Brine shrimp lethality assay as a prescreening system for anti-Trypanosoma cruzi activity

Phytomedicine Vol. 2 (1), pp. 47-50,1995 © 1995 by Gustav Fischer Verlag, Stuttgart· Jena . New York

Brine shrimp lethality assay as a prescreening system for anti-Trypanosoma cruz; activity C. L. ZANI1, P. P. G. CHAVES1, R. QUEIROZ1, A. B. DE OLiVEIRA2, J. E. CARDOS02, A. M. G. ANJOS3 and T. S. M. GRANDI3 Laborat6rio de Quimica de Produtos Naturais, Centro de Pesquisas "Rene Rachou" -FIOCRUZ. Cx. Postal 1743, CEP 30190-002, Belo Horizonte, MG - Brazil. 2 Faculdade de Farrnacia, Univ. Federal de Minas Gerais. Av. Olegario Maciel 2360, CEP 30.180, Belo Horizonte, MG - Brazil. 3 Departamento de Botanica, Universidade Federal de Minas Gerais, Pampulha, CEP 30.000, Belo Horizonte, MG - Brazil. 1

Summary With the aim of finding an acceptable method for selecting plant extracts to be assayed against the infective blood form of Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanosomiasis), two different strategies were compared: a) screening only medicinal species and b) pre-screening random collected species in the brine shrimp lethality assay (BSLA). Fiftytwo plants belonging to the Asteraceae family, including eighteen medicinal species, were collected and their ethanol extracts assayed against both T. cruzi and Artemia salina (brine shrimp). The proportion of trypanocidal extracts among the medicinal species and among the random collection did not differ significantly. On the other hand, the proportion of trypanocidal extracts among those that presented LCso of less than 100 ppm to A. salina was four times higher than among the medicinal species. Keywords: Asteraceae, Trypanosoma cruzi, Chagas' disease, trypomastigote, brine shrimp, Artemia salina.

Introduction Chagas' disease (American trypanosomiasis) is caused by the protozoan Trypanosoma cruzi and afflicts more than 16 million people in South-America (WHO, 1992). With the control of the insect vector population, transfusion with infected blood is becoming the major mode of transmission of this disease (Dias, 1993). Beside donor selection, chemosterilization of the blood for transfusion with gentian violet (Nussenzweig et aI., 1953) is the only prophylactic measure available. However, due ro some undesired effects its use is in decline and a suitable substitute is needed. An accessible approach for drug discovery in developing countries is the screening of the local flora using appropriate bioassays. However, the lack of ethnopharrnacological information directly related to Chagas' disease and the limited data on potentially useful structural class of natural

products make difficult a rational design of a screening program. Furthermore, manipulation of the infective trypomastigote form of T. cruzi is risky and its maintenance expensive, inhibiting the use of bioassays with this parasite. The aim of the present work is to find a good procedure to reduce the number of extracts ro be assayed in the complex assay with the infective trypomastigote form of T. cruzi. Using fifty-two ethanol extracts obtained from Brazilian Asteraceae species, two different strategies were compared: a) screening of medicinal species only (random vs. ethnomedical approach) and b) pre-screening with a surrogate assay. Among the possible candidates to serve as a pre-screening assay, the brine shrimp (Artemia salina Leach) lethality assay (BSLA) was chosen because of its simplicity and ability to correlate well with other important biological models (McLaughlin, 1991).

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Table 1. List of Asteraceae species used in the present study, indicating the results of the bioassays with Artemia salina (BSLA) and Trypanosoma cruzi trypomastigotes, entry' BHCBb species

