0944-7113/03/10/02-03-209 $ 15.00/0
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Introduction
Plants of the Annonaceae family are very important sources of edible fruits and material for perfumery, and are used in folk medicine in various capacities, such as antitumoral, parasiticidal and antidiarrhoeal agents (Cor-rea, 1926; Leboeuf et al. 1982). From the phytochemical viewpoint, plants from this family have been investigat-ed intensively, motivatinvestigat-ed initially by the isolation of nu-merous alkaloids (Leboeuf et al. 1982) and, afterwards, due to the detection of Annonaceous acetogenins. The structural diversity of this class of natural products, aside from their large range of biological activities, such as cy-totoxic, antitumoral and pesticidal effects (Cavé et al. 1997), have stimulated phytochemical studies of some genera of this family, especially Annona, Rollinia, Go-niathalamus and Uvaria. A good example of a powerful antitumoral agent is bullatacin, an acetogenin isolated from A. bullata A. Rich., which showed an in-vivo activ-ity 300 times stronger than that of taxol against L1210 murine leukemia (Ahammadsahib et al. 1993).
Our team has been investigating plants of the An-nonaceae family for ten years, focusing on the genera
Xylopia, Annona and Rollinia (Takahashi et al. 1995; Santos et al. 1996). Plants from these genera are native to Brazil, especially Minas Gerais state, where they are used popularly as an antidiarrheal agent, an insecticide, a parasiticide, and against snake bite (Correa, 1926). Annona crassiflora Mart seeds were subjected to bioassay-directed fractionation, and seven of the sec-ondary metabolites isolated were found to be aceto-genins (Pimenta, 1995; Santos et al., 1996). The pre-sent paper reports on the screening of other parts of A. crassiflora, using the brine shrimp lethality bioas-say, as well of three other species of the genus Annona used in traditional medicine in Brazil.
The brine shrimp lethality bioassay is an efficient, rapid and inexpensive test that requires only a relative-ly small amount of sample (2–20 mg). This bioassay has a good correlation with cytotoxic activity in some human solid tumors and with pesticidal activity, and has led to the discovery of the annonaceous aceto-genins as a new class of natural pesticides and active antitumoral agents (McLaughlin et al. 1998).
Phytomedicine 10: 209–212, 2003 © Urban & Fischer Verlag
http://www.urbanfischer.de/journals/phytomed
Phytomedicine
SHORT COMMUNICATION
Biological screening of Annonaceous Brazilian Medicinal
Plants using
Artemia salina
(Brine Shrimp Test)
L. P. Santos Pimenta, G. B. Pinto, J. A. Takahashi, L. G. F. e Silva
1, and M. A. D. Boaventura
Departamento de Química – ICEx – Universidade Federal de Minas Gerais, Belo Horizonte, M.G., Brazil
Summary
Eighteen different extracts from five Annona species collected in Minas Gerais state, Brazil, were submitted to the brine shrimp lethality test in order to detect potential sources of novel cytotoxic, antitumor, pesticidal and anti-Trypanosoma cruzi compounds. All of the Annonaceous species test-ed showtest-ed good larvicidal activity as compartest-ed to a reference compounds and literature data.
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Materials and Methods
Plant materials
Four plants were collected Annona crassiflora Mart. (seeds, leaves and wood) as collected in Itatiaiu˛cu, Minas Gerais, Brazil, March 1998. Annona nutans R. E. Fries (seeds) and Annona cherimola Mill. (leaves) were collected in Curvelo, Minas Gerais, Brazil, from June to August, 1998. Voucher specimens were deposited at the Instituto de Ciências Biológicas Herbarium (BHCB), UFMG, Belo Horizonte, MG, Brazil. Annona hypo-glauca Mart. (wood) was collected in the Amazon re-gion in March 1987. A voucher specimen was deposited at Instituto de Pesquisa da Amazônia (INPA’s) herbari-um, Manaus, Brazil.
