Biological screening of Annonaceous braz
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 Silva1, 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 tested showed good larvicidal activity as compared to a reference compounds and literature data.
Key words: Annona, Annonaceae, brine shrimp larvicidal activity, acetogenins and alkaloids
j 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 (Correa, 1926; Leboeuf et al. 1982). From the phytochemical
viewpoint, plants from this family have been investigated intensively, motivated initially by the isolation of numerous 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 cytotoxic, antitumoral and pesticidal effects (Cavé et al.
1997), have stimulated phytochemical studies of some
genera of this family, especially Annona, Rollinia, Goniathalamus 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 activity 300 times stronger than that of taxol against L1210
murine leukemia (Ahammadsahib et al. 1993).
Our team has been investigating plants of the Annonaceae 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 secondary metabolites isolated were found to be acetogenins (Pimenta, 1995; Santos et al., 1996). The present paper reports on the screening of other parts of
A. crassiflora, using the brine shrimp lethality bioassay, 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 relatively 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 acetogenins as a new class of natural pesticides and active
antitumoral agents (McLaughlin et al. 1998).
0944-7113/03/10/02-03-209 $ 15.00/0
210
L. P. Santos Pimenta et al.
j 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 hypoglauca Mart. (wood) was collected in the Amazon region in March 1987. A voucher specimen was deposited
at Instituto de Pesquisa da Amazônia (INPA’s) herbarium, Manaus, Brazil.
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
fractions
xobtained
% dry wt.
Annona
crassiflora
Seeds
ACEHS
ACF01S
10.3
16.7
Leaves
ACEHL
ACF01L
ACF02L
ACF03L
ACF04L
18.7
41.0
0.2
4.5
40.1
Wood
ACF01W
ACF02W
ACF03W
8.6
0.6
8.0
Annona nutans
Seeds
ANF01S
ANF02S
ANF03S
ANF04S
15.0
8.9
1.4
5.8
Annona
hypoglauca
Wood
AHF01W
AHEBW
AHF05
AHF06
20.0
5.0
2.2
2.0
Annona
cherimola
Leaves
AChEHL
AChF01L
10.0
15.0
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 extracted 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. crassiflora 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 obtained. 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) fractions.
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. crassiflora (ACF01S and ACF01W), A. cherimola (AChF01L)
and A. mutans (ANF01S, ANF02S, ANF03S) showed a
positive test, characteristic of an α,β-unsaturated-γ-lactone moiety, found commonly with annonaceous acetogenins (Cavé et al. 1997). All extracts and fractions
were also analyzed using TLC plates sprayed with Dragendorff’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 dissolved 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. Controls containing 50 µl of DMSO in seawater were included 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% (LC50 value)
and 95% confidence intervals were calculated by Probit analysis (Finney, 1971).
Biological screening of Annonaceous Brazilian Medicinal Plants
211
Table 2. Brine-shrimp larvicidal activity of some extracts and fractions of Annonaceous
Brazilian plants.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Extracts and fractions tested
LC50 value in µg/ml
(95% confidence interval)
ACF01S (seeds) of A. crassiflora
ACEHS (seeds) of A. crassiflora
ACEHL (leaves) of A. crassiflora
ACF01L (leaves) of A. crassiflora
ACF01W (wood) of A. crassiflora
ACF02W (wood) of A. crassiflora
ACF03W (wood) of A. crassiflora
ACF02L (leaves) of A. crassiflora
ACF03L (leaves) of A. crassiflora
ANF01S (seeds) of A. nutans
ANF02S (seeds) of A. nutans
ANF03S (seeds) of A. nutans
ANF04 (seeds) of A. nutans
F01W (wood) of A. hypoglauca
EBW (wood) of A. hypoglauca
AHF05 (wood) of A. hypoglauca
AChEH (leaves) of A. cherimola
AChG01 (leaves) of A. cherimola
Lapachol
23.85 (2.93 < LC < 44.78)
754.8 (675.5 < LC < 834.6)
622.1 (540 < LC < 703.6)
ND1
1.29 (0.70 < LC < 1.87)
22.09 (9.75 < LC < 34.44)
28.50 (26.27 < LC < 30.70)
21.72 (16.78 < LC < 26.67)
2.73 (2.24 < LC < 3.22)
0.20 (0.19 < LC < 0.60)
1.42 (1.14 < LC < 1.69)
0.47 (0.35 < LC < 0.58)
1333 (954.4 < LC < 1714)
ND1
76.78 (13.88 < LC < 139.7)
27.76 (17.16 < LC < 45.28)
NT
0.88 (–27.09 < LC < 28.85)
68.09 (57.25 < LC < 79.17)
ND1 = LC > 500 µg/ml . NT = Not tested.
