Cristiane de Melo Cazal

Isolation of xanthyletin, an inhibitor of ants’ symbiotic fungus, by high-speed counter-current chromatography

Xanthyletin, an inhibitor of symbiotic fungus (Leucoagaricus gongylophorus) of leaf-cutting ant (Atta sexdens rubropilosa), as well as suberosin, seselin and xanthoxyletin were isolated from Citrus sinensis grafted on Citrus limonia. A two-phase solvent system composed of hexane/ethanol/acetonitrile/water (10:8:1:1, v/v) was used for the high-speed counter-current chromatographic isolation of xanthyletin with high yield and over 99% purity as determined by liquid and gas chromatography with mass spectrometry detection. Identifications were performed by UV spectra, IR spectra, (1)H NMR and (13)C NMR.

Isolation of secondary metabolites from Hortia oreadica (Rutaceae) leaves through high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) with a two-phase solvent system (hexane-ethanol-acetonitrile-water 10:8:1:1, v/v) was applied to examine the leaves of Hortia oreadica, which afforded the known limonoid guyanin (1), the alkaloids rutaecarpin (2) and dictamnine (6), the dihydrocinnamic acid derivatives methyl 5,7-dimethoxy-2,2-dimethyl-2H-1-benzopyran-6-propanoate (3), 5,8-dimethoxy-2,2-dimethyl-2H-1-benzopyran-6-propanoic acid (4), together with the new E-3,4-dimethoxy-alpha(3-hydroxy-4-carbomethoxyphenyl)cinnamic acid (5). The recovery of compounds 1-6 was determined by comparison with LC-atmospheric pressure chemical ionization MS/MS data: 66.2%, 93.1%, 102.5%, 101.2%, 99.0% and 84.9%, respectively. Compound 3 showed IC(50) of 23.6microM against Plasmodium falciparum and 15.6microM against Trypanosoma brucei rhodesienses and was not toxic to KB cells (IC(50)>100microM).

Evaluation of Effect of Triterpenes and Limonoids on Cell Growth, Cell Cycle and Apoptosis in Human Tumor Cell Lines

Six triterpenes and eight limonoids were evaluated for their capacity to inhibit the growth of three human tumour cell lines, breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and melanoma (A375-C5). The mechanisms involved in the observed cell growth arrest of the four most potent compounds were carried out by studying their effect in cell cycle profile and programmed cell death. The results showed that one triterpene (odoratol) and two limonoids (gedunin and cedrelone) caused cell cycle arrest while only the limonoids gedunin and cedrelone were found to be very potent inducers of apoptosis.

High-speed counter-current chromatographic isolation of ricinine, an insecticide from Ricinus communis.

The alkaloid ricinine, an insecticide for leaf-cutting ant (Atta sexdens rubropilosa), was obtained from Ricinus communis. A two-phase solvent system composed of CH(2)Cl(2)/EtOH/H(2)O (93:35:72, v/v/v) was used for high-speed counter-current chromatographic (HSCCC) isolation of ricinine in high yield and with over 96% purity, as determined by liquid and gas chromatography-mass spectrometry (LC-MS and GC-MS). Identification of ricinine was performed by comparison of (1)H NMR, (13)C NMR and LC-MS/MS data.

Chemical Composition and Biological Activities of the Essential Oils from the Fresh Leaves of Citrus limonia Osbeck and Citrus latifolia Tanaka (Rutaceae)

This study aimed at determining, for the first time, the chemical composition of the essential oils from fresh leaves of Citrus limonia and Citrus latifolia and evaluating its in vitro antibacterial, trypanocidal, leishmanicidal and cytotoxic effects. The essential oils from fresh leaves of C. latifolia and C. limonia were extracted by hydro distillation and analyzed by GC-MS. The oil yield was 0.8% for C. latifolia and 0.5% for C. limonia, whereas 17 and 18 chemical components were identified for C. latifolia and for C. limonia, respectively. Major components were limonene (46.3 %), nerol (10.0 %) and 1,8-cineole (13.4 %). In vitro antibacterial activity was evaluated against the bacteria Streptococcus mitis, Streptococcus mutans, Streptococcus sanguinis, Streptococcus sobrinus and Bacteroides fragilis by the broth dilution method. Protozoa of the Trypanosomatidae family cause neglected tropical diseases, such as trypanosomiasis, whose treatment has depended on few available drugs. Therefore, the essential oils from fresh leaves of C. limonia and C. latifolia were also evaluated against the Y strain of Trypanosoma cruzi trypomastigotes, resulting in IC50 = 88.2 μg/mL and 51.7 μg/mL, respectively. Regarding the evaluation of the cytotoxic activity against fibroblasts LLCMK2 line, the essential oil from C. limonia (CC50 = 422.9 μg/mL) and C. latifolia (CC50 = 502.9 μg/mL) showed moderate cytotoxicity. The leishmanicidal activity of the essential oils from fresh leaves of C. limonia and C. latifolia were evaluated against promastigote forms of Leishmania amazonensis, resulting in IC50 = 2.63 μg/mL and 2.03 μg/mL, respectively. Thus, the essential oils from fresh leaves of Citrus limonia and Citrus latifolia had moderate antibacterial and trypanocidal activities and significant leishmanicidal activity.

