Antileishmanial activity of the hydroalcoholic extract of miconia langsdorffii, isolated compounds, and semi-synthetic derivatives

Article abstract of DOI:10.3390/molecules16021825

The in vitro activity of the crude hydroalcoholic extract of the aerial parts of Miconia langsdorffii Cogn. was evaluated against the promastigote forms of L. amazonensis, the causative agent of cutaneous leishmaniasis in humans. The bioassayguided fractionation of this extract led to identification of the triterpenes ursolic acid and oleanolic acid as the major compounds in the fraction that displayed the highest activity. Several ursolic acid semi-synthetic derivatives were prepared, to find out whether more active compounds could be obtained. Among these ursolic acid-derived substances, the C- 28 methyl ester derivative exhibited the best antileishmanial activity.

Full text of DOI:10.3390/molecules16021825

Molecules 2011, 16, 1825-1833; doi:10.3390/molecules16021825
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antileishmanial Activity of the Hydroalcoholic Extract of
Miconia langsdorffii, Isolated Compounds, and Semi-Synthetic
Derivatives
1
1
1
Juliana A. Peixoto , Márcio Luis Andrade e Silva , Antônio E. M. Crotti , Rodrigo Cassio Sola
1
2
1
3
Veneziani , Valéria M. M. Gimenez , Ana H. Januário , Milton Groppo , Lizandra G.
1
1
4
5
Magalhães , Fransérgio F. dos Santos , Sérgio Albuquerque , Ademar A. da Silva Filho and
1
,
Wilson R. Cunha *
1
Núcleo de Pesquisa em Ciências Exatas e Tecnológicas da Universidade de Franca, Av. Dr.
Armando Salles de Oliveira 201 - Parque Universitário, CEP 14404-600, Franca, SP, Brazil;
E-Mails: ju_andradepeixoto@yahoo.com.br (J.A.P.); mlasilva@unifran.br (M.L.A.S);
millercrotti@unifran.br (A.E.M.C.); rcsvenez@unifran.br (R.C.S.V.); anahjanuario@unifran.br
(
A.H.J.); lizandraguidi@unifran.br (L.G.M.); fransergiofs@hotmail.com (F.F.S.)
2
3
Centro Universitário Claretiano, Rua Dom Bosco 466, CEP 14300-000, Batatais, SP, Brazil;
E-Mail: vmmgimenez@yahoo.com.br
Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto,
Universidade de São Paulo, Av. do Café s/n CEP 14040-901 Ribeirão Preto, SP, Brazil;
E-Mail: groppo@ffclrp.usp.br
4
5
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café
s/n, CEP 14040-903, Ribeirão Preto, SP, Brazil; E-Mail: sdalbuqu@fcfrp.usp.br
Departamento Farmacêutico, Universidade Federal de Juiz de Fora, Rua José Lourenço Kelmer s/n,
Campus Universitário, CEP 36036-900, Juiz de Fora, MG, Brazil;
E-Mail: silvafilhoaa@gmail.com
*
Author to whom correspondence should be addressed; E-Mail: wrcunha@unifran.br;
Tel.: +55-16-37118878; Fax: +55-16-37118878.
Received: 8 February 2011; in revised form: 14 February 2011 / Accepted: 21 February 2011 /
Published: 22 February 2011
Abstract: The in vitro activity of the crude hydroalcoholic extract of the aerial parts of
Miconia langsdorffii Cogn. was evaluated against the promastigote forms of L.
amazonensis, the causative agent of cutaneous leishmaniasis in humans. The bioassay-
guided fractionation of this extract led to identification of the triterpenes ursolic acid and
oleanolic acid as the major compounds in the fraction that displayed the highest activity.