j Results and Discussion
j References
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 (LC50 value > 1000 µg/ml). LC 50 values
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 resulting 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 activity. These fractions showed positive test results for alkaloids. The crude extracts which showed positive tests
with Kedd’s reagents also showed good larvicidal activity, with the LC50 values ranging between 0.13 and 28.5
µg/ml. These results agree with the presence of annonaceous acetogenins and reveal these plants as potential
pesticidal and antitumoral agents.
Ahammadsahib KI, Hollingworth RM, McGovren JP, Hui
YH, McLaughlin JL (1993) Mode of action of bullatacin:
A potent antitumor and pesticidal annonaceous acetogenin. 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. Phytochemistry 21: 2783–2813
McLaughlin JL, Rogers LL, Anderson JE (1998) The Use of
Biological Assays to Evaluate Botanicals. Drug Information 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 sementes de Annona crassiflora objectivando o isolamento
de acetogeninas tetra-hidrofurânicas. PhD Thesis, UFMG,
Belo Horizonte, MG, p 268
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 helpful discussions.
212
L. P. Santos Pimenta et al.
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 Xylopia 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
j Address
L. P. Santos Pimenta, Departamento de Química –
ICEx – Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, 31270-901, Belo Horizonte, M.G.,
Brazil
Tel.: ++55-31-34995754; Fax: ++55-31-34995700;
e-mail: [email protected]
© 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 Silva1, 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 tested showed good larvicidal activity as compared to a reference compounds and literature data.
Key words: Annona, Annonaceae, brine shrimp larvicidal activity, acetogenins and alkaloids
j 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 (Correa, 1926; Leboeuf et al. 1982). From the phytochemical
viewpoint, plants from this family have been investigated intensively, motivated initially by the isolation of numerous 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 cytotoxic, antitumoral and pesticidal effects (Cavé et al.
1997), have stimulated phytochemical studies of some
genera of this family, especially Annona, Rollinia, Goniathalamus 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 activity 300 times stronger than that of taxol against L1210
murine leukemia (Ahammadsahib et al. 1993).
Our team has been investigating plants of the Annonaceae 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 secondary metabolites isolated were found to be acetogenins (Pimenta, 1995; Santos et al., 1996). The present paper reports on the screening of other parts of
A. crassiflora, using the brine shrimp lethality bioassay, 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 relatively 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 acetogenins as a new class of natural pesticides and active
antitumoral agents (McLaughlin et al. 1998).
0944-7113/03/10/02-03-209 $ 15.00/0
210
L. P. Santos Pimenta et al.
j 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 hypoglauca Mart. (wood) was collected in the Amazon region in March 1987. A voucher specimen was deposited
at Instituto de Pesquisa da Amazônia (INPA’s) herbarium, Manaus, Brazil.
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
fractions
xobtained
% dry wt.
Annona
crassiflora
Seeds
ACEHS
ACF01S
10.3
16.7
Leaves
ACEHL
ACF01L
ACF02L
ACF03L
ACF04L
18.7
41.0
0.2
4.5
40.1
Wood
ACF01W
ACF02W
ACF03W
8.6
0.6
8.0
Annona nutans
Seeds
ANF01S
ANF02S
ANF03S
ANF04S
15.0
8.9
1.4
5.8
Annona
hypoglauca
Wood
AHF01W
AHEBW
AHF05
AHF06
20.0
5.0
2.2
2.0
Annona
cherimola
Leaves
AChEHL
AChF01L
10.0
15.0
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 extracted 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. crassiflora 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 obtained. 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) fractions.
