SciELO - Scientific Electronic Library Online

 
vol.48 special issue 2Study of gels of molybdenum with cerium in the preparation of generators of 99Mo - 99mTcIn Vitro antileishmanial properties of neutron-irradiated meglumine antimoniate author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand

Journal

Article

Indicators

Related links

Share


Brazilian Archives of Biology and Technology

Print version ISSN 1516-8913On-line version ISSN 1678-4324

Braz. arch. biol. technol. vol.48 no.spe2 Curitiba Oct. 2005

https://doi.org/10.1590/S1516-89132005000700008 

NUCLEAR PHARMACY

 

In Vitro study of Vellozia pusilla pohl (Velloziaceae), a Brazilian plant species: antitumoral activity and labeling of blood elements

 

 

Ana Letícia Almeida DantasI, II, *; Lígia Maria Marino ValenteII; Lilia Aparecida Salgado de MoraisIII; Glaucio FelicianoIV; Mário Bernardo-FilhoIV

IInstituto de Radioproteção e Dosimetria - IRD -CNEN; Serviço de Monitoração Interna; Av. Salvador Allende, s/n; Recreio dos Bandeirantes; 22780-160; adantas@ird.gov.br; Rio de Janeiro - RJ - Brasil
IIUniversidade Federal do Rio de Janeiro; Instituto de Química; Departamento Química Orgânica; Centro de Tecnologia; Bloco A; 21910-240; Rio de Janeiro - RJ - Brasil
IIIInstituto de Tecnologia em Fármacos; Fundação Oswaldo Cruz; Av. Brasil, 4365; Manguinhos; 21045-900; Rio de Janeiro - RJ - Brasil
IVUniversidade do Estado do Rio de Janeiro; Instituto de Biologia Roberto Alcântara Gomes; Departamento de Biofísica e Biometria; Av. 28 de setembro, 87; 20551-030; Rio de Janeiro - RJ - Brasil

 

 


ABSTRACT

Vellozia pusilla Pohl is a species of the botanic family Velloziaceae that occurs in the subtropical regions of South America and, although it lives under conditions of high solar irradiation and low water availability, shows great longevity. The methanol extract of roots, stem and leaf sheaths of this species showed an antitumoral activity through the inhibition of the enzyme Topoisomerase I when analyzed by an in vitro bioassay employing DNA repair or recombination deficient mutants of the yeast Saccharomyces cerevisiae. In the evaluation of the effect of Vellozia pusilla methanol extract on the labeling of RBC, blood of mice was treated with 99mTc tracer solutions. The percentage of radioactivity (%ATI) bound to plasma (P) and blood cells (BC) was determined. The %ATI in the insoluble fraction of plasma (IF) was also evaluate, and the results showed that there was a decrease in %ATI in this fraction that represents the plasmatic proteins.

Key words: Drug interaction, antitumoral activity, technetium-99m, blood elements, Velloziaceae


RESUMO

Vellozia pusilla Pohl é uma espécie de planta da família Velloziaceae que ocorre em regiões subtropicais da América do Sul e, apesar de viver sob condições de alta incidência solar e baixa disponibilidade de água, apresenta grande longevidade. O extrato metanólico das raízes, caule e folhas desta espécie apresentou atividade antitumoral através da inibição da enzima Topoisomerase I quando utilizado o bioensaio in vitro que emprega cepas mutantes Saccharomyces cerevisiae que apresentam deficiência na reparação ou recombinação do DNA. Na avaliação do efeito do extrato metanólico de Vellozia pusilla na marcação dos elementos sangüíneos com tecnécio 99m, sangue de rato foi tratado com solução de 99mTc como traçador sendo determinado o percentual de radioatividade (%ATI) no plasma (P) e nas células vermelhas (BC). As frações solúvel e insolúvel do plasma também foram avaliadas. Os resultados mostraram que houve um decréscimo de %ATI na fração insolúvel do plasma que é representada pelas proteínas plasmáticas.  


