Anti-trypanosomal activity of 1,2,3,4,6-penta-O-galloyl-β -D-glucose isolated from Plectranthus barbatus Andrews (Lamiaceae)

Abstract

MeOH extract from the leaves of Plectranthus barbatus Andrews (Lamiaceae), showed in vitro anti-trypanosomal activity. The bioassay-guided fractionation resulted in the isolation of a gallic acid derivative, identified as 1,2,3,4,6-penta-O-galloyl-β-D-glucose (PGG), after thorough NMR and MS spectral analysis. Finally, this compound was tested against trypomastigote forms of T. cruzi and displayed an EC50 value of 67 µM, at least 6.6-fold more effective than the standard drug benznidazole. This is the first occurrence of PGG in the Plectranthus genus and the first anti-parasitic activity described for PGG in the literature.

Full text

Quim. Nova, Vol. 35, No. 11, 2229-2232, 2012
Artigo
*e-mail: psartorelli@unifesp.br
#Artigo em homenagem ao Prof. Otto R. Gottlieb (31/8/1920-19/6/2011)
ANTI-TRYPANOSOMAL ACTIVITY OF 1,2,3,4,6-PENTA-O-GALLOYL-b-D-GLUCOSE ISOLATED FROM
Plectranthus barbatus Andrews (Lamiaceae)#
Roberta T. dos Santos, Liliane L. Hiramoto, João Henrique G. Lago e Patrícia Sartorelli*
Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo. R. Prof. Artur Ridel, 275,
09972-270 Diadema – SP, Brasil
André G. Tempone
Departamento de Parasitologia, Instituto Adolfo Lutz, Av. Dr. Arnaldo, 351, 8o andar, 01246-000 São Paulo – SP, Brasil
Erika G. Pinto
Departamento de Parasitologia, Instituto Adolfo Lutz, Av. Dr. Arnaldo, 351, 01246-000 São Paulo – SP / Instituto de Medicina
Tropical de São Paulo, Universidade de São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 470, 05403-000 São Paulo – SP, Brasil
Harri Lorenzi
Instituto Plantarum de Estudos da Flora, Av. Brasil, 2000, 13460-000 Nova Odessa – SP, Brasil
Recebido em 25/5/12; aceito em 17/9/12; publicado na web em 9/11/12
MeOH extract from the leaves of Plectranthus barbatus Andrews (Lamiaceae), showed in vitro anti-trypanosomal activity. The
bioassay-guided fractionation resulted in the isolation of a gallic acid derivative, identified as 1,2,3,4,6-penta-O-galloyl-b-D-glucose
(PGG), after thorough NMR and MS spectral analysis. Finally, this compound was tested against trypomastigote forms of T. cruzi and
displayed an EC50 value of 67 µM, at least 6.6-fold more effective than the standard drug benznidazole. This is the first occurrence
of PGG in the Plectranthus genus and the first anti-parasitic activity described for PGG in the literature.
Keywords: Plectranthus barbatus; 1,2,3,4,6-penta-O-galloyl-b-D-glucose; anti-trypanosomal activity.
INTRODUCTION
The genus Plectranthus, synonym Coleus (Lamiaceae), contains
about 350 species with occurrence in Africa, Asia and Australia.1 In
Brazil, it is an introduced genus, whose occurrence has been described
in Northeast and Southeast regions.2 Due to its ethnobotanical uses,
Plectranthus can be classified as an important genus from Lamiaceae,1
affording wide biologically active substances.3-6
P. barbatus, popularly known as “falso boldo”, is one of the most
relevant specie of the Plectranthus genus and has been used in tradi-
tional medicine for the treatment of digestive and respiratory disorders
as well as for heart and central nervous diseases.7-9 Pharmacological
studies have been conducted with extracts of P. barbatus, in which
antioxidant,10 antimicrobial,11 antifungal12 and antiacetylcholineste-
rase potentials were detected.13
Chemically, this specie has been characterized by the presence
of diterpenoids14, especially modified abietanoids.8,9,15 Some typical
abietane derivatives isolated from leaves of P. barbatus, classified
according to their structural characteristics, are barbatusin, ciclobuta-
tusin, plectrinone A, and coleun U.5,16,17 Other described constituents
include the phenolic derivatives rosmarinic acid and 7-O-glucoronide
4’-methyl ether.6
Chagas disease is one of most neglected diseases in several un-
derdeveloped and developing countries and affects approximately ten