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. crassiflora (ACF01S and ACF01W), A. cherimola (AChF01L)
and A. mutans (ANF01S, ANF02S, ANF03S) showed a
positive test, characteristic of an α,β-unsaturated-γ-lactone moiety, found commonly with annonaceous acetogenins (Cavé et al. 1997). All extracts and fractions
were also analyzed using TLC plates sprayed with Dragendorff’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 dissolved 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. Controls containing 50 µl of DMSO in seawater were included 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% (LC50 value)
and 95% confidence intervals were calculated by Probit analysis (Finney, 1971).
Biological screening of Annonaceous Brazilian Medicinal Plants
211
Table 2. Brine-shrimp larvicidal activity of some extracts and fractions of Annonaceous
Brazilian plants.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Extracts and fractions tested
LC50 value in µg/ml
(95% confidence interval)
ACF01S (seeds) of A. crassiflora
ACEHS (seeds) of A. crassiflora
ACEHL (leaves) of A. crassiflora
ACF01L (leaves) of A. crassiflora
ACF01W (wood) of A. crassiflora
ACF02W (wood) of A. crassiflora
ACF03W (wood) of A. crassiflora
ACF02L (leaves) of A. crassiflora
ACF03L (leaves) of A. crassiflora
ANF01S (seeds) of A. nutans
ANF02S (seeds) of A. nutans
ANF03S (seeds) of A. nutans
ANF04 (seeds) of A. nutans
F01W (wood) of A. hypoglauca
EBW (wood) of A. hypoglauca
AHF05 (wood) of A. hypoglauca
AChEH (leaves) of A. cherimola
AChG01 (leaves) of A. cherimola
Lapachol
23.85 (2.93 < LC < 44.78)
754.8 (675.5 < LC < 834.6)
622.1 (540 < LC < 703.6)
ND1
1.29 (0.70 < LC < 1.87)
22.09 (9.75 < LC < 34.44)
28.50 (26.27 < LC < 30.70)
21.72 (16.78 < LC < 26.67)
2.73 (2.24 < LC < 3.22)
0.20 (0.19 < LC < 0.60)
1.42 (1.14 < LC < 1.69)
0.47 (0.35 < LC < 0.58)
1333 (954.4 < LC < 1714)
ND1
76.78 (13.88 < LC < 139.7)
27.76 (17.16 < LC < 45.28)
NT
0.88 (–27.09 < LC < 28.85)
68.09 (57.25 < LC < 79.17)
ND1 = LC > 500 µg/ml . NT = Not tested.
j Results and Discussion
j References
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 (LC50 value > 1000 µg/ml). LC 50 values
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 resulting 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 activity. These fractions showed positive test results for alkaloids. The crude extracts which showed positive tests
with Kedd’s reagents also showed good larvicidal activity, with the LC50 values ranging between 0.13 and 28.5
µg/ml. These results agree with the presence of annonaceous acetogenins and reveal these plants as potential
pesticidal and antitumoral agents.
Ahammadsahib KI, Hollingworth RM, McGovren JP, Hui
YH, McLaughlin JL (1993) Mode of action of bullatacin:
A potent antitumor and pesticidal annonaceous acetogenin. 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. Phytochemistry 21: 2783–2813
McLaughlin JL, Rogers LL, Anderson JE (1998) The Use of
Biological Assays to Evaluate Botanicals. Drug Information 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 sementes de Annona crassiflora objectivando o isolamento
de acetogeninas tetra-hidrofurânicas. PhD Thesis, UFMG,
Belo Horizonte, MG, p 268
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 helpful discussions.
212
L. P. Santos Pimenta et al.
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 Xylopia 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
j Address
L. P. Santos Pimenta, Departamento de Química –
ICEx – Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, 31270-901, Belo Horizonte, M.G.,
Brazil
Tel.: ++55-31-34995754; Fax: ++55-31-34995700;
e-mail: [email protected]