 

 

INTRODUCTION

The Velloziaceae constitutes a family of tropical plants occurring in South America and Africa. The Brazilian species occurring in the subtropical regions, especially in the state of Minas Gerias, Bahia, Goiás, Mato Grosso, Rio de Janeiro and coastal Santa Catarina. These plants grow in a characteristic ecosystem fully exposed on mountain sides in rocky or sandy soils. Although plants in the Velloziaceae family live under conditions of high solar irradiation and low water availability, they show surprising longevity (Ayensu, 1973). Previous studies on the specie Vellozia pusilla Pohl have afforded several terpenoids, including isopimarane nor-diterpenoid and diterpenoids, cleistantane diterpenoids and lupenone (Pinto et al.,1988; Dantas et al., 2003). There are reports concerning the use of some species of Velloziaceae in Brazilian and African folk medicine (Ayensu, 1973). Some Brazilian species of the genus Vellozia have been shown to be active against Schistossoma mansoni, Trypanossoma cruzi and Erlich carcinoma (Sampaio et al. 1984). A bioactive extract of Vellozia candida yielded a moderately DNA-damaging 6,7-seco-rosane diterpenoid through a bioassay-guided fractionation (Valente et al. 1997).

Based on the knowledge that many tumor cells have defects in their ability to repair damage to DNA as compared with normal cells, agents with selective toxicity towards repair-deficient cells might be potential anticancer agents. In this paper, studies of the MeOH extract of Vellozia pusilla of this botanical family was carried out using mechanism-based yeast bioassay that utilizes DNA repair- or recombination-deficient mutants of Saccharomyces cerevisiae yeast (Johnson et al., 1986) with the aim of discovering DNA-damaging and/or topoisomerase I and II inhibitor agents. This mechanism-based screening depends on differential responses of DNA repair-deficient and repair-proficient yeast strains to the sample under investigation (Gunatilaka et al. 1994). The methanol extract of Vellozia pusilla Pohl, collected in 1978, was tested and showed a significant activity. Based on these results, another collection was carried out in 1993, and the new methanol extract presented about the same bioactivity. This type of activity is similar to that of compounds like Camptothecin and Topotecan, both used in chemotherapy for solid tumor and hematologic malignancies (Cohen et al., 1999). The DNA Topoisomerase enzymes play a fundamental process in the cells, including replication and recombination and are an important therapeutical target in cancer chemotherapy.

Technetium 99m (99mTc) has been the radionuclide most utilized in clinical nuclear medicine procedures for diagnosis. It has also been used in basic research to study the influence of drugs on the labeling of red blood cells (RBC) and plasma proteins and in the biodistribution of this radionuclide in vivo. The labeling technique involves pre-tinning the RBC with stannous ions, followed by exposure to technetium 99m pertechnetate, which is reduced within the cell and remains trapped intracellulary by binding to the beta chain of hemoglobin. The use of medicinal plants or natural products has increased all over the world, and there are some studies about the effect of the medicinal plants on the labeling of RBC (Braga et al., 2000). In the present work, the effect of Vellozia pusilla extract on the labeling of RBC, plasma and cellular proteins with 99mTc was evaluated. Mouse blood was treated with 99mTc tracer solutions. The percentage of radioactivity (%ATI) bound to plasma (P) and blood cells (BC) was determined. The %ATI in the insoluble fraction of plasma was also evaluated.  

 

MATERIALS AND METHODS

Plant material

The plants were collected at Serra do Cipó and Diamantina, Minas Gerais, Brazil. Voucher specimens were deposited at the Departamento de Botânica of the Universidade de São Paulo, SP, Brazil.

Extraction

The shade-dried and powdered whole plant materials were sequentially extracted with hexane, AcOEt and MeOH at room temperature. After the evaporation of the solvents under low pressure, some of the dried extracts were submitted to the in vitro bioassays.

