Print version ISSN XOn-line version ISSN X
Rev. bras. farmacogn. vol.18
suppl.0 Jo?o Pessoa Dec. 2008
http://dx.doi.org/10.-695X2
Screening for antifungal,
DNA-damaging and anticholinesterasic activities of Brazilian plants from the
Atlantic Rainforest - Ilha do Cardoso State Park
Avalia&&o das atividades antif&ngica,
no reparo do DNA e anticolinester&sica de plantas brasileiras da Mata
Atl&ntica - Parque Estadual da Ilha do Cardoso
Elaine Monteiro Cardoso-LopesI;
Rosana Cristina CarreiraI; D&bora Gomes AgripinoI;
Luce Maria Brand&o TorresI; In&s CordeiroI;
Vanderlan da Silva BolzaniII; S&nia Machado de Campos DietrichI;
Maria Claudia Marx YoungI,
IInstituto de Bot&nica, CP 3005,
0 S&o Paulo-SP, Brazil
IIInstituto de Qu&mica, Universidade Estadual Paulista, CP
355, 1 Araraquara-SP, Brazil
Crude extracts from 17 plant species collected
from an Atlantic Forest region in the State of S&o Paulo (Brazil) have
been screened for antifungal, DNA-damaging and acetylcholinesterase inhibiting
activities. Of the 34 extracts obtained from leaves and stems of plants assayed
for antifungal activity with Cladosporium sphaerospermum and C. cladosporioides
26.5% were active. However, only the extract of leaves of Cabralea canjerana
showed a strong inhibition of both fungi. The DNA-damaging assay with mutant
strains of Saccharomyces cerevisiae resulted in 11.7 % of the extracts
being active whereas 100% of them showed selectivity for the DNA-repair mechanism
of topoisomerase II. Of the 17 species analysed, 12 showed anticholinesterasic
activity in TLC assay. However, only extracts from Tetrastylidium grandifolium
(stems) and Sloanea guianensis (leaves and stems) inhibited acetylcholinesterase
activity more than 50% in quantitative assay.
Keywords: Atlantic Rainforest, antifungal
activity, acetylcholinesterase inhibitors, plant screening.
Extratos brutos de 17 esp&cies de plantas
coletadas em regi&o de Mata Atl&ntica no Estado de S&o Paulo
(Brasil) foram avaliadas para as atividades: antif&ngica, no reparo do
DNA e inibi&&o da acetilcolinesterase. Dos 34 extratos obtidos
de folhas e galhos das plantas analisadas para a atividade antif&ngica
com Cladosporium sphaerospermum e C. cladosporioides, 26,5% foram
ativos. Todavia, apenas o extrato das folhas de Cabralea canjerana inibiu
fortemente o crescimento dos dois fungos. No ensaio de reparo do DNA com linhagens
mutantes de Saccharomyces cerevisiae, 11,7% dos extratos foram ativos,
sendo que, 100% destes foram seletivos para o mecanismo de reparo do DNA envolvendo
topoisomerase II. Das 17 esp&cies analisadas, 12 demonstraram atividade
anticolinester&sica no ensaio qualitativo sobre cromatografia de camada
delgada (CCD). No entanto, apenas os extratos de Tetrastylidium grandifolium
(galhos) e Sloanea guianensis (folhas e galhos) apresentaram atividade
anticolinester&sica maior que 50% no ensaio quantitativo.
Unitermos: Floresta Mata Atl&ntica,
atividade antif&ngica, inibidores da acetilcolinesterase, triagem de
INTRODUCTION
The Atlantic Rainforest is considered one of
the priorities for conservation of biodiversity in the American Continent (Myers
et al., 2000). Today, the Atlantic Forest is restricted to ca 98.800
km2 of remnants, i.e. 7.6% of its original extension. This
complex biome extends for more than 17 Brazilian States, displaying several
phytophysiognomies determined by coastal proximity, relief, types of soil and
pluviometric regimes (Rizzini, 1979). Very few surveys of medicinal plants have
been performed in areas of Mata Atl&ntica as well as in other Brazilian
biomes such as "Caatinga", "Pantanal" and "Cerrado"
(Vieira, 1999). Recently, an ethnobotanical survey has been performed in rural
and urban areas of three cities located in the Atlantic forest region of the
State of S&o Paulo in which 628 medicinal uses were described for 114
plant species. The survey demonstrated that the majority of the plants were
employed for respiratory and gastrointestinal diseases and as analgesics. The
majority of the plants cultivated for medicinal usage were exotic (Di Stasi
et al., 2002).