Molecules 2011, 16
1826
Several ursolic acid semi-synthetic derivatives were prepared, to find out whether more
active compounds could be obtained. Among these ursolic acid-derived substances, the C-
8 methyl ester derivative exhibited the best antileishmanial activity.
2
Keywords: antileishmanial activity; Miconia langsdorffii; oleanolic acid; ursolic acid
1
. Introduction
Leishmaniasis is a group of tropical diseases caused by a number of species of protozoan parasites
belonging to the genus Leishmania, and it is an ailment that affects around 12 million people in 88
countries. It is estimated that there are about two to three million new leishmaniasis cases each year,
and that some 350 million people are at risk of infection [1,2]. Over the last 20 years there has been an
increase in the number of cases of all leishmaniasis forms worldwide. Urban development,
deforestation, environmental changes, immune status, and treatment failure are probably the factors
responsible for the spread of the disease [2,3]. Historically, the chemotherapy of leishmaniasis has
been based on the use of toxic heavy metals, particularly antimony compounds. When this kind of
treatment is not effective, other medications are utilized, including pentamidine and amphotericin B.
All these pharmaceuticals require administration by injection and clinical supervision or
hospitalization during treatment, due to the severity of the possible side effects [4]. Thus, there is a
clear need for safer and more effective treatments against the various leishmaniasis forms.
In recent years there has been an intense search for antileishmanial compounds obtained from
natural sources, which has led to the identification of several classes of active plant metabolites [2,5].
Melastomataceae is one of the largest angiosperm families, with ca. 4,570 species distributed
throughout tropical and subtropical areas in the world [6]. Approximately one quarter of these species
belong to the genus Miconia, with 1,056 species occurring from the South of Mexico to the North of
Argentina and Uruguay [7]. Previous studies on Miconia species have revealed the presence of
triterpenes, coumarins, and benzoquinones [8-12]. Miconia extracts and their isolated compounds have
been demonstrated to display remarkable biological activities, such as trypanocidal [12,13],
antitumoral [14], antimutagenic [15], antimicrobial [16,17], analgesic, and anti-inflammatory
actions [18,19].
As part of our ongoing research on plant-derived metabolites for the treatment of diseases caused by
protozoan parasites [12,13,20], evaluation of the in vitro antileishmanial activity of the hydroalcoholic
extract of Miconia langsdorffii Cogn. is reported herein. The bioassay-guided fractionation and HPLC
analysis of the obtained fractions has also been accomplished, in order to identify the main compounds
involved in this biological activity. Some semi-synthetic derivatives were prepared as well, so as to
investigate possible structure-activity relationships.
2
. Results and Discussion
Some extracts and several compounds isolated from Miconia species have been investigated for
their biological properties, including their antiparasitic activity [12,13]. However, this is the first time

Molecules 2011, 16
1827
that M. langsdorffii has been investigated with this aim. Regarding the in vitro antileishmanial assay,
the crude hydroalcoholic extract of M. langsdorffii (HEML) displayed moderate activity against the
promastigote forms of Leishmania amazonensis, with an IC50 value of 175.4 μg/mL. Among the
HEML chromatographic fractions, F2 exhibited the highest antileishmanial activity, with an IC50 value
of 4.5 μg/mL. On the basis of these results, F2 was further chromatographed by HPLC, which enabled
isolation of ursolic acid (1) and oleanolic acid (2) (Figure 1). Both these acids are triterpenoids that are
widely distributed in the plant kingdom, and they have been frequently isolated from Miconia species
as mutually isomeric mixtures [12,13,19]. These triterpene acids display several biological
activities [21].
Figure 1. Chemical structures of ursolic acid (1), oleanolic acid (2), and the semi-synthetic
ursolic acid derivatives 1a, 1b, 1c, 1d and 1e.
2
CO H
2
CO R
2
1
HO
R O
2
R1
H
R2
H
1
1a
COCH
3
H
1b
CO(CH ) CH
H
2
10
3
1c
CO(CH ) CH
H
2
16
3
1
d
H
H
CH3
K
1
e
The antileishmanial activity of the isolated compounds was compared with that of the
corresponding original fraction, as shown in Table 1. The isolated compounds 1 and 2 gave IC50 values
of 360.3 μM and 439.5 μM, respectively (Table 1); in other words, 1 displayed better activity than 2.
Moreover, the original fraction F2 was more active in the antileishmanial assay compared with its
isolated compounds, so it may be suggested that minor compounds present in this fraction could be
playing an important role in the in vitro antileishmanial activity of F2. Also, compared with the
isolated compounds 1 and 2, a mixture of 1 and 2 displayed increased antileishmanial activity, with an
IC50 of 199.6 μg/mL. This is an indication of a possible synergistic effect. Similar results had been
previously observed when a mixture of 1 and 2 showed higher anti-inflammatory effect compared with
the isolated compounds in the paw edema assay [19]. However, the results obtained for 1 and 2
contrast with the data previously reported by Torres-Santos and co-workers [22], who described high
antileishmanial activity for these compounds against the promastigote forms of L. amazonensis (strain
LV79, MPRO/BR/72M 1841). The divergent results may be related to the diversity of parasite strains,
parasitic load, stages of parasite life cycle. Differences in the applied experimental conditions can also