million people worldwide, mostly in Latin America.18 It is caused by
the protozoan parasite Trypanosoma cruzi (T. cruzi), which is trans-
mitted to humans by triatomine insects primarily through posterior
transmission from fecal matter during feeding, by blood transfusion,
and even via congenital transmission.19 The therapeutic arsenal is very
limited. At present, there are only two effective drugs for the treatment
of Chagas disease: nifurtimox and benznidazol (nitroheterocycle
derivatives).20 These drugs show high cytotoxicity and the main side
effects for nifurtimox are anorexia, loss of weight, psychological
changes, besides digestive manifestations such as nausea, vomiting
and diarrhoea. Benznidazol is also highly toxic and the symptoms
of adverse reactions are hypersensitivity, dermatitis with cutaneous
eruptions, besides articular and muscular pain.21 Also, these drugs are
ineffective against the chronic phase of the disease demonstrating the
urgent need for new drugs.20 In our continuing search for anti-parasitic
natural products from Brazilian plants,22-24 the crude MeOH extract
from leaves of P. barbatus displayed in vitro anti-trypanosomal ac-
tivity and was subjected to bioactivity-guided fractionation. Using
several chromatographic procedures, the compound 1,2,3,4,6-penta-
-O-galloyl-b-D-glucose (PGG) was isolated for the first time in the
Plectranthus genus, identified after NMR and LRESIMS analysis.
RESULTS AND DISCUSSION
Preliminary testing of crude MeOH extract obtained from leaves
of P. barbatus revealed considerable anti-trypanosomal activity. With
the aim of isolating active compounds, this extract was partitioned
using EtOAc and organic active phase was subjected to several steps
of bioactivity-guided fractionation using SiO2 and Sephadex LH-20.
An amorphous white solid was thus isolated, found to be composed
of a pure substance after TLC and HPLC analysis.
This compound showed a molecular formula of C41H32O26 eviden-
ced by a pseudo-molecular ion peak at m/z 938.6 [M-H]- observed in the
LRESIMS (negative mode) spectrum. The 1H NMR spectrum showed
signals attributed to seven protons of the glucopyranosyl unit at d 6.14
(d, 1H, J = 8.4 Hz, H-1), d 5.52 (dd, 1H, J = 8.4; 9.9 Hz, H-2), d 5.81
(t, 1H, J = 9.9, H-3), d 5.49 (t, 1H, J = 9.6 Hz, H-4), d 4.42 (d, 1H, J =
10.5 Hz, H-5), d 4.32 (m, 2H, H-6). This spectrum also showed singlets
at d 7.01, 6.95, 6.88, 6.85, 6.80 which could be assigned to H-2’/H-6’
of five tetrasubstituted aromatic rings (Gal 1-Gal 5), suggesting that
the glucopyranosyl unit could be esterified with five galloyl units. The

dos Santos et al.
2230 Quim. Nova
13C NMR and DEPT 135° spectra showed signals of methynic carbons
at d 93.9 (C-1), d 72.4 (C-2), d 74.2 (C-3), 69.9 (C-4), 74.5 (C-5), and
methylenic carbon at d 63.3 (C-6), characteristic of a glucopyranosyl
unit.25 Additionally, were observed peaks were observed at the d 110-
168 range, assigned to galloyl units.26 The complete structure of the
bioactive compound was determined by analysis of the HMBC spec-
trum, which showed cross peaks between the signals at d 7.01 (H-2´
and H-6´) and d 166.4 (C-7') as well as between the peaks at d 6.14
(H-1) and d 166.4 (C-7'). Finally, the comparison of NMR data with
those described in the literature25-27 (Table 1) allowed the identification
of 1,2,3,4,6-penta-O-galloyl-b-D-glucose (PGG) Figure 1, whose
occurrence has been described for the first time in the Plectranthus
genus. This compound was previously identified in various medici-
nal plants, including species of Rhus, Schinus, Galla and others.27,28
Pharmacologically, this substance showed diverse biological activities,
such as antioxidant,25 anticancer,29 and inhibitory action against the
invasion of mouse melanoma.30 Additionally, an inhibitory effect of
the a-glycosidase of PGG was described, suggested by the presence
of galloyl substituents at the glucose unit26.
Ethnopharmacological studies have revealed anti-parasitic activi-
ty of the Plectranthus species, especially an anti-leishmanial effect.
