SUPPLEMENTARY MATERIAL
In vivo antimalarial efficacy of acetogenins, alkaloids and flavonoids
enriched fractions from Annona crassiflora Mart.
Lúcia Pinheiro Santos Pimentaa*, Giani Martins Garciab, Samuel Geraldo
do Vale Gonçalvesa, Bárbara Lana Dionísioa, Érika Martins Bragac,
Vanessa Carla Furtado Mosqueirab
a
Departamento de Química – Instituto de Ciências Exatas – Universidade Federal de
Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG, Brazil; 31270-901.
b
Departamento de Farmácia – Escola de Farmácia – Universidade Federal de Ouro
Preto, Campus Morro do Cruzeiro, Ouro Preto, MG, Brazil; 35400-000.
c
Departamento de Parasitologia - Instituto de Ciências Biológicas – Universidade
Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG, Brazil;
31270-901
*Corresponding author. Tel.: +55 (31) 3409 5754, Fax: +55 (31) 3409 5700
E-mail address: lpimenta@qui.ufmg.br
In vivo antimalarial efficacy of acetogenins, alkaloids and flavonoids enriched
fractions from Annona crassiflora Mart.
Annona crassiflora and Annonaceae plants are known to be used to treat
malaria by traditional healers. In this work, the antimalarial efficacy of
different fractions of A. crassiflora, particularly acetogenin, alkaloids and
flavonoid rich-fractions, was determined in vivo using Plasmodium bergheiinfected mice model and toxicity was accessed by brine shrimp assay. The A.
crassiflora fractions were administered at doses of 12.5 mg/kg/day in a fourday test protocol. The results showed that some fractions from woods were rich
in acetogenins, alkaloids and terpenes, and other fractions from leaves were
rich in alkaloids and flavonoids. The parasitaemia was significantly (p<0.05,
p<0.001) reduced (57-75%) with flavonoid and alkaloid-rich leaf fractions,
which also increased mean survival time of mice after treatment. Our results
confirm the usage of this plant in folk medicine as antimalarial remedy.
Keywords: Annona crassiflora; antimalarial efficacy; flavonoids; aporphine
alkaloids
Experimental section
General
Chloroquine diphosphate was purchased from Sigma-Aldrich (St. Louis, MO). N,Ndimethylacetamide, dimethyl sulfoxide and PEG 300 were provided by Vetec. The
solvents were from Quimex, Vetec, Carlo Erba and J. T. Baker (Brazil). The UV data
were obtained in a Hitachi 2010 spectrophotometer and IR spectra were measured on
a Perkin Elmer spectrometer version 3.02.1.
Plant material and extraction procedure
Wood and leaves of Annona crassiflora Mart. were collected in Itatiaiuçu,
Minas Gerais, Brazil, July 2007. A voucher specimen was deposited at the Instituto de
Ciências Biológicas Herbarium (BHCB) (no 22988), UFMG, Belo Horizonte, MG,
Brazil. Plant parts were dried at 40 oC and extracted at room temperature with
solvents which were removed under vacuum to give the crude dry extracts. Leaves of
A. crassiflora (610.24 g) were successively and exhaustively extracted with hexane
and ethanol leading to the hexanic (ACLH; 13.49 g) and ethanolic (ACLF01; 268.63
g) extracts. The wood of A. crassiflora (4,690 g) was extracted only with ethanol
(ACWF01; 519.78 g). Ethanolic extracts of leaves (7.00 g) and wood (94.44 g) of A.