Natural products research employs a number of
relevant bioassays in order to find out biologically active chemicals. For the
initial screening of crude extracts, simple, rapid, inexpensive bioassays with
good reproducibility are used. These bioassays generally require a small amount
of sample. Methods using thin-layer chromatography (TLC) bioautography which
possess good compatibility with a large number of samples have many utility
to screening extracts (Marston et al., 2002).
A vast number of native Brazilian plant species
have not yet been chemically or biologically evaluated, the interdisciplinary
BIOTA/SP program "Conservation and Utilization of S&o Paulo Biodiversity"
has been assembled aiming at description of the biodiversity in the State in
terms of taxonomy and biological properties. The project "Conservation
and sustainable use of the diversity from Cerrado and Atlantic Forest: chemical
diversity and prospecting for potential drugs" aims at searching lead-containing
molecules from plant species occurring in the State of S&o Paulo, especially
in the "Cerrado" and Atlantic Forest. The project was conceived to
identify antioxidant, anti-inflammatory, antifungal, anticancer, antimalarial
and acetylcholinesterase inhibiting lead- containing compounds from plant species
of these biomes ().
Following the objectives of this project, crude extracts from leaves and stems
of Atlantic Forest plants were screened for antifungal, DNA-damaging and acetylcholinesterase
inhibiting activities.
MATERIAL AND METHODS
Study area
The Ilha do Cardoso with 22.500 ha is situated
in the Canan&ia municipality, State of S&o Paulo, Brazil (25°03'05"
- 25°18'18" S and 47°53'48" - 48°05'42" W). The island is
the central region is dominated by a massif of over 800 m, composed of metamorphic
rocks (gneisses and micaschists) with magmatic outcrops (granites). Climate
is megathermic, superhumid, with no water shortage and considerable excess in
summer. Average annual temperature is 21.2 °C (Fiuza de Mello and Mantovani,
1994). It might be said that the Ilha do Cardoso offers a sample of all vegetation
types occurring along the coast of Brazil: pioneer dune vegetation, "restinga"
vegetation, coastal plain rainforest, Serra do Mar tropical rainforest and mangrove
(Barros et al., 1991).
Plant material
The plants analyzed were collected in Ilha do
Cardoso, in June 2002, identified and a voucher specie of each was deposited
at the Herbarium (SP) of the Instituto de Bot&nica de S&o Paulo,
Preparation of the extract
The plants were dried in the shadow at room temperature.
The dry material was separated (stems and leaves) and ground. The ground material
(30-60 g) was extracted with 60-80 mL ethanol 92?GL in an automatic
extractor (ASE 300, Dionex) at 70 ?C with an extraction cycle of
15 min. The extracts were concentrated under vacuum in a rotatory evaporator
and dried in a steam bath at 50 ?C. The yields varied from 2-10 g.
Biological assays by TLC bioautography
Antifungal activity
Cladosporium cladosporioides (Fresen)
de Vries SPC 140 and Cladosporium sphaerospermum Penzig (SPC 491) from
the live collection of the Instituto de Bot&nica. were grown in potato
dextrose agar for 12 days until sporulation. Ten microliters of a solution corresponding
to 400 µg of crude extracts
were applied on Al-backed silica Gel GF254 TLC layers (Merck) and
run with CHCl3: MeOH (9:1 v/v). The solvent was then completely removed
and the plates were sprayed with a spore suspension of either C. sphaerospermum
or C. cladosporioides in a glucose and salt solution (Homans and Fuchs,
1970; Rahalison et al., 1994) and incubated for 48 h at 28 ?C. After
incubation, clear inhibition zones appeared against the dark background of the
chromatogram. Nystatin (1.0 µg)
and miconazole (0.5 µg)
were used as standards.