In vitro antiparasitic activity and chemical composition of the essential oil from Protium ovatum leaves (Burceraceae)

Leishmaniasis and trypanosomiasis are globally widespread parasitic diseases which have been responsible for high mortality rates. Since drugs available for their treatment are highly hepatotoxic, nephrotoxic and cardiotoxic, adherence to therapy has been affected. Thus, the search for new, more effective and safer drugs for the treatment of these diseases is necessary. Natural products have stood out as an alternative to searching for new bioactive molecules with therapeutic potential. In this study, the chemical composition and antiparasitic activity of the essential oil from Protium ovatum leaves against trypomastigote forms of Trypanosoma cruzi and the promastigote forms of Leishmania amazonensis were evaluated. The essential oil was promising against trypomastigote forms of T. cruzi (IC50= 28.55 μg.mL-1) and L. amazonensis promastigotes (IC50 = 2.28 μg.mL-1). Eighteen chemical constituents were identified by Gas Chromatography coupled to Mass Spectrometry (GC-MS) in the essential oil, whose major constituents were spathulenol (17.6 %), caryophyllene oxide (16.4 %), β-caryophyllene (14.0 %) and myrcene (8.4 %). In addition, the essential oil from P. ovatum leaves had moderate cytotoxicity against LLCMK2 adherent epithelial cell at the concentration range under analysis (CC50 = 150.9 μg.mL-1). It should be highlighted that this is the first report of the chemical composition and anti-Trypanosoma cruzi and anti-Leishmania amazonensis activities of the essential oil from Protium ovatum leaves.

Effects of Limonoid Cedrelone on MDA-MB-231 Breast Tumor Cells in vitro

Cancer is the second leading cause of death, preceded only by cardiovascular diseases, and there is epidemiological evidence that demonstrate this tendency is emerging worldwide. Brazil has an extensive vegetal biodiversity with more than 55,000 species listed. Such biodiversity collaborates with the finding of compounds which could be the basis for the design of new anti-tumor drugs, with fewer side effects than the conventional chemotherapy used currently. Cedrelone is a limonoid isolated from Trichilia catigua (Meliaceae) which is a native Brazilian plant. This study demonstrates that cedrelone inhibits proliferation, adhesion, migration and invasion of breast tumor cells from the line MDA-MB-231. The effects of cell migration and invasion on MDA-MB-231 cell may be explained, at least in part, by the ability of cedrelone to inhibit MMP activity. We also demonstrate that cedrelone is able to induce apoptosis in MDA-MB-231 cells. There are only a few works investigating the effect of limonoids in cellular processes closely related to tumor progression such as adhesion, migration and invasion. To the best of our knowledge, this is the first work describing the effects of a limonoid on tumor and non-tumor cell adhesion process.

Chemical composition, anti-Trypanosoma cruzi and cytotoxic activities of the essential oil from green fruits of Protium ovatum (BURSERACEAE)