P. amboinicus has been used in popular medicine to treat cutaneous
leishmaniasis in a rural endemic area of Bahia state, Brazil.31 Its
activity against L. chagasi promastigotes has also been described
from MeOH extract of P. barbatus.32
In our assays, trypomastigotes of T. cruzi showed susceptibility
to the isolated PGG, with an EC50 value of 66.66 µM (Table 2).
Considering the activity against the parasite, it could also be shown
that PGG was 6.6-fold more effective than the standard drug benzni-
dazole, although the cytotoxicity against mammalian cells, resulted in
a CC50 of 39.52 µM (Table 2). In summary, our results demonstrated
that PGG displayed activity against T. cruzi parasites and suggested
the performance of additional studies on reducing its cytotoxicity with
the aim of using this compound as a prototype for the development
of new drugs to treat Chagas disease.
EXPERIMENTAL
General procedures
1H and 13C NMR spectra were recorded on a Bruker DPX-300
spectrometer operating at 300 and 75 MHz, respectively, using
CD3OD (Tedia Brazil) as the solvent and internal standard (dH 3.21
and dC 49.2). LRESIMS (negative mode) was performed on an Esquire
3000 Plus spectrometer. Analytical HPLC analysis was performed on
a Dionex Ultimate 3000 chromatograph, using a Luna Phenomenex
C18 column (3 µm, 150 × 5 mm) and an UVD-DAD detector. Silica
gel (Merck, 230-400 mesh) and Sephadex LH-20 (Amersham
Biosciences) were used for column chromatographic separation, while
silica gel 60 PF254 (Merck) was used for analytical (0.25 mm) TLC.
Plant material
Leaves of P. barbatus were collected at the Instituto Plantarum de
Estudos da Flora, Brazil, in Nova Odessa/São Paulo state, in October
2010. The studied specie was identified by MSc. H. Lorenzi and a
voucher specimen has been deposited in the Herbarium Plantarum
under number H. Lorenzi 771.
Extraction and isolation
Leaves of P. barbatus (227 g) were dried, milled and extracted
at room temperature with 3 X 1 L of MeOH. Concentration in vacuo
Table 2. Anti-trypanosomal activity and cytotoxicity of PGG and benznidazole
Compound
EC50 µM
T. cruzi tripomastigotes
(95% C.I.)
CC50 µM
NCTC
(95% C.I.)
PGG 66.66
(57.00-77.96)
39.52
(33.10-47.17)
Benznidazole 441.15
(406.54-478.85)
470.38
(415.38-532.69)
EC50: 50% Effective Concentration; CC50: 50% Cytotoxic Concentration;
95% C.I. : 95% confidence interval. For EC50 values was determined % T.
cruzi survival: 159.57 mM: 15.5 ± 1.92; 79.79 mM: 27.53 ± 0; 39.89 mM:
95.14 ± 2.85; 19.95 mM: 100 ± 0.42. (Values expressed as mean ± SEM of
two determinations)
Table 1. 1H, 13C and HMBC data of 1,2,3,4,6,-penta-O-galloyl-b-D-glucose
(CD3OD, 300 and 75 MHz)
Position dHdCHMBC (H → C)
16.14 (1H, d, 8.4 Hz) 93.4 C2, C3, C7’ (Gal 1),
25.52 (1H, dd J = 8.4,
9.9 Hz)
72.4 C1, C3, C4, C7’ (Gal 2)
35.81 (1H, t, 9.9 Hz) 74.2 C1, C2, C4, C5, C7’ (Gal 3)
45.49 (1H, t, 9.6 Hz) 69.9 C2, C3, C5, C6, C7’ (Gal 4)
54.42, (1H, d, 10.5 Hz) 74.5 C3, C4, C6
6a/6b 4.32 (2H, m)63.3 C4, C5, C7’ (Gal 5)
Gal 1
1’ - 119.8 EC50
2’/6’ 6.95 (2H, s)110.7 C1’,C3’, C4’, C5’, C6’, C7’
3’/5’ - 141.0 -
4’ - 146.7 -
7’ - 166.4 -
Gal 2
1’ - 120.3 -
2’/6’ 6.85 (2H, s)110.5 C1’, C3’, C4’, C5’, C6’, C7’
3’/5’ - 140.5 -
4’ - 146.5 -
7’ - 167.2 -
Gal 3
1’ - 120.5 -
2’/6’ 6.80 (2H, s)110.5 C1’, C3’, C4’, C5’, C6’, C7’
3’/5’ - 140.3 -
4’ - 146.4 -
7’ - 167.4 -
Gal 4
1’ - 120.3 -
2’/6’ 6.88 (2H, s)110.6 C1’, C3’, C4’, C5’, C6’, C7’
3’/5’ - 140.6 -
4’ - 146.6 -
7’ - 167.1 -
Gal 5
1’ - 121.1 -
2’/6’ 7.01 (2H, s)110.4 C1’, C3’, C4’, C5’, C6’, C7
3’/5’ - 140.2 -