Antitumoral in vitro bioassay

Four yeast strains have been used: RAD+, rad6, rad52 and rad52.top1. The assay was performed by introducing the test sample (crude extract) dissolved in a 1:1 v/v mixture of MeOH/DMSO into a 100 µL well in agar plates separately impregnated with normal "wild-type" RAD+ yeast cells (strain derived from a mutation that makes the cell wall of the yeast more permeable to drugs but capable of DNA repair) and with mutant yeast cells and incubating the plates for 48 hrs at 30 ºC. If the test sample contained a DNA-damaging agent or a topoisomerase inhibitor, then it would inhibit the growth of one or more mutant strains, producing a zone of inhibition. First, a single dose test of the crude extracts was carried out at the concentration of 2000 µg/mL. Those extracts that showed an inhibition zone >10 mm for the mutant strains and <10 mm for RAD+ were submitted to a dose response curve where four or five different doses were used for each sample, with the dose range dependent on the magnitude of the inhibition zone previously observed. The results are expressed as IC12 values that represent the concentration of the test sample (in µg/mL) required to produce an inhibition zone 12 mm in diameter. An active extract should have an IC12 for the wild-type RAD+ strain equal to or greater than three times the IC12 for one of the mutant strains. For topoisomerase I inhibitors, the IC12 value for rad52.top1 should be at least threefold larger than that for rad52, while for topoisomerase II inhibitors the reverse should be true. The activity of yeast in each plate was tested by the use of a known anticancer drug (positive control): for RAD+, rad52 and rad52.top1, camptothecin (Johnson et al. 1986) and for rad6, streptonigrin (Gutanilaka et al. 1994).

Labeling of Blood Elements

The experimental procedure was accomplished without sacrificing the animals. Samples (0.5 mL) of mouse blood were incubated with the solutions of the methanol extract of Vellozia pusilla in NaCl 0.9 % for 60 minutes. The concentrations of the extract were 100; 50.0; 25.0; 12.5 and 6.25% of the stock solution containing 5.0 mg/mL. After this period, 1.2 ppm stannous chloride (SnCl2) and 100 µL of 99mTc tracer solution (3.7 MBq) were added (this solution was recently milked from a 99Mo/99mTc generator IPEN/CNEN). After incubating for 10 minutes, these samples were centrifuged and plasma (P) and blood cells (BC) were separated. Aliquots of 20 µL of P and BC were precipitated with 1.0 mL of 5% trichloroacetic acid (TCA), and the soluble (SF) and insoluble fractions (IF) were isolated. The 99mTc radioactivity was counted in a NaI (Tl) well counter (Automatic Gamma Counter, 1272 Clinigamma, LKB, Wallac, Finland). The percentage of radioactivity (%ATI) bound to P and BC was determined.  

 

RESULTS AND DISCUSSION

The hexane extract of V. pusilla from the 1978 collection showed the same moderate anti-tumor activity that was confirmed for the similar extract from the 1993 collection. The results obtained from the comparison of the activity of the methanol extract of a Vellozia pusilla and Camptotecin compound, when analyzed by in vitro bioassay employing DNA repair or recombination deficient mutants of Saccharomyces cerevisiae (rad+, rad 52y and rad 321) are presented in Table 1. The MeOH extract of V. pusilla presented an IC12 value for rad52.top1 three to four times larger than that for rad52 and, although exhibiting a high degree of inhibition for the RAD+ strain, might be considered a potential topoisomerase I inhibitor.

 

 

The analysis of the influence of drugs on the labeling of red blood cells and plasma proteins (Table 2) showed that there is a decrease in %ATI in IF of plasma, which represents the plasmatic proteins, for all the concentrations of extract solutions.

 

 

The lower concentration was probably sufficiently to alter the value from about 70 to 55 %. These results indicated an interaction between the substances in the methanol extract and the plasmatic proteins blocking the bonds with 99mTc. There was no interaction of the red blood cells with the substances in the extract, as shown by the values of (BC).

