemistry: Potential Leads for Drug Design. Curr. Med. Chem . 2005 , 12, 173-190. Rhodes, M.; Robins, R. High-performance liquid chromatographic methods for the analysis and purification of quassinoids from Quassia amara L. J. Chromato . 1984 , 283, 436-440. Ospina, C. A.; Pagán, M.; Carvajal, A.; Claudio, K; Rivera, J.; Ortiz, I.; Hernández, J. In “Cytotoxic Screening of Tropical Plants Using Brine Shrimp Lethality Test”.; Montes, E. L.; Eds.; Cuadernos de Investigación Number 7; Instituto de Investigaciones Interdisciplinarias: Cayey, 2009 ; 1-20. ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],1 H NMR Spectrum (400 MHz) for crude extract of the leaves of S. tulae 1 H NMR Spectrum (400 MHz) for the chloroform extract of the leaves of S. tulae (SHC2 sample) ,[object Object],[object Object],[object Object],[object Object],Chloroform extraction was first separated through a silica gel column chromatography. The number of the sample indicates the fraction obtained from the column. Compounds were separated using a mixture of solvents of CHCl 3 / MeOH 9:1. Plates were revealed in an Iodine chamber and observed under UV light of 254 nm. Fractions were mix according to separations observed in the TLC plates. Samples SHC2 and SHC3 were analyzed with NMR spectra. Grant # 5 R25 GM059429 C-C σ bonds Oxygenated C’s C-C σ bonds Oxygenated C’s Alkenes Oxygenated C’s C-C σ bonds *All extracts were evaluated at a single doses of 100 μg/mL. Simarouba tuale was the species selected for this study since it is the only endemic representative plant of the family Simaroubaceae in Puerto Rico. This family of plants is known for its therapeutical properties in America. The Brine Shrimp Lethality Test was a preliminary method chosen to identify possible bioactivity in S. tulae extracts, since previous literature had reported a correlation between this test and the antitumor activity (Anderson et al 1993 ). Moreover is important to identify, characterize and study separately metabolites in the extracts of S. tulae since synergy effect could be hiding some compounds activity. Results of the cytotoxicity screening shows that most of the extracts of Simarouba tulae are seem to be actives except for hexane extraction. Testing in breast cancer cell lines MCF-7 and ZR-75-1 showed growth inhibition from 57 to 90% and 79 to 90% respectively. The crude extraction was one of the most toxic in the Brine Shrimp Lethality Test and the most active inhibiting breast cancer cell growth (90% of growth inhibition in MCF-7 and ZR-75-1 cell lines). Chemical analyses of the extractions from S.tulae leaves has not been finished yet since samples, already evaluated in the NMR , need further separations in order to be purified. Although purification of compounds has not been achieved yet, the NMR spectra for the chloroform extraction show clear signals in regions belonging to oxygenated carbons and aliphatic carbons (characteristic signals of quassinoids). The NMR spectra of the crude sample from leaves shows the same pattern and also a tall, clear signal in the region of alkenes. Patterns found in the NMR spectra for crude and chloroform extraction could suggest the presence of triterpenes such as quassinoids . Further chemical analyses are needed to be done with the objective to identify compounds such as quassinoids or others in the endemic plant Simarouba tulae and eventually to contribute to the chemotaxonomy of the Puertorrican flora. Extract LC 50 * Cytotoxic?** Ethyl Acetate 35.4 µg/ml Yes Crude 2.0 µg/ml Yes Methanol 38.4 µg/ml Yes Cloroform 160.7 µg/ml Yes Hexane > 200.0 µg/ml No Simarouba Tulae Extract (leaves) Breast Cancer Cell MCF-7 % of growth Inhibition Breast Cancer Cell ZR-75-1 % growth inhibition Crude Extract 90 90 Hexane Extract 57 89 Chloroform Extract 83 79 Ethyl Acetate Extract 74 85

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