BSLN

1.* 19056 Acanthospermum australe (Loef.) Kant 2.* 19017 Achyrocline alata DC. 3." 19067 Achyroclinesatureoides (Lam.) DC. + 4.* 19031 Ageratum conyzoides L. 5. 19043 Alomia myriadenia Baker + 6. 19019 Aspiliaserrulata Baker 7.* 19048 Baccharis dracunculifolia DC. 8. 19063 Baccharis platypoda DC. 9.* 19077 Baccharis trtmera (L.) DC 10. 19062 Baccharis trinervis Pers. 11. 19028 Bidensgardneri Baker 12.* 19014 BidenspilosaL. 13. 19023 Bidens rubifolius HBK 14. 19052 Blainvil/ea biaristata DC. 15. 19045 Cetratherum punctatum Casso 16.* 19046 Cosmos sulfureus Cav. 17. 19076 Dasyphyl/um macrocephala Baker 18. 19037 Eclipta paniculata Kuntz 19.* 19032 Elephantopusmol/is HBK 20.* 19034 EmiliasonchifoliaDC. 21. 19073 Eremanthus glomerulatus Less. 22. 19058 Eremanthusspbaerocephalus Baker 23. 19029 Eupatorium aff. capilare DC. 24. 19065 Eupatorium amphidictyum DC. 25. 19051 Eupatorium bupleurifoliumDC. 26.* 19050 Eupatorium laeuigatum Lam. 27.* 19022 Eupatorium squalidum DC. 28.* 19078 Gochnatia polymorpha (Less.] Cabr. 29. 19040 [aegeria hirta (Lag.) Less. 30.* 19057 ]ungia floribunda Less. 31. 19020 Lynchnophora pinasterMart. 32.* 19047 Mikania cordifolia (L.f.) Wild. 33. 19061 Mikania lutzelburghii Mattf. 34. 19069 Mikania obtusata DC. + 35. 19060 Mikania sessilifolia DC. + 36." 19036 Pluchea quitoc L. 37.* 19030 Porophillum ruderale (Jacq.) Casso 38. 19025 Pterocaulon alopecuroides (Lam.) DC. 39. 19035 Senecio confusus Britten 40. 19064 Stenocline chionaea DC. 41. 19059 Symphyopappus polystachyus Baker 42." 19070 Taraxacum officinale (With.) Wiggers43. 19026 Tithonia rotundifolia Baker 44. 19072 Trixis uauthieri DC. 45. 19016 Verbesina clausseni Sch.-Bip. 46. 19027 Vernonia ammophila Gardn. 47. 19021 Vernonia crotonoides (DC.) Sch.-Bip. 48. 19066 Vernonia linearis Spreng. 49. 19053 Vernonia remotiflora Rich. 50. 19042 Vernonia varroniaefolia DC. 51. 19075 Viguiera ouatifolia Baker + 52. 19033 Wedelia paludosa DC.

Materials and Methods

Plant material Fifty-two herbaceous species belonging to the Asteraceae (Table 1) were collected in the vicinities of Belo Horizonte, Minas Gerais State, Brazil, from 1988 to 1990. The plants were authenticated and voucher specimens deposited at the Federal University of Minas Gerais Herbarium (see voucher number in Table 1). The aerial parts of the plants were dried in the shade, ground in a knife mill or in a homogenizer and extracted twice (24 h) with ethanol at room temperature. The solvent was removed by rotary evaporation under reduced pressure at temperatures below 45 "C. The resulting crude extracts were kept in the dark in a freezer (-20 "C) until assayed. Immediately before running the bioassay, sufficient amounts were transferred to a vial, and the residual solvent was removed under high vacuum.

Brine shrimp lethality assay (BSLA)

+

An established protocol (McLaughlin, 1991) was employed. Briefly, the crude extracts were assayed at 1000, 100 and 10 ppm, using 10 second instar larvae of the brine shrimp (Artemia salina, Leach) in triplicate. After 24 h contact at room temperature, the number of surviving organisms was recorded and the LC so calculated using a computer program kindly supplied by Dr. J. L. McLaughlin. The extracts presenting LC so values of less than 100 ppm were considered active and are indicated in Table 1 with a plus sign.

Biologicalassay with trypomastigotes +

+

+

Blood infected with trypomastigotes of T. cruzi strain was obtained by cardiac puncture of experimentally infected Swiss albino mice and diluted with normal murine blood to 2 x 10 6 trypomastigotes ml:'. Stock solutions were prepared by dissolving 2.5 mg of each extract in 1.0 ml of dimethyl sulfoxide. This solution (10 ~.L1) was added to the wells of a microplate containing 90).11 of infected blood, producing a final concentration of 250 ug/ml, Controls with dimethyl sulfoxide (DMSO) (10%) and gentian violet (250 ug/rnl] were run in parallel. After 24 h at 4 °C the trypanocidal activity was evaluated microscopically by counting 50 fields at 400x magnification using 5).11 of blood and a 22 x 22 mm coverslip (Brener, 1962). Those extracts that reduced the parasitemia by more than 70% when compared with the control with DMSO were considered active and are indicated in Table 1 with a plus sign.