Molecules 2011, 16
1828
lead to different values of IC50 of amphotericin B, which was used as positive control in both studies;
also reinforce the divergent results obtained for the compounds.
In order to obtain more active compounds, semi-synthetic derivatives of ursolic acid 1a-1e were
prepared (Figure 1). Among all the semi-synthetic derivatives, 1d gave the highest antileishmanial
activity, with an IC value of 174.9 μM. Comparing the IC values of 1 and its derivative 1a, a
5
0
50
decrease in antileishmanial activity can be noticed. The activity detected for 1a is in agreement with
the report by Gnoatto and co-workers [23], who also observed reduced activity for 1a against the
promastigote forms of L. amazonensis (MHOM/BR/1987/BA) compared with 1. However,
Mallavadhani and co-workers [24] have shown that lipophilicity is an important parameter in the
development of biological agents, so molecules with carbon chains above C-10 are fairly lipophilic and
good candidates for pharmacological evaluation. In view of these observations, two lipophilic 3-O-
fatty acid ester chain derivatives from 1 were prepared herein, namely 1b and 1c, which were then
screened for their leishmanicidal activity. Our results demonstrate that a larger carbon chain seems to
increase the leishmanicidal activity.
Table 1. In vitro leishmanicidal activity of the pure compounds isolated from M.
langsdorffii and of ursolic acid derivatives.
%
8
Lysis ± S.D. / concentration (μM)
Compounds
IC (μM)
5
0
32
128
1
2
+ 2
8.6 ± 1.2
14.8 ± 2.4
15.3 ± 1.7
12.8 ± 2.3
8.2 ± 0.7
23.4 ± 0.3
21.7 ± 0.8
8.9 ± 1.2
15.7 ± 2.6
23.7 ± 4.2
26.1 ± 3.2
22.6 ± 0.9
17.9 ± 2.0
44.3 ± 1.9
25.0 ± 2.4
12.1 ± 1.0
28.7 ± 4.6
39.3 ± 1.0
52.5 ± 3.9
33.8 ± 1.9
20.4 ± 0.8
48.4 ± 0.3
53.8 ± 2.1
16.2 ± 2.7
360.3
439.5
199.6
406.0
340.4
240.4
174.9
458.7
1
1
a
b
c
d
e
1
1
1
1
Positive control: amphotericin B (IC50 = 13.7 μM).
Interestingly, comparing the antileishmanial activity of 1a, 1b, 1c, and 1d, the increase in the
lipophilicity at C-17 is more relevant for the antileishmanial activity than enhanced lipophilicity at C-
3
. On the other hand, more polar groups at C-17 appear to cause a loss of antileishmanial action, as
compound 1e is less active than 1. Nevertheless, a larger number of ursolic acid derivatives, bearing
different patterns of substituting groups, should be further evaluated, in order to determine the
antileishmanial structure-activity relationship. Moreover, as previously suggested, this class of
compounds should also be considered for further antiprotozoal studies [22,23,25].
3. Experimental
3
.1. Plant Material
Miconia langsdorffii Cogn. (Melastomataceae) was collected in March, 2009 on the farm Fazenda
Palmyra, Serra Azul, state of São Paulo, Brazil (21°18'39,46" S, 47°33'25,55 W). A voucher specimen
collector V.M.M. Gimenez 4405) was deposited in the Herbarium of Departamento de Biologia,
Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, University of São Paulo-USP (SPFR
(

Molecules 2011, 16
1829
herbarium) under the record number 12288. The species was identified using the treatments of
Cogniaux [26] and Goldenberg [7,27], and the identification was compared with those for other
exsicatae deposited at the SPFR herbarium.
3.2. Extraction and Fractionation
The aerial parts of M. langsdorffii were dehydrated at 40 °C, followed by pulverization (0.5 kg) and
extraction with ethanol/H O (96:4 v/v) by maceration (5 L, 3 days × 3), at room temperature. The
2
solvent was removed under vacuum in a rotary evaporator, yielding 7.81 g of the dry crude
hydroalcoholic extract. Part of this extract (6.7 g) was chromatographed over 300 g silica gel 60
(
0.063–0.200 mm, Merck) by vacuum liquid chromatography, furnishing six fractions of 1000 mL
each (F1: n-hexane/AcOEt 75:25 v/v; F2: n-hexane/AcOEt 50:50 v/v; F3: AcOEt; F4:AcOEt/EtOH
5:25 v/v; F5: AcOEt/EtOH 50:50 v/v; F6: EtOH). All the obtained fractions were analyzed by HPLC.
7
1
13
Fraction (F2) exhibited significant antileishmanial effect, and its NMR- H and C data revealed the
presence of a mixture of ursolic acid (1) and oleanolic acid (2) [28]. A part of this combined fraction
(
500 mg) was filtered through 60 g of a mixture of Celite/Norit (3:1w/w) and eluted with AcOEt,
furnishing 350 mg of a mixture containing 65% of 1 and 35% of 2. These compounds were separated
by HPLC, affording 1 as a white amorphous solid (R : 20.67 min) and 2 also as a white amorphous
solid (R : 21.52 min) [28].
t
t
3
.3. Preparation of C-3 Ester Derivatives of Ursolic Acid
In order to obtain some triterpene acid derivatives, 1 (20 mg) was treated with excess acetic
anhydride in pyridine [12], to give the 3-acetoxyl derivative 1a (15 mg). Also, 1 (50 mg) was treated
with appropriate long chain fatty acids (44 mg lauric acid or 62.6 mg stearic acid), N,N’-
dicyclohexylcarbodiimide (DCC, 113.5 mg), and 4-dimethylaminopyridine (DMAP, 67 mg) in 1,2-
dichloroethane. The reaction mixture was stirred at room temperature for 24 h. Next, the mixture was
filtered, followed by evaporation under reduced pressure [24]. The residue was purified by silica gel
column chromatography with n-hexane, which gave the corresponding fatty acid esters 1b (35 mg) and
1
c (38 mg).
3
.4. Preparation of the C-28 Methyl Ester Derivative of Ursolic Acid
1
(about 20 mg) was treated with CH
derivative 1d (17 mg), which was purified by column chromatography on silica gel 60
0.063–0.200 mm, Merck, Darmstadt, Germany).
2 2 2
N in Et O [12], yielding the respective C-28 methyl ester
(
3
.5. Preparation of the Potassium Salt Derivative of Ursolic Acid
In order to obtain the potassium ursolate derivative, a solution of 1 (20 mg) was treated with 2%
KOH in Me
resulting material was chromatographed over Sephadex LH-20 using MeOH as eluent, which furnished
5 mg potassium ursolate (1e) as a white powder [29].
2 2 2
CO-H O (1:1 v/v) for 30 min, at room temperature. After evaporation of Me CO, the
1

