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Revista do Instituto de Medicina Tropical de São Paulo

On-line version ISSN 1678-9946

Rev. Inst. Med. trop. S. Paulo vol.44 no.2 São Paulo Mar./Apr. 2002

https://doi.org/10.1590/S0036-46652002000200010 

BRIEF COMMUNICATION

ANTI-TRYPANOSOMAL ACTIVITY OF PENTACYCLIC TRITERPENES ISOLATED FROM Austroplenckia populnea (CELASTRACEAE)

 

Lucienir Pains DUARTE(1), Sidney Augusto VIEIRA FILHO(1,3), Grácia Divina de Fátima SILVA(1), José Rego de SOUSA(1) & Artur da Silveira PINTO(2)

 

 


SUMMARY

Four pentacyclic triterpenes isolated from Austroplenckia populnea and four compounds of known anti T. cruzi or anti-malarial activity were tested. Of those triterpenes tested 20a-hydroxy-tingenone showed high activity, epikatonic acid was less active, while populnilic and populninic acids were inactive against the trypanosome of the subgenus Schizotrypanum tested. Benzonidazole, nifurtimox, ketoconazole and primaquine presented a remarkable dose-dependent inhibitory effect reaching practically to a total growth inhibition of the parasite at the end of incubation time. The trypanosome tested appear to be a suitable model for preliminary screen for anti T. (S.) cruzi compounds.

KEYWORDS: Austroplenckia populnea; Pentacyclic triterpenes; Anti-trypanosomal activity; Growth inhibition.


 

 

Austroplenckia populnea Reiss (Celastraceae) is a tropical tree commonly found in the Minas Gerais State, Brazil. "Mangabarana, "Mangabeira-Brava", and "Marmelinho do Campo" are its popular names. The tea of its leaves is used as antidysenteric2 and anti-rheumatic6 in the popular medicine. Extracts and several constituents obtained from A. populnea have been investigated for biological activity including antitumor activity8, as well as, antibacterial activity11,16.

On the other hand, Chagas' disease remains a major public health problem in Latin America where it has been estimated that 16-18 millions people are chronically infected by Trypanosoma cruzi, it's etiological agent17.

Since reduviid bugs (mainly the domiciate species Triatoma infestans), are intermediate hosts for T. cruzi, control is largely carried out by use of residual insecticides in endemic areas. However, efficacy of this activity is limited by economic-social factors1. The chronicity combined with the involvement of the host's immune response in the pathogenesis of Chagas' disease makes the development of a effective vaccine particularly difficult. On the other hand, chemotherapy using the nitrofuran derivative nifurtimox or the nitroimidazole derivative benzonidazole because of their clinical effects continue inexpressive and controversial14, claiming for new drugs.

In order to avoid handling the pathogenic T. cruzi for preliminary screening of anti-T. cruzi organic compounds GABORAK et al.3, had earlier suggested the use of other members of the Schizotrypanum genus instead of, since a number of compounds of known anti-T. (S.) cruzi activity had showed similar activity when tested in vitro against non-pathogenic trypanosomes.

In the present study we used a trypanosome of the genus Schizotrypanum [T. (S) cruzi] isolated from a Phyllostomus hastatus bat collected in Minas Gerais State, Brazil9, to test pentacyclic triterpenes isolated from A. populnea. This isolate was unable in produce detectable parasitemia in mice9, and distinct from T. cruzi in their isoenzymatic and lectin agglutination profiles10,15. In addiction, trials in vitro are of permanent interest because the identification of active compounds may be useful for the development of new agents for the treatment of Chagas' disease.

A. populnea Reiss (Celastraceae) was collected in Nova Lima region, Minas Gerais State, Brazil. After botanical identification by Dr. Luiz Pedersoli, Departament of Botany, Universidade Federal de Minas Gerais, a voucher specimen (No. 10473) representing this collection has been deposited at the Herbarium of the Natural History Museum of the Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil.

The cleaned bark wood (3.0 kg) and roots (270 g) of A. populnea was dried at room temperature and than milled in powder. The bark wood was submitted to extraction using methanol. This extract (656 g) was further fractionated with hexane, benzene and ethyl acetate. The roots were extracted with petroleum ether, dichloromethane and methanol. The phytochemical methods were described previously12,13. From the petroleum ether extract of the roots was isolated 48.0 mg of the 20a-hydroxy-tingenone12, and from the bark wood extracts were isolated 209.4 mg of populninic acid 361.0 mg of epikatonic acid and 46.0 mg of populnilic acid13.