4’ - 146.6 -
7’ - 168.1 -

Anti-trypanosomal activity of 1,2,3,4,6-penta-O-galloyl-b-D-glucose 2231
Vol. 35, No. 11
yielded 9.1 g of crude extract. Part of this material (9 g) was dissolved
in MeOH:H2O 8:2 and subsequently extracted using hexane, EtOAc
and CHCl3. After evaluation of trypanocidal potential, the active
EtOAc phase (4.0 g) was subjected to silica gel column chromatog-
raphy eluted with increasing amounts of EtOAc in hexane to give 77
fractions (10 mL). After TLC analysis, these fractions were combined
into 23 groups (A1-A23), which displayed activity against T. cruzi
trypomastigote forms. As bioactivity was detected at group A18, part
of this material (700 mg) was introduced to a Sephadex LH-20 column
which was eluted with MeOH. This procedure afforded 40 fractions
(5 mL each) which were pooled into 11 groups (A18/1-A18/11) after
TLC analysis. As the activity was detected at the A18/1 group, this
was analyzed by HPLC (MeOH:H2O 7:3) to afford 48 mg of pure
1,2,3,4,6-penta-O-galloyl-b-D-glucose.
1,2,3,4,6-penta-O-galloyl-b-D-glucose
White amorphous solid. NMR data: see Table 1. LRESIMS m/z
938.6 [M – H]–.
Animals
BALB/c mice and Golden hamsters (Mesocricetus auratus)
were supplied by the animal breeding facility at the Adolfo Lutz
Institute of São Paulo and maintained in sterilized cages under a
controlled environment, receiving water and food ad libitum. Animal
procedures were performed with the approval of the Research Ethics
Commission.
Anti-trypanosomal activity
Trypomastigotes of T. cruzi (Y strain) were obtained from LLC-
MK2 culture (ATCC CCL 7), counted on a Neubauer hemocytometer
and applied to 1 x 106/wells in 96-well plates. The substance was
dissolved in DMSO and further incubated in various concentrations
ranging from 150 to 0.073 µg/mL for 24 h at 37 °C with 5% CO2 in
a humidified incubator, using benznidazole as the standard drug. The
DMSO concentration did not exceed 0.5% (v/v) of the final volume
of wells to avoid damage to parasites. The viability of parasites was
measured by the trypomastigote cellular conversion of MTT solu-
tion - bromide 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium
bromide) - in insoluble formazan by mitochondrial enzymes.33 The
extraction of formazan was performed with 10% (v/v) SDS for 18
h (100 mL/well) at 24 °C.34 MTT and the test compound (without
parasites) was added to an internal control to investigate possible
oxidative reaction.
Determination of cytotoxicity against mammalian cells
Mouse subcutaneous connective tissue cells, NCTC clone 929,
(ATCC CCL-1™) were seeded at 4 x 104 cells/well in 96-well micro-
plates and incubated with compounds to the highest concentration of
200 µg/mL for 48 h at 37 ºC in a 5% CO2 humidified incubator. The
viability of cells was determined by the MTT assay.35 Benznidazole
was used as the standard drug. Control cells were incubated in the
presence of DMSO and without drugs.
Statistical analysis
Data represent the mean and standard deviation of duplicate
samples from two independent assays. The IC50 values were calcu-
lated using sigmoidal dose-response curves in Graph Pad Prism 5.0
software. The Mann-Whitney t-test (unpaired two-tailed) was used
for significance testing (p < 0.05).
ACKNOWLEDGMENTS
This work was funded by grants provided by FAPESP and CNPq.
J. H. G. Lago and A. G. Tempone are grateful to CNPq to Research
Fellow. R. T. dos Santos and L. L. Hiramoto are indebted to CAPES
and PIBIC/UNIFESP by scholarships.
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