Acetylcholinesterase activity
The procedure recently reported by Marston et
al. (2002) was used for this bioassay. Briefly, acetylcholinesterase type V
(Sigma, product no. C
U) was dissolved in Tris-hydrochloric acid
buffer (pH 7.8) and stabilized by the addition of bovine serum albumin fraction
V (0.1%, Sigma, product no. A-4503). TLC layers were spotted with 200 µg/spot
of plant extract and galanthamine (Sigma, 1 µg/spot)
and eserine (Sigma, 0.3 µg/spot)
were used as positive controls. TLC layers were developed with CHCl3:MeOH
(9:1 v/v) and subsequently dried. The plates were then sprayed with the enzyme
solution (6.66 U/ml), thoroughly dried and incubated at 37 °C for 20 min (moist
atmosphere). Enzyme activity was detected by spraying with a solution consisting
of 0.25% of 1-naphtyl acetate in EtOH (5 ml) plus 0.25% aqueous solution of
Fast Blue B salt (20 ml). Potential acetylcholinesterase inhibitors appeared
as clear zones on a purple colored background.
Microplate assay
The method used is extremely sensitive and is
applicable to low concentrations of enzyme. The principle of the method is the
measurement of the rate of production of thiocholine as acetylthiocholine is
hydrolyzed. This is accomplished by the continuous reaction of the thiol with
5,5'-dithiobis-2-nitro-benzoic acid (Ellman et al., 1961).
Acetylcholinesterase activity was measured using
a 96-well microplate reader (Rhee et al,. 2001) based on Ellman's method (Ellman
et al., 1961). In this method the enzyme hydrolyzes the substrate acetylthiocholine
resulting in the production of thiocholine which reacts with 5,5'-dithio-bis(2-nitrobenzoic
acid) (DTNB) to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate
which can be detected at 405 nm. In the 96-well plates, 25 µl
of 15 mM acetylthiocholine iodide (ATCI) in water, 125 µl
of 3 mM DTNB in buffer C, 50 µl
of buffer B, 25 µl of
plant extract sample (10 mg/mL in MeOH diluted ten times with buffer A, to give
a concentration of 1 mg/mL) were added and the absorbance was measured at 405
nm every 30 s for three times. Then 25 µl
of 0,22 U/mL of the enzyme were added and the absorbance was again read every
10 min for two times. Any increase in absorbance due to the spontaneous hydrolysis
of the substrate was corrected by subtracting the rate of the reaction before
the addition of the enzyme from the rate of the enzyme reaction. The percentage
of inhibition was calculated by comparison with the rates for the sample to
a blank (10% MeOH in Buffer A). The following buffers were used. Buffer A: 50
mM Tris-HCl, pH 8; buffer B: 50 mM Tris-HCl, pH 8, containing 0.1% bovine serum
albumin V fraction (BSA); buffer C: 50 mM Tris-HCl, pH 8, containing 0.1 M NaCl
and 0.02 M MgCl2.6H2O.
DNA-Damaging assay
Due to the large number of extracts that required
testing, a pre-screen using strains of topoisomerase-deficient Saccharomyces
cerevisiae (Rad52Y, topoisomerase I, and RS321N, topoisomerase II) was conducted
by means of an agar well diffusion assay. Provided there is no inhibition of
growth of the wild type (RAD+) strain, differential inhibition of growth in
any of the mutant strains (Rad52Y or RS321N) acts as an indicator of DNA-damaging
activity (Gunatilaka et al., 1992).
Individual strains of yeast were seeded onto
2% of YPD (Yeast extract, Peptone and Dextrose) agar plates. Samples of plant
extracts were solubilized in 1:1 DMSO-MeOH to a concentration of 4 mg/ml and
100 µl were placed in
agar wells made by the removal of 6-mm plugs from agar plates containing media.
Activity was measured as the diameter of the zone (mm) of inhibition around
each well. Camptothecin (5 µg/ml)
and streptonigrin (4 µg/ml)
were used as positive controls for the strains Rad 52Y and RS321, respectively.
