Trypanocidal constituents in plants: 7. Mammea-type coumarins

Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 103(5): 431-436, August 2008 431 Trypanocidal constituents in plants. 7. Mammea-type coumarins Ricardo Reyes-Chilpa/+, Elizabeth Estrada-Muñiz1, Elisa Vega-Avila2, Fumiko Abe3, Junei Kinjo3, Simón Hernández-Ortega Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, México, 04510 DF, México Sección Externa de Toxicologia, CINVESTAV-IPN, México, DF, México 2Departamento de Ciencias de la Salud, Universidad Autónoma Metropolitana, México, DF, México 3Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan 1 Calophyllum brasiliense and Mammea americana (Clusiaceae) are two trees from the tropical rain forests of the American continent. A previous screening showed high trypanocidal activity in the extracts of these species. Several mammea-type coumarins, triterpenoids and biflavonoids were isolated from the leaves of C. brasiliense. Mammea A/ AA was obtained from the fruit peels of M. americana. These compounds were tested in vitro against epimastigotes and trypomastigotes of Trypanosoma cruzi, the etiologic agent of Chagas disease. The most potent compounds were mammea A/BA, A/BB, A/AA, A/BD and B/BA, with MC100 values in the range of 15 to 90 μg/ml. Coumarins with a cyclized γ,γ-dimethylallyl substituent on C-6, such as mammea B/BA, cyclo F + B/BB cyclo F, and isomammeigin, showed MC100 values > 200 μg/ml. Several active coumarins were also tested against normal human lymphocytes in vitro, which showed that mammea A/AA and A/BA were not toxic. Other compounds from C. brasiliense, such as the triterpenoids, friedelin, canophyllol, the biflavonoid amentoflavone, and protocatechuic and shikimic acids, were inactive against the epimastigotes. The isopropylidenedioxy derivative of shikimic acid was inactive, and its structure was confirmed by X-ray diffraction. Our results suggest that mammea-type coumarins could be a valuable source of trypanocidal compounds. Key words: Trypanosoma cruzi - Calophyllum brasiliense - Mammea americana - coumarins - Chagas disease The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease (American trypanosomiasis), which affects 16-18 million people in Latin America and is responsible for the deaths of more than 45,000 patients per year (WHO 1997). It is transmitted to humans by triatomine bugs or through infected blood transfusions (Pizzolatti et al. 2002). The life cycle of T. cruzi is complex, and it alternates between vertebrate and invertebrate hosts. When the triatomine bug is feeding on human blood, it defecates and releases metacyclic trypomastigotes near the bite wound. This infective agent is capable of reaching intracellular locations and transforming into amastigotes. After reproduction, the amastigotes turn back into trypomastigotes, and the cells burst. The released parasites can either infect other cells or be consumed by other triatomine bugs. In the intestine of an invertebrate host, the blood trypomastigotes transform into epimastigotes, which divide and give rise to infective metacyclic-trypomastigotes (Brener 1973, Contreras et al. 2002). Medication for Chagas disease is usually effective when given during the acute stage of infection. Once the disease has progressed to later stages, no medication has proven to be completely effective. Moreover, synthetic drugs such as nifurtimox, a 5-nitrofuran, and + Corresponding author: Received 20 March 2008 Accepted 21 July 2008 benznidazole, a 2-nitro-imidazole (Lampit®, Bayer, and Rochagan®, Roche, respectively) are used for the treatment of this disease. Unfortunately, both drugs have limited efficacy during the chronic phase of the disease and present several undesirable side effects (Gutteridge 1985, Umezawa et al. 2001, Coura & Castro 2002). Chemoprophylatic measures against Chagas disease are scarce, present variable efficacy, or are not adopted due to their costs. Currently, gentian violet is used to sterilize blood banks in endemic areas (Pizzolatti et al. 2002, Coura & Castro 2002). An urgent need to develop new drugs exists, and natural products can provide useful drug leads as well as the scientific basis for the rational use of medicinal plants. Calophyllum brasiliense Camb. is a large tree, measuring up to 40 m in height, from the tropical rain forest. It is distributed throughout Latin America, from Brazil to Mexico, and is used in folk medicine. In Mexico, its most common name is bari, and the infusion of the cortex is consumed by women over nine days for “cleaning” the womb after child-birth, while the seeds provide an oil used for lighting and for healing skin afflictions (Soto & Sousa 1995). In Colombia, the peasants call this tree árbol de aceite (oil tree), and the yellow latex exuded from the cortex is applied topically for healing the navels of newborn children (García-Barriga 1992). In Brazil, it is known as guanandi, and has been used in folk medicine for the treatment of rheumatism, varicose hemorrhoids and chronic ulcers (Corrêa 1978). In the Amazon basin, the bark infusion is used by the Karaja ethnic group for the treatment of diarrhea, and in Guyana, C. brasiliense is mixed with Coutarea hexandra to treat diabetes and worms (Mesía-Vela et al. 2001). online | memorias.ioc.fiocruz.br 432 Trypanocidal coumarins • R Reyes-Chilpa et al. C. brasiliense heartwood contains xanthones with trypanocidal (Abe et al. 2002) and antifungal (ReyesChilpa et al. 1997) properties. The methanol extract from the leaves contains hyperin (hyperoside), amentoflavone, quercetin, gallic acid and protocatechuic acid (da Silva et al. 2001). The analysis of the hexane extracts of the leaves has demonstrated the existence of two chemotypes of C. brasiliense in Mexico. Only the organic extracts from C. brasiliense chemotype 1 showed trypanocidal activity during preliminary screening (Abe et al. 2002). The leaves of this chemotype contain mammea-type coumarins that were cytotoxic to human tumor cells in vitro (Reyes-Chilpa et al. 2004). The mixture of two of these coumarins, mammea A/BA and A/ BB, was better than vincristine at inhibiting the growth of implanted tumors in mice (Ruiz-Marcial et al. 2007). The leaves of chemotype 2 contain chromanones and triterpenoids as the major constituents, and tetracyclic dipyranocoumarins in low concentrations, which inhibit the reverse transcriptase of the human immunodeficiency virus type 1 in vitro (Huerta-Reyes et al. 2004). It has recently been reported that (-) -mammea A/BB from the leaves of C. brasiliense showed potent activity against the promastigote and amastigote forms of Leishmania (L.) amazonensis (Brenzan et al. 2007). Mammea A/BB is also known to be molluscicidal against Biomphalaria glabrata (Gasparotto et al. 2005). Mammea americana L. is a tree measuring up to 20 m in height from the tropical rain forest. Its original distribution was through the Antilles Islands and South America, but it was p (...truncated)