+

a The asterisk in the entry number indicates medicinal species (see Table2). b BHCB: Herbarium of Departamento de Botanica,Instituto de Ciencias Biologicas, Universidade Federalde Minas Gerais, Brazil; C Brine shrimp lethality assay: + means LCso of less than

100 ppm; d Assay with the trypomastigote form of T. cruzi: + means more than 70% reduction in the number of parasites in infected blood.

Brine shrimp lethality assay as a pre-screening system for anti-Trypanosoma cruzi activity

Results Among the fifty-two Asteraceae species collected for this study, eighteen (35%) are reported as being medicinal. They are indicated with an asterisk in Table 1 while their traditional uses are summarized in Table 2. All extracts were evaluated for their activity against the trypomastigote blood form of T. cruzi and the nauplii of Artemia salina. Table 1 shows that five of them (10%) reduced the number of T. cruzi in the blood by more than 70% and were considered trypanocidal. Five (10%) presented an LCso of less than 100ppm and were considered toxic to A. salina. Eleven percent of the eighteen medicinal species were trypanocidal while 9% among the non-medicinal were considered active against T. cruzi. From the five extracts that were toxic to A. salina, two (40%) were also active against the protozoan T. cruzi (Ta-

bid).

Table 2: Medicinal use of Asteraceae species collected in this study Species

use"

Acanthospermum australe

diarrhea, fever

(Loef.) Kunt.

Achyrocline alata DC. Achyroclinesatureoides (Lam.) DC.

Ageratum conyzoides L. Baccharis dracunculifolia DC. Baccharis trimera (L.) DC. Bidens pilosa L. Cosmos sulfureus Cav. Elepbantopusmol/is HBK Emiliasonchifolia DC. Eupatorium laevigatum Lam. Eupatorium squalidum DC. Gochnatia polymorpha (Less.) Cabr. fungia {loribunda Less. Mikania cordifolia (L.f.) Wild.

Discussion One third of the plants collected are described as medicinal, confirming previous findings of several surveys of medicinal plants from different regions in Brazil where plants from the Asteraceae appear in high proportions (Cervi er al., 1989; Gavilanes et al., 1982; Silberbauer-Gottsberger, 1982). The results show that the proportion of active extracts among those toxic to A. salina (40%) is approximately four times higher than that observed for the medicinal group (11 %), meaning that there is a higher probability of finding trypanocidal extracts within those that are toxic to brine shrimp than within the medicinal plants. This analysis is corroborated by comparing the calculated kappa, the inter-ratter agreement index (Fleiss, 1981), for trypanocidal activity vs. ethnomedical information and trypanocidal activity vs. brine shrimp toxicity. A kappa index of only 0.45 for the first case indicates that ethnomedical information is poorly correlated with the trypanocidal activity. On the contrary, a calculated kappa of 0.8 expresses an excellent degree of agreement beyond chance between trypanocidal activity and brine shrimp toxicity. Although a small number of plants were examined, with only a small proportion being trypanocidal, the data suggest that a preliminary screening of a random collection using A. salina could reduce by 90% the number of extracts that would have to be assayed with T. cruzi. Interestingly, a bioassay-guided fractionation of the ethanol extract of Mikania obtusata using T. cruzi and A. salina (Alves et al., 1994) yielded the diterpene ent-kaur-16en-19-oic acid as the toxic principle to both organisms (LCso of 1.6 mM against T. cruzi and 6.6 mM against A. salina). The toxic effect of this compound against A. salina was previously described by Hui et al. (1989). This