crassiflora (ACLF01 and ACWF01), were dissolved in ethanol/water (7:3) and
successively extracted with hexane. After solvent removal, the hexanic (ACLF02 and
ACWF02) or lipophilic fractions were obtained. The defatted extracts were submitted
to extraction in acid moiety (1% HCl aqueous solution/CHCl3) yielding the organic
layer (ACLF03 and ACWF03) and the acidic hydroalcoholic (ACF04L and
ACWF04) fractions. The ACLF04 and ACWF04 were basified with 6N NH4OH and
extracted with CHCl3. The organic layer was washed with water, dried and
concentrated to give an alkaloidal mixture named ACLF05 and ACWF05. During
washing of the leaf chloroformic layer, a solid precipitated from ACLF05, which was
removed by filtration and called ACLF05s. The aqueous basic layers from leaves and
wood (ACLF06, ACWF06) were successively extracted with ethyl acetate and nbuthanol affording the ethyl acetate fractions ACLF07, ACWF09, and the nbuthanolic fractions ACLF08, ACWF08, respectively. From the extraction of
ACLF06 and ACWF06 with ethyl acetate precipitated a solid that were named
ACLF07s and ACWF07s, respectively. The wood aqueous fraction was neutralized
and named ACWF09. The parts of plants used in each case, their yields in % dry wt.
and the yields from acidic extraction are given in Table S1.
Chromatography analysis
All extracts and fractions were submitted to analytical TLC analysis. The
plates containing the extracts and fractions from A. crassiflora were sprayed with
Kedde’s reagent in order to characterise an ,-unsaturated--lactone moiety,
commonly found on annonaceous acetogenins (Cavé, Cortes, Figadère, Laurens, &
Pettit, 1997). All extracts and fractions were also analysed by TLC plates sprayed by
Dragendorff’s reagent, indicative of the presence of alkaloids, and by Natural
Products-PEG reagent at UV-365 nm, where intense fluorescence produced indicated
detection of flavonoids (Wagner, Bladt, & Zgainski, 1984). As nearly all fractions
gave positive reaction to Dragendorf’s reagent, the TLC analysis was performed to
compare the wood fractions with the standard compounds liriodenine and
atherospermidine isolated from A. crssiflora wood.
Biological screening
The brine shrimp test (BST) (Pimenta, Pinto, Takahashi, Silva, & Boaventura, 2003)
Artemia salina encysted eggs (10 mg) were incubated in 100 ml of seawater
under artificial lighting at 28oC, pH 7-8. After incubation for 24 h, nauplii were
collected with a Pasteur pipette 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 (dimethyl sulfoxide) (2 mg/400 l or 2 mg/1000 l) and serially
diluted (10, 20, 30 and 50 l/5ml) in seawater. About 10-20 nauplii were added to
each set of tubes containing the samples. Controls containing 50 l of DMSO in
seawater were included in each experiment. As a positive control, lapachol dissolved
in DMSO was used. Twenty-four hours later, the number of survivors was counted,
recorded and the lethal concentration 50% (LC50) and confidence intervals 95% were
calculated by Probit analysis (Finney, 1976).
Hemolitic activity of A. crassiflora fractions
Hemolysis was tested by colorimetric measurement of hemoglobin release
after red blood cell (RBC) incubation with the different fractions (Aditya, Patankar,
Madhusudhan, Murthy, & Souto, 2010). Whole blood from healthy human donors
was collected using tubes containing 0.5 ml 3.8% citrate. The tubes were centrifuged
(200 × g, 15 min at 4°C) and the supernatant discarded. The cell pellet was diluted
with isotonic PBS (157 mM) and concentration adjusted to give an optical density of
400 to 540 nm. Two controls were used: T0 (no hemolysis) constituted by RBC in
PBS and T100 (total hemolysis) related to RBC in distilled water. Samples of wood
fractions were added at various dilutions in PBS (ACWF02, ACWF03, ACWF05).
All the preparations were diluted and incubated for 30 min at 37°C. After incubation,
hemolysis was stopped at +4°C and intact cells were removed by centrifugation (5
min at 100 × g). Turbidity was removed by filtrating the supernatant in 0.45 µm filter
(Millex®, Millipore). The supernatant was collected (300 µl) and analyzed by UV-VIS
spectroscopy at 540 nm in 80 µl flow cell sipper in spectrophotometer (Helious-α,
Thermo Spectronic, USA). The results were expressed as percentage of hemolysis by
the equation where T is the optical density of the sample supernatant: % hemolysis =
(T - T0)/T100
Animals
The in vivo experiments were approved by the Ethics Committee on Animal
Experimentation of the Universidade Federal Ouro Preto, Brazil, and are in
compliance with the Guide for the Care and Use of Laboratory Animals
recommended by the Institute of Laboratory Animals Resources (Committee on Care
and Use of Laboratory Animals, 1985). Female out bred Swiss albino mice weighing
20 to 24 g were supplied by the Animal Facility of Universidade Federal de Ouro
Preto. They were kept in a normal diurnal cycle and had free access to food and water
throughout the experiments.