Preparation of the crude extracts
Plant parts were dried at 40 °C and extracted at room temperature with solvents, which were removed in vacuo to yield the crude extracts. Seeds and leaves of A. crassiflora, A. mutans and A. cherimola were ex-tracted successively and exhaustively using hexane (EH) and ethanol (F01), yielding the hexanic (ACHS, ACEHL, AChEHL) and ethanolic (ACF01S, ACF01L, ANF01S, AchF01L) extracts. The wood of A. crassi-flora was extracted only with ethanol (ACF01W). The wood of A. hypoglauca was extracted using benzene (AHEB) and ethanol (AHF01W). The parts of plants used in each case and the yields in % dry wt. are shown in Table 1. Ethanolic extracts of leaves and wood of A. crassiflora (ACF01L and ACF01W) and seeds of A. mutans were dissolved in ethanol/water (7:3) and extracted successively with hexane and chloroform. After solvent removal, the hexanic (F02), chloroformic (F03) and hydroalcoholic (F04) fractions were ob-tained. The benzenic extract of wood of A. hypoglauca (AHEB) was partitioned between CH2Cl2 and water, and the CH2Cl2 layer was concentrated in vacuo to yield the fraction AHF03. This fraction was partitioned between hexane and MeOH · H2O (9:1), furnishing the hydroalcoholic (AHF05) and hexanic (AHF06) frac-tions.
Chromatography analysis
All extracts and fractions were submitted to analytical TLC analysis. The plates were sprayed with Kedde’s reagent and the extracts and fractions from A. crassiflo-ra (ACF01S and ACF01W), A. cherimola (AChF01L) and A. mutans (ANF01S, ANF02S, ANF03S) showed a positive test, characteristic of an α,β-unsaturated-γ-lac-tone moiety, found commonly with annonaceous aceto-genins (Cavé et al. 1997). All extracts and fractions were also analyzed using TLC plates sprayed with Dra-gendorff’s reagent. All of them showed positive spots,
indicating the presence of alkaloids (Wagner et al. 1984).
Biological screening
Artemia salina encysted eggs (10 mg) were incubated in 100 ml of seawater under artificial light at 28 °C, pH 7–8. After incubation for 24 h, nauplii were collected with a Pasteur piipette and kept for an additional 24 h under the same conditions to reach the metanauplii stage. The samples (triplicate) to be assayed were dis-solved in DMSO (dimethylsulfoxide) (2 mg/400 µl or 2 mg/1000 µl) and diluted serially (10, 20, 30 and 50 µl/5 ml) in seawater. About 10–20 nauplii were added to each set of tubes containing the samples. Con-trols containing 50 µl of DMSO in seawater were in-cluded in each experiment. Lapachol dissolved in DMSO was used as a positive control. Twenty-four hours later, the number of survivors was counted, recorded and the lethal concentration 50% (LC50value) and 95% confidence intervals were calculated by Pro-bit analysis (Finney, 1971).
210 L. P. Santos Pimenta et al.
Table 1. Crude extracts and fractions from solvent partition
(quantity obtained from 100 g of dried plant material, % dry wt.).
Plant names Part used Extract and % dry wt. fractions
xobtained
Annona Seeds ACEHS 10.3
crassiflora ACF01S 16.7
Annona nutans Seeds ANF01S 15.0
ANF02S 8.9 ANF03S 1.4 ANF04S 5.8
Annona Wood AHF01W 20.0
hypoglauca AHEBW 5.0
AHF05 2.2 AHF06 2.0
Annona Leaves AChEHL 10.0
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Results and Discussion
The majority of the extracts and fractions tested showed good brine shrimp larvicidal activity according to Meyer et al. (1982), who classified crude extracts and pure substances into toxic (LC50 value < 1000 µg/ml) and non-toxic (LC50value > 1000 µg/ml). LC50values for the annonaceous extracts are reported in Table 2. A. nutans proved to be the most larvicidal sample and its whole extract was more active than its fractions result-ing from solvent extractions. In contrast, the ethanolic extracts of A. crassiflora leaves did not show activity, but the fractions resulting from solvent extraction (ACF02L and ACF03L) showed good larvicidal activi-ty. These fractions showed positive test results for alka-loids. The crude extracts which showed positive tests with Kedd’s reagents also showed good larvicidal activ-ity, with the LC50values ranging between 0.13 and 28.5 µg/ml. These results agree with the presence of annona-ceous acetogenins and reveal these plants as potential pesticidal and antitumoral agents.
Acknowledgment
This work was supported by FAPEMIG and PRPq/UFMG, which also granted a fellowship to G. V. Pinto. The authors are also grateful to Dr. Jo˜ao Máximo de Síqueira for his help-ful discussions.