1MSc, Instituto Federal Goiano, Rio verde-GO, Brazil. E-mail: elisangelaborges49@hotmail.com 2PhD, Instituto Federal Goiano, Rio verde-GO, Brazil. E-mail: cassiacefetrv@gmail.com 3PhD, Universidade de Franca, Franca-SP, Brazil. E-mail: viviane.sampaio@unifran.edu.br 4PhD, Instituto Federal do Sudeste de Minas Gerais. Barbacena-MG, Brazil. E-mail: criscazal@yahoo.com.br 5MSc, Instituto Federal do Mato Grosso do Sul, Coxim-MS, Brazil. E-mail: allex_fonseca@yahoo.com.br 6 PhD, Instituto Federal do Sul de Minas Gerais, Pouso Alegre-MG, Brazil. E-mail: mayker.miranda@ifsuldeminas.edu.br Abstract Chagas disease is a major neglected tropical disease caused by Trypanosoma cruzi. It has been treated with the antiparasitic drugs nifurtimox and benzonidazole, which cause several side effects. The market for natural products has considerably grown lately and the use of plants has become an alternative to the development of novel drugs to cure the disease. Therefore, this study aimed at describing the chemical analysis of the essential oil from green fruits of Protium ovatum and evaluating their trypanocidal and cytotoxic potential. The essential oil was obtained by Clevenger hydrodistillation whereas its chemical composition was determined by gas chromatography coupled to mass spectrometry (GC-MS). The major compounds found in the essential oil from green fruits of P. ovatum were β-myrcene (62.0 %), α-pinene (11.3 %) and limonene (7.3 %). To the best of our knowledge, this was the first time that the chemical composition of the essential oil from green fruits of P. ovatum was described. Results showed that the essential oil had strong trypanocidal activity against trypomastigote forms of theY strain of Trypanosoma cruzi (IC50 = 1.2 μg/mL). In addition, the essential oil from green fruits of P. ovatum did not display cytotoxicity against LLCMK2 adherent epithelial cell at the concentration range under analysis (CC50 = 550.3 μg/mL). As a result, it is an excellent option for the development of novel antiparasitic drugs.

Characterization of Nanospheres Containing Zanthoxylum riedelianum Fruit Essential Oil and Their Insecticidal and Deterrent Activities against Bemisia tabaci (Hemiptera: Aleyrodidae)

The aim of our study was to produce and characterize poly-ε-caprolactone (PCL) nanospheres containing essential oils from Zanthoxylum riedelianum fruit and to evaluate their stability gains as well as their insecticidal and deterrent activities against whitefly (Bemisia tabaci). The PCL nanospheres exhibited a homogeneous spherical morphology, with particle diameters between 106.7 nm and 129.2 nm, pH of approximately 6, zeta potential (ZP) lower than −19.0 mV and encapsulation efficiency higher than 98%. Only 43% of the nanoencapsulated essential oil (NSEO) was degraded in response to ultraviolet light, whereas the essential oil (EO) degraded by 76% over the same period. In a free-choice test, the NSEO and EO reduced the number of whitefly eggs by approximately 70%. NSEO and EO at 1.5% killed 82.87% and 91.23% of 2nd-instar nymphs of whitefly, respectively. Although NSEO displayed lower insecticidal activity, it offers a greater advantage over the free EO, due to protection conferred by polymer against photodegradation. Therefore, its usage may optimize the maintenance of essential oils in the field through photoprotection and controlled release. Our results suggest that the EO of Z. riedelianum fruit can be used for B. tabaci management strategy; nevertheless, the benefits of NSEO require further evaluation at the field level.

ÓLEOS ESSENCIAIS DOS FRUTOS E FOLHAS DE Kielmeyera coriacea: ATIVIDADE ANTITUMORAL E ESTUDO QUÍMICO

Kielmeyera coriacea e uma planta tipica do Cerrado conhecida como pau-santo sendo muito utilizada pela medicina popular no tratamento de diversas doencas. O presente trabalho teve como objetivo caracterizar quimicamente os oleos essenciais dos frutos e das folhas de K. coriacea e avaliar sua atividade antitumoral frente as linhagens de celulas tumorais humanas MDA-MB-231(tumor mama) e DU-145 (tumor de prostata) na busca de novos compostos bioativos de origem natural. Os oleos essenciais foram extraidos por hidrodestilacao em aparato clevenger. O rendimento dos oleos essenciais das folhas e frutos foi de 0,38 % e 0,0041 %, respectivamente. Os compostos majoritarios presentes no oleo essencial das folhas de K. coriacea , foram: 2- Acetato de (2 E )-tridecenol ( 9,06 % ), Acido 1,2-benzenodicarboxilico ( 8,28 % ) e E - nerolidol ( 7,70 % ). E no oleo essencial dos frutos os constituintes majoritarios identificados foram: D-germacreno (18,94 %), neo-intermedeol (12,18 %) e biciclogermacreno (12,00 %). Este e o primeiro relato da composicao quimica do oleo essencial dos frutos de Kielmeyera coriacea . O oleo essencial dos frutos inibiu a ploriferacao celular das linhagens tumorais DU-145 e MDA-MB-231 e das celulas nao tumorais (FH), enquanto que o oleo essencial das folhas nao exibiu atividade frente a nenhuma linhagem tumoral testada. Destaca-se ainda que ambos os oleos essenciais analisados foram citotoxicos frente as celulas normais (FH).