The trypanosome strain [T.(S) cruzi] used in the experiments was isolated from a P. hastatus bat collected in Serrania, Minas Gerais State, Brazil. Isolation was performed by hemoculture in brain-hearth-infusion (BHI) culture medium supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS), 2% of 10% rabbit haemoglobin solution. Clone was obtained by successive plating technique in the same culture medium added with 0.75 agar. Flagellate was maintained by serial passage every 10 days and also by criopreservation in liquid nitrogen after addition of 10% glycerol (v/v) to culture.

Four pentacyclic triterpenes 20a-hydroxy-tingenone, epikatonic acid, populnilic acid and populninic acid were assayed (Table 2). Benzonidazole, nifurtimox, ketoconazole and primaquine drugs of known anti-trypanosomal, anti-fungical and anti-malarial activity were tested for comparison (Table 1).

 

 

 

 

T. (S) cruzi growth inhibition experiments were carried out in 16 x 150 mm screw-capped tubes containing of BHI medium added with 10% of foetal bovine serum (FBS) and 2% of a 10 mg/mL hemine (Sigma Co. US) solution, pH 7.2. The compounds (10 and 50 mg/mL) tested were previously solubilized in DMSO (Dimethyl sulfoxide), filtered in Millepore (0.2 mm) membrane and aseptically added to the tubes (0.1 mL/tube). In no-added control test tubes 0.1 mL of the solvent was added. Final volumes of T.(S) cruzi culture medium/tube was always 2.5 mL.Inocula consisted of 0.1 mL of a exponential growth phase culture which correspond to about 4.8 x 102 cells/mL. Cultures were incubated at 28C for 2-12 days. Growth was estimated with heamocytometer (Improved Double Neubauer). Cell motility and cellular integrity was observed by microscopical observation in fresh-smears.

The results obtained in these experiments were given in tables 1 and 2. Table 1 presents the effects of the well-known T. (S.) cruzi growth inhibitors drugs nifurtimox, benzonidazole, ketoconazole and primaquine1, on the epimastigote forms of the T. (S.) cruzi-like trypanosome used. It can be seen that, for all assayed drugs occurred a remarkable dose-dependent inhibitory effect reaching practically to a total parasite growth inhibition, at the end of the incubation time. Those results are agreements with the GABORAK et al.3 observation, and we can conclude that the trypanosome used appear to be a suitable model for the preliminary screen of anti-T. cruzi drugs, mainly in less-equipped laboratory in developing countries.

The activity of those pentacyclic triterpenes from A. populnea tested may be classified as follows; a) highly active: 20a-hydroxy-tingenone, b) less active: epikatonic acid; and c) inactive: populnilic acid and populninic acid (Table 2). Tingenone (Maitenine) other natural quinonoid5, that differs from 20a-hydroxy-tingenone only by the presence of a methyl instead of the hydroxyl group at 20-position, inhibited T. (S.) cruzi growth mainly by DNA double-strand intercalation mechanism17. Otherwise, the presence of a carboxylic group at the 20-position, such as in epikatonic acid, populnilic acid or populninic acid rises loss of activity, possible by difficulties in cross cell cytoplasmatic membrane. Frequently changes in critical radicals affect markedly the drug activity, such as in primaquine in which the 6-methoxy group was found to be responsible for the toxic effect, since analogues which have different substituents at this position are inactive against T. (S.) cruzi4.

Of those pentacyclic triterpenes obtained from A. populnea tested, 20a-hydroxytingenone show high activity; epikatonic acid less active and populninic acid and populnilic acid were inactive against the trypanosome used. In addiction the T. (S.) cruzi trypanosome from P. hastatus showed to be a good biological approach for T. cruzi screening of organic compounds.

 

RESUMO

Atividade anti-tripanosomicida de triterpenes pentacíclicos isolados de Austroplenckia populnea (Celastraceae)

Foram testados quatro triterpenos pentacíclicos isolados de Austroplenckia populnea e quatro compostos de conhecida atividade anti-T. cruzi ou anti-malárica. Dos triterpenos testados 20a-hidroxi-tingenona mostrou atividade elevada, ácido epicatônico foi menos ativo, enquanto ácido populnílico e populnínico foram inativos contra o tripanossoma do subgênero Schizotrypanum testado. Benzonidazole, nifurtimox, cetoconazole e primaquina apresentaram efeito inibitório dose-dependente atingindo praticamente a inibição total do crescimento do parasita no final do tempo de incubação. O tripanossoma testado mostrou ser um modelo adequado para uma seleção preliminar de compostos anti. T. (S.) cruzi.

 

 

ACKNOWLEDGEMENTS

The authors thank Conselho Nacional de Desenvolvimento Científico, Tecnológico (CNPq), Coordenação de Aperfeiçoamento do Pessoal de Ensino Superior (CAPES) and Fundação de Apoio a Pesquisa do Estado de Minas Gerais (FAPEMIG) for financial support.

