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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 105(1): 45-51, February 2010 45
online | memorias.ioc.fiocruz.br
Antimycobacterial neolignans isolated from Aristolochia taliscana
Rosalba León-Díaz1, Mariana Meckes1, Salvador Said-Fernández2, Gloria Maria Molina-Salinas2,
Javier Vargas-Villarreal2, Javier Torres3, Julieta Luna-Herrera4, Adelina Jiménez-Arellanes1/+
1Unidad Investigación Médica en Farmacología de Productos Naturales 3Unidad de Investigación Médica en Enfermedades Infecciosas y
Parasitarias, Hospital de