Results from bioautographic assays were expressed
as reference values (Rf) of zones of growth inhibition for antifungal activity
and inhibition zones that measure acetylcholinesterase inhibition and photographed
using an Epson Photo PC 3000 Z Mod G 790A and Camag Reprostar 3 System.
The quantitative results of acetylcholinesterase
inhibition were represented as means of one typical experiment performed in
triplicate. The values were analysed with the program GraphPad Prism Software,
San Diego CA, version 3.0.
RESULTS AND DISCUSSION
To screen a diverse number of natural products,
an effective and fast assay system is needed. In this work 34 extracts were
screened for antifungal, AChE inhibitors and antitumoral activities, aiming
at finding others compounds with not much side effects and good therapeutic
The results of this screening are summarized
in . From the 34 extracts assayed for
antifungal activity with the filamentous fungi Cladosporium sphaerospermum
and C. cladosporioides, nine species showed a positive moderate to
strong response to either one or for both species of fungi. From the 17 plant
species evaluated, the extracts from leaves of Tetrastylidium grandifolium
and Phyllocalyx membranaceus showed activity only against C. cladosporioides.
Antifungal activity against C. sphaerospermum
was only observed in the leaves of Brosimum guianense and in the
stems of Platymiscium floribundum, Endlicheria paniculata and Guatteria
australis.
Of 17 species extracts tested, only Myrsine
umbellata (leaves), Cabralea canjerana (leaves) and Roupala paulensis
(leaves) showed positive response against both fungi Cladosporium sphaerospermum
and C. cladosporioides.
The isoflavonoids 7-hydroxy-6,4-dimethoxy-isoflavonequinone,
2-hydroxy-6,4,6,4-tetramethoxy-[7-O-7]-bisisoflavone, and seven other
known flavonoids, 3-hydroxy-9-methoxypterocarpan (medicarpin), 3,10-dihydroxy-9-methoxypterocarpan,
3,9-dimethoxypterocarpan (homopterocarpin), 2,3,9-trimethoxypterocarpan, 3,4-dihydroxy-9-methoxypterocarpan
(vesticarpan), 2,4,4-trihydroxychalcone (isoliquiritigenin), and 7,4-dihydroxyflavanone
(liquiritigenin) were isolated from the heartwood of Platymiscium floribundum.
The flavonoids homopterocarpin, vesticarpan and liquiritigenin showed cytotoxic
activity against five human cancer cell lines in vitro (Falc&o
et al., 2005). From the trunk of Platymiscium floribundum were isolated
five pterocarpans: (+)-2,3,9-trimethoxypterocarpan, (+)-3,9-dimethoxypterocarpan
[(+)-homopterocarpin], (+)-3,10-dihydroxy-9-methoxypterocarpan, (+)-3,4-dihydroxy-9-methoxypterocarpan
[(+)-vesticarpan] and (+)-3-hydroxy-9-methoxypterocarpan [(+)-medicarpin]. All
tested compounds showed strong antimitotic effects on sea eggs assay, the (+)-2,3,9-trimethoxypterocarpan
was the most active (Milit&o et al., 2005). The compounds (+)-2,3,9-trimethoxypterocarpan
and (+)-3,9-dimethoxypterocarpan [(+)-homopterocarpin] induce necrosis while
(+)-3,4-dihydroxy-9-methoxypterocarpan [(+)-vesticarpan] and (+)-3-hydroxy-9-methoxypterocarpan
[(+)-medicarpin] trigger apoptosis in HL-60 human leukemia cells (Milit&o
et al., 2006). Trevisan and Macedo (2003) in a recent study with Brazilian plants
showed good results against acetylcholinesterase inhibition for the species
Amburana cearensis, Lippia sidoides, Paullinia cupana, Solanum asperum
and Plathymiscium floribundum.