Results observed in the two experiments suggest a significant competition between 99mTc and the compounds present in the extract. The activity of the extract in the inhibition of the enzyme Topoisomerase I in the in vitro bioassay and the results observed in the distribution of the radioactivity in labeling of blood cells suggested a specific interaction with proteins. Future work will be performed with compounds like camptothecin with the aim of correlating these results  

 

ACKNOWLEDGMENTS

The authors are grateful to Dr. Bernardo M. Dantas for revising the manuscript.  

 

REFERENCES

Ayensu, E. S. (1973), Biological and morphological aspects of the Velloziaceae. Biotropica, 5, 135-149.        [ Links ]

Braga, A. C. S.; Oliveira, M. B. N.; Feliciano, G. D.; Reiniger, I. W.; Oliveira, J. F.; Silva, C. R. and Bernardo-Filho, M. (2000), The effect of drugs on the labeling of blood elements with technetium-99m. Current Pharmaceutical Design, 6 : (11), 1179-1191.        [ Links ]

Cohen, D. P.; Adams, D. J.; Flowers, J. L.; Wall, M. E.; Wani, M. C.; Manikumar, G.; Colvin, O. M. and Silber, R. (1999), Pre-clinical Evaluation of SN-38 and Novel Camptothecin Analogs Against Human Chronic B-cell Lymphocytic Leukemia Lymphocytes. Leukemia Research, 23 : (11), 1061-1070.        [ Links ]

Dantas, A. L. A. (1997), Estudo do extrato metanólico bioativo e obtenção de padrões a partir do extrato hexânico não ativo de vellozia pusilla Pohl (Velloziaceae), Dissertação (Mestrado), Instituto de Química, UFRJ, Rio de Janeiro. 145 pp.        [ Links ]

Dantas, A. L. A.; Valente, L. M. M. and Pinto, A. C. (2003), A new isopimarane diterpenoid fom Vellozia pusilla Pohl (Velloziaceae). J. Braz. Chem. Soc., 14, 126-128.        [ Links ]

Gunatilaka, A. A. L.; Kingston, D. G. I. and Johnson, R. K. (1994), Mechanism-based isolation and structures of some anticancer active natural products. Pure & Appl. Chem., 66, 2219-2222.        [ Links ]

Johnson, R. K.; Bartus, H. F.; Hofmann, G. A.; Bartus, J. O.; Mong, S. M.; Faucettte, L. F.; McCabe, F.L.; Chan, J. A. and Mirabelli, C. K. (1986), Discovery of new DNA-reactive drugs. In: Hanka, L. J.; Kondo, T. and White, R. J. (Eds.). In vitro and in vivo models for detection of new antitumor drugs.         [ Links ]In: International Congress of Chemotherapy, 14., Kyoto. Proceedings… Kyoto. pp. 15-26.        [ Links ]

Pinto, A. C.; Valente, L. M. M. and Silva, R. S. (1988), "Norditerpenoids from Vellozia pusilla", Phytochemistry, 27: (12), 3913-3915.        [ Links ]

Sampaio, M. R. F. P.; Nakamura, I. T.; Oliveira Jr, B. S. and Oliveira, M. M. (1984), Modificação do método de DiPaolo & Moore para testes de extratos e substâncias anticancerígenas – Parte I. Arq. Inst. Biol., 51, 35-39.        [ Links ]

Valente, L. M. M.; Gunatilaka, A. A. L.; Kingston, D. G. I.; Patitucci, M. L. and Pinto, A. C. (1997), A bioactive seco-rosane diterpenoid from Vellozia candida. J. Nat. Prod., 60, 478-481.        [ Links ]

 

 

Received: July 05, 2005;
Revised: July 14, 2005;
Accepted: August 01, 2005

 

 

* Author for correspondence

Creative Commons License All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License