 

REFERENCES

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2. CORREA, M.P. - Dicionário das plantas úteis do Brasil. Rio de Janeiro, Ministério da Agricultura, 1969. p. 152.         [ Links ]

3. GABORAK, M.; DARLING, J.L. & GUTTERIDGE, W.E. - Comparative drug sensitivities of culture forms of Trypanosoma cruzi and Trypanosoma dionisii. Nature (Lond.), 268: 339-340, 1977.         [ Links ]

4. GLOBE, F.C. - Activity of certain 6-Methoxy-8-aminoquinolines against Trypanosoma cruzi. Antibiot. and Chemother., 41: 265-270, 1985.         [ Links ]

5. GOIJMAN, S.G.; TURRENS, J.F.; MARINI-BETTOLO, G.B. & STOPPANI, A.O.M. - Effect of tingenone, a quinonoid triterpene, on growth and macromolecule biosynthesis on Trypanosoma cruzi. Experientia (Basel), 41: 646-648, 1985.         [ Links ]

6. GONZALEZ, J.G.; DELLE MONACHE, G.; DELLE MONACHE, F. & MARINI-BETTOLO, G.B. - Chuchuhuasha: a drug used in folk medicine in the Amazonian and Andean areas. A chemical study of Maytenus laevis. J. Ethnopharm., 5: 73-77, 1982.         [ Links ]

7. HOARE, C.A. - The trypanosomes of mammals: a zoological monography. Oxford, Blackwell, 1972.         [ Links ]

8. MONACHE, F.D. - Populnonic acid, a new triterpenic acid with friedelane carbon skeleton. Gazz. Chim. ital., 102: 636-645, 1972.         [ Links ]

9. PINTO, A.S.; RENAULT, C.P.; TAFURI, W.S. & CHIARI, E. - Trypanosoma cruzi-like trypanosomes isolated from the bat Phyllostomus hastatus. Mem. Inst. Oswaldo Cruz, 82(supl.): 53, 1987.         [ Links ]

10. PINTO, A.S.; TEIXEIRA, L.F.M.; SOUTO-PADRON, T. & ANDRADE, A.F.B. - Cell surface analysis of trypanosomes of the sub-genus Schizotrypanum isolated from bat. Parasitology, 3: 141-146, 1995.         [ Links ]

11. RAO, K.S. - Antibacterial activity of some medicinal plants of Papua New Guinea. Int. J. Pharmacognosy, 34: 223-225, 1996.         [ Links ]

12. SOUSA, J.R.; JANNOTTI, N.K.; SILVA, G.D.F. & PINHEIRO, J.A. - Friedelane triterpene related to populnonic acid. Gazz. Chim. ital., 118: 821-822, 1988.         [ Links ]

13. SOUSA, J.R.; SILVA, G.D.F.; PERDESOLI, J.L. & ALVES, R.J. - Friedelane and oleanane triterpenoids from bark wood of Austroplenckia populnea. Phytochemistry, 29: 3259-3261, 1990.         [ Links ]

14. TAYLOR, E.R.R. - Bureau of hygiene and tropical diseases, trypanosomiasis. Trop. Dis. Bull., 8: 3, 1986.         [ Links ]

15. TEIXEIRA, L.F.M.; GONÇALVES, A.M.; ROMANHA, A.J.; STEINDEL, M. & PINTO, A.S. - Schizodeme and zymodeme analysis of trypanosomes of the subgenus Schizotrypanum from the bat. Parasit. Res., 79: 497-500, 1993.         [ Links ]

16. VIEIRA FILHO, S.A.; DUARTE, L.P.; PAES, H.C.S. et al. - Antibacterial activity of pentacyclic triterpenes from Austroplenckia populnea. Acta Horticulturae, 501: 199-203, 1999.         [ Links ]

17. WORLD HEALTH ORGANIZATION – Control of Chagas disease. Report of a WHO Expert Committee. Wld. Hlth.Org. techn. Rep. Ser., (811): 27-31, 1991.         [ Links ]

 

Received: 17 July 2001

Accepted: 21 February 2002

 

 

(1) Núcleo de Estudo de Plantas Medicinais (NEPLAM), Departamento de Química, ICEx –UFMG, MG, Brasil.
(2) Departamento de Microbiologia, ICB, UFMG, Caixa postal 486, 31270-010 Belo Horizonte, MG, Brasil.
(3) DEFAR, Escola de Farmácia, UFOP.

Correspondence to: Artur da Silveira Pinto, E-mail address: dasilvei@mono.icb.ufmg.br 

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