Antimalarial activity in P. berghei-infected mice
The 4-day suppressive test was used for monitoring in vivo activity of the
extracts and fractions, as described by Peters et al. (1986), for determination of
antimalarial activity against chloroquine-sensitive Plasmodium berghei NK65 strain.
An infective inoculum was prepared from a previously infected donor mouse with
rising parasitaemia (20 %). On day 0 the mice were infected intravenously with a
million of infected erythrocytes of P. berghei in 0.2 ml of phosphate-buffered saline.
They were randomly divided in groups of 5 mice and treated by intraperitoneal route
once daily (12.5 mg/kg) with different fractions of A. crassiflora for four consecutive
days (days 0 to 3). The fractions were dissolved in N,N-dimethylacetamide and
polyethyleneglycol (PEG 300) at 1:2 proportion and further diluted 5-fold in saline to
obtain the desired concentration in 0.2 ml for injection. Two control groups were used
each time, one treated with chloroquine diphosphate (15 mg/kg/day) as positive
control and one not treated or treated with saline, as specified in the results. Thin
blood smears were made from tail blood on days 4, 7, 10, 15, 22 and 30 after
infection, methanol-fixed and stained with Giemsa. At least 3000 cells were checked
to calculate parasitaemia percent. Drug activity was determined on the basis of
average parasitaemia per group of mice. The percent reduction of parasitaemia in
treated groups as compared to untreated groups was calculated as follows: %
parasitaemia in control group (Pcg) - % parasitaemia in test group/Pcg × 100. Overall
mortality was monitored daily until day 30 after infection.
Statistics
All RBC counts and parasitaemia levels are expressed as mean values ±
standard deviations. The parasitaemia data were analysed by using the one-way
analysis of variance (ANOVA) test using Prisma® 5.0 software. The Kruskal-Wallis
was used for survival comparative analysis.
Table S1. Crude extracts* and fractions from acidic extraction#
Plant names
Part used
Extract and fractions
% dry wt.
obtained
Annona crassiflora
ACLH
2.21
ACLF01
44.02
ACLF02
2.37
ACLF03
2.77
ACLF05
6.93
ACLF06
63.55
ACLF07
2.12
ACLF05s
6.93
ACLF07s
2.12
ACWF01
11.08
ACWF02
3.05
ACWF03
5.40
ACWF05
0.26
ACWF07
8.79
ACWF08
0.58
ACWF09
37.91
ACWF07s
7.73
ACWF03s
0.21
Leaves
Wood
* quantity obtained from 100g of dried plant material, % dry wt. # quantity obtained
from 100g of dried extract, % dry wt.
Table S2. Brine shrimp larvicidal activity of some extracts and fractions of Annona
crassiflora.
LC50 in g/mL
Extracts and fractions tested
(95% confidence interval)
1
AC L F01 (leaves)
1029.97 (769.61<LC<1396.55)
2
AC L F02 (leaves)
189.17 (159.93<LC<223.76)
3
AC L F03 (leaves)
1.45 (1.22<LC<1.74)
4
AC L F05 (leaves)
3.98 (3.25<LC<4.87)
5
AC L F06 (leaves)
1351.55 (889.32<LC<2054.04)
6
AC L F07 (leaves)
488.68 (406.35<LC<587.67)
7
ACWF01 (wood)
3.41 (2.39<LC<4.87)
8
ACWF02(wood)
11.64 (6.58<LC<22.55)
9
ACWF03 (wood)
1.29 (0.70<LC<1.87)
10
ACWF05(wood)
11.57 (4.05<LC<33.04)
ND1
11
ACWF07(wood)
1
ND= Not determined
.
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