The DNA-damaging assay with mutant strains of
S. cerevisiae resulted in 11.7% active extracts (inhibition zone &
8 mm) with selectivity for the DNA-repair mechanisms of topoisomerase II. The
four active extracts from three species, were selective for topoisomerase II
(Myrsine umbellata (stems), Platymiscium floribundum (leaves and
stems) and Sloanea guianensis (stems).
The remarkable number of flavonoids isolated
from Platymiscium floribundum may be responsible for antifungal and DNA-damaging
activities, since P. floribundum showed strong cytotoxic activity (Milit&o
et al., 2005; Milit&o et al., 2006).
The crude alkaloids from Guatteria australis
were active against K1 strain of Plasmodium falciparum with an IC(50)=0,3
microg/ml. The alkaloid fraction from G. australis had antiplasmodial
activity with IC(50)=5 microg/ml (Fischer et al., 2004).
Several plant-derived drugs, such as rivastigmine
and galanthamine, inhibit AChE, hence increasing the endogenous levels of acetylcholine
to boost cholinergic neurotransmission (Lopez et al., 2002). Although plant
alkaloids are best known for inhibiting AChE enzyme, recent studies indicate
new classes of AChE-inhibiting compounds, such as essential oils (Miyazawa et
al., 1997; Perry et al., 2000; Savelev et al., 2004), coumarins (Rollinger et
al., 2004; Miyazawa et al., 2001), flavonoid glycosides (Hillhouse et al., 2004),
stilbene oligomers and xanthones (Urbain et al., 2004; Howes et al., 2003).
Barbosa-Filho et al. (2006) showed in a review that 309 plants belong to 92
botanical families were tested against acetylcholinesterase inhibition whereas
156 of them were active (ca 50%). Presently, the use of inhibitors of
acetylcholinesterase still seems to be the most efficient approach to treat
the symptoms of neurodegenerative diseases, such as Alzheimer disease (Viegas
et al., 2004).
Of the 17 species analysed, 12 showed anticholinesterasic
activity by the TLC assay. However, only three species had higher than 50% inhibitory
effect on acetylcholinesterase, Tetrastylidium grandifolium (stems),
Sloanea guianensis (leaves and stems) and Chrysophyllum flexuosum
(stems). From the leaves of the latter three triterpene lactones have already
been isolated: butanolide [2,3]-β-amirine,
butanolide [2,3]-β-amirine
and butanolide-[2,3]-lupeol. However, no report on acetylcholinesterase inhibiting
activity has been described for this species (Marqui et al., 2006).
CONCLUSION
The therapeutic arsenal for the treatment of
some diseases as skin mycoses, cancer and Alzheimer disease is limited and very
expensive for the last two of them. Of the 34 extracts from 17 plant species
assayed, 22 showed some biological activity. Some of these species have already
been analysed for chemical constituents, some of which potentially bearing biological
activity. As the present assays were performed with crude extracts, it is now
necessary to perform this evaluation bio-guided fractionation of the active
extracts which are now in our laboratories. Thus, the Brazilian biodiversity
holds a tremendous resource of new products, which has still hardly been explored.
Its great potential come only be exploited by chemical prospecting of nature's
genetically encoded combinatorial library.
ACKNOWLEDGMENTS
The RS321N, rad52Y, and RAD+ strains of Saccharomyces
cerevisiae were kindly donated from Dr. David G. I. Kingston (Virginia Polytechnic
Institute and State University) and Dr. Randall K. Johnson (SmithKline Beecham
Pharmaceuticals).
This work was funded by grants by FAPESP (Proc.
-7) and CAPES. This work was also supported by the Funda&&o
de Amparo & Pesquisa do Estado de S&o Paulo (FAPESP) within the
BIOTA/FAPESP - The Biodiversity Virtual Institute Program (, 2006). M. C. M. Young, V. S. Bolzani and S. M. C. Dietrich
are grateful to CNPq for research fellowships. Elaine Monteiro Cardoso-Lopes
thanks PRODOC/CAPES for providing a post-doctoral fellowship through the graduate
programme on "Biodiversidade Vegetal e Meio Ambiente" of Instituto
de Bot&nica (S&o Paulo).
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Received 9 April 2008
Accepted 18 September 2008
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