Molecules 2011, 16
.6. HPLC Analysis
Both analytical and semi-preparative HPLC analyses were carried out on a Shimadzu LC-6AD
1830
3
system equipped with a DGU-20A5 degasser, an SPD-20A series UV-VIS detector with a CBM-20A
module, and a Reodyne manual injector. Analytical and semi-preparative separations were
accomplished using Shimadzu Shim-pack ODS columns (250 × 4.60 mm and 250 × 20 mm,
respectively) equipped with a pre-column of the same material. Methanol was HPLC grade (J. T.
Baker), and ultrapure H O was obtained by passing redistilled H O through a Direct-Q UV3 system
2
2
(
(
Millipore). The mobile phase used for chromatographic purification of 1 and 2 was MeOH/H O
2
85:15 v/v) with 0.1% AcOH.
3.7. Structural Identification
The structures of all the compounds were determined by spectroscopic methods in comparison with
1
13
previously published data [12,24,28,29]. H (400 MHz) and C (100 MHz) NMR spectra were
recorded on a Brüker DPX-400 spectrometer in DMSO-d or CDCl using TMS as internal standard.
High-resolution ESI-MS were registered on a Bruker Ultro-TOF mass spectrometer.
6
3
3
.8. Antileishmanial Assay
The bioassays were performed using Leishmania amazonensis (MHOM/BR175/M2904).
Promastigote forms of L. amazonensis were incubated in M199 medium (invitrogen), supplemented
with L-glutamine (2 mM), NaHCO
3
(10 mM), penicillin (100 UI/mL), streptomycin (100 µg/mL), and
6
5% bovine fetal serum (Gibco). After 6 days of initial inoculation, promastigote forms (2 × 10
parasites/mL) were incubated in 96-well microtiter plates containing the tested samples. The crude
extract, fractions, and isolated compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted
into the medium, to give final concentrations of 8, 32, and 128 (in µg/mL for extract and fractions and
μM for pure compounds). The plates were incubated at 22 °C for 24 h, and the lysis percentage was
determined by an MTT colorimetric method [30]. The bioassays were performed in triplicate, using
M199 medium with 5% DMSO as negative control and amphotericin B as positive control group.
3
.9. Statistical Analysis
The obtained data are represented as mean ± S.D. The data were statistically analyzed by one-way
ANOVA, followed by Tukey’s multiple comparison test. The IC50 (minimum concentration necessary
for inhibition of 50% of microorganism growth) values were calculated using sigmoid dose–response
curves.
4
. Conclusions
In summary, our results indicate that the antileishmanial activity of ursolic acid (1) and oleanolic
acid (2) is not so strong as compared with those of the antileishmanial drugs currently employed in the
clinical setting. However, it has been reported that these compounds are not so toxic [21,25], which
makes these acids and their derivatives particularly interesting for the development of new

Molecules 2011, 16
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antiprotozoal agents. It is noteworthy that in light of the results presented here, further biological
studies are needed, so that the mechanisms of antiprotozoal action of these compounds as well as their
structure-activity relationships can be elucidated.
Acknowledgements
The authors are grateful to FAPESP and CNPq for financial support.
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2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
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