49

Pluchea quitoc L. Poropbillum ruderale (Jacq.) Casso

Taraxacum officinale (With.) Wiggers

stomachic, sedative antispasmodic, antinflammatory, antibacterial tonic, diuretic, fever, cold tonic, febrifuge, stimulant diabetes, febrifuge, leprosy, anthelmintic hepatitis, diabetes, vermifuge hepatitis astringent, febrifuge, diuretic anti-asthmatic, febrifuge, antiseptic antiseptic, resolutive bequic, emollient lung affections edema, warts bequic, vulnerary, snake bites, rheumatism hepatitis sedative, diaphoretic, vulnerary febrifuge, diuretic, tonic, rheumatism

Source: Anais do VII Simposio de Plantas Medicinais do Brasil. Oreades 8 (14-15): 15-559, 1982; Correa, M,.P. Dicionario das Plantas Dteis do Brasil e das Exoticas Cultivadas. Vol. 1-6, 1984. Ministerio da Agricultura-IBDF, Rio de Janeiro.

a

Table 3. Proportion of trypanocidal extracts among the random and medicinal species. Group

Number of species

Number of trypanocidal extracts

Proportion

random medicinal

34 18

3 2

9% 11%

Fisher exact test

p=1

finding indicates that, at least with Asteraceae species, there is a good possibility that the BSLA could be used both in a preliminary screen and to monitor the fractionation of trypanocidal extracts. However, in order to validate this approach, results obtained from larger plant collections and fractionation of other extracts toxic to both organisms will have to be carried out. Acknowledgements We are grateful to Dr. J. L. McLaughlin, Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy, Pardue University, for the computer program to calculate the LC 50 values for the BSLA.

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References Alves, T. M. A., Chaves, P. P. G., Nagem, T.]., Murta, S. M. E, Ceravolo, I. P., Romanha, A.]. and Zani, C. L.: Trypanocidal comp onents from Mikania obtusata D. C. Planta Medica 61:

85-86, 1995. Brener, Z.: Therapeutic activity and criterion of cure on mice experiment ally infected with Trypanosoma cruzi. Rev. Inst. Med. Trap. Sdo Paulo 4: 389-396, 1962. Cervi, A: c., Negrelle, R. R. B. and Sbalchiero, D.: Especies vegetars utilizadas na terapeutica popular no municipio de Curitiba ' Parana, Brasil. Estudos de Biologia (Curitiba) 5-4 2, 1989. Dias, ]. C. P.: Transfusional Chagas disease in Brazil. Mem. Inst. Oswaldo Cruz 88: (suppl), 63-65, 1993. Fleiss, J. L.: Statistical Methods for Rates and Proportions. John Wiley, New York, 1981. Gavilanes, M. L., Brandao, M. and Cardoso, c.: Levantamento das plantas daninhas utilizadas como medicinais, de uso popular. Oreades (Belo Horiz ont e) 8: 34-47,1982. Hui, Y.-H., Rupprecht, J. K., Liu, Y. M., Anderson, J. E., Smith, D. L., Chang, C.-]. and McLaughlin, J. L.: Bullatacin and bullatacinone: Two highly potent bioactive acetogenins from Annona bullata. ]. Nat. Prado 52: 463-4 77,1 989.

McLaughlin , ]. M.: Crown gall tum ors on potato discs and brine shrimp le~hali~: two simple bioassays for higher plant screening and fractionation. In, Methods ill Plant Biochemistry, vol. 6, Ed. K. Ho stettmann , pp. 1-32. Academic Press, San Diego CA

1991.

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Nussenzweig, V., Sonntag, R., Freitas, J. L. P. d., Neto, V.A., Biancalana, A. and Kloetzel, J.: Acdo da violeta de genciana sobre 0 T. c~uzi in vitro: sua importancia na esterilizacao do sangue destinado a transfusao. Rev. Paul. Med. 42: 85-86, 1953. Silberbauer-Gottsberger, I.: 0 cerrado como poten cial de plant as medicinais e tocicas. Oreades (Bela Horizonte) 8, 15-30, 1982. WHO: New Initiati ve will halt Chagas disease in six years. TDR Ne ws 3-5, 1992.

Address C. L. Zani, Laborat6rio de Quimica de Produtos Naturais, Centro de Pesquisas " Rene Rachou "-Fiocruz. Cx. Posta11743, CEP 30190-002, Belo Horizonte, MG - Brazil.