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References
Ahammadsahib KI, Hollingworth RM, McGovren JP, Hui YH, McLaughlin JL (1993) Mode of action of bullatacin: A potent antitumor and pesticidal annonaceous aceto-genin. Life Sci 53: 1113–1120
Cavé A, Cortes D, Figadère B, Laurens A, Pettit GR (1997) In: Progress in the Chemistry of Organic Natural Products. Springer-Verlag Wien, Austria, pp 81–288
Correa Pio (1926) Dicionário de Plantas Utéis e Espécies Exóticas e Cultivadas, Rio de Janeiro
Finney DJ (1971) Probit Analysis. Cambridge University Press. Cambridge
Leboeuf M, Cavé A, Bhaumik PK, Mukherjee B, Bukherjee R (1982) The Phytochemistry of Annonaceae. Phytochem-istry 21: 2783–2813
McLaughlin JL, Rogers LL, Anderson JE (1998) The Use of Biological Assays to Evaluate Botanicals. Drug Informa-tion Journal 32: 513-524
Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL (1982) Brine Shrimp: a convenient general bioassay for active plant constituents. Planta Med 45: 31–34
Pimenta LPS (1995) Estudo Químico Bio-monitorado das se-mentes de Annona crassiflora objectivando o isolamento de acetogeninas tetra-hidrofurânicas. PhD Thesis, UFMG, Belo Horizonte, MG, p 268
Biological screening of Annonaceous Brazilian Medicinal Plants 211 Table 2. Brine-shrimp larvicidal activity of some extracts and fractions of Annonaceous
Brazilian plants.
Extracts and fractions tested LC50value in µg/ml
(95% confidence interval)
1 ACF01S (seeds) of A. crassiflora 23.85 (2.93 < LC < 44.78) 2 ACEHS (seeds) of A. crassiflora 754.8 (675.5 < LC < 834.6) 3 ACEHL (leaves) of A. crassiflora 622.1 (540 < LC < 703.6) 4 ACF01L (leaves) of A. crassiflora ND1
5 ACF01W (wood) of A. crassiflora 1.29 (0.70 < LC < 1.87) 6 ACF02W (wood) of A. crassiflora 22.09 (9.75 < LC < 34.44) 7 ACF03W (wood) of A. crassiflora 28.50 (26.27 < LC < 30.70) 8 ACF02L (leaves) of A. crassiflora 21.72 (16.78 < LC < 26.67) 9 ACF03L (leaves) of A. crassiflora 2.73 (2.24 < LC < 3.22) 10 ANF01S (seeds) of A. nutans 0.20 (0.19 < LC < 0.60) 11 ANF02S (seeds) of A. nutans 1.42 (1.14 < LC < 1.69) 12 ANF03S (seeds) of A. nutans 0.47 (0.35 < LC < 0.58) 13 ANF04 (seeds) of A. nutans 1333 (954.4 < LC < 1714) 14 F01W (wood) of A. hypoglauca ND1
15 EBW (wood) of A. hypoglauca 76.78 (13.88 < LC < 139.7) 16 AHF05 (wood) of A. hypoglauca 27.76 (17.16 < LC < 45.28) 17 AChEH (leaves) of A. cherimola NT
18 AChG01 (leaves) of A. cherimola 0.88 (–27.09 < LC < 28.85) 19 Lapachol 68.09 (57.25 < LC < 79.17)
Santos LP, Boaventura MAD, Sun N-J, Cassady JM, Oliveira AB (1996) Araticulin, a bis-tetrahydrofuran polyketide from Annona crassiflora seeds. Phytochemistry 42: 705 Takahashi JA, Boaventura MAD, Bayma JC, Oliveira AB
(1995) Frutoic acid, a dimeric kaurane diterpene from
Xy-lopia frutescens. Phytochemistry 40: 607–609
Wagner H, Bladt S, Zgainski EM (1984) Alkaloid Drugs. In: Plant Drug Analysis A Thin layer Chromatography Atlas. Springer-Verlag Berlin Heidelberg, pp 51–92
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Address
L. P. Santos Pimenta, Departamento de Química – ICEx – Universidade Federal de Minas Gerais, Av. An-tônio Carlos, 6627, 31270-901, Belo Horizonte, M.G., Brazil
Tel.: ++55-31-34995754; Fax: ++55-31-34995700; e-mail: lpimenta@dedalus-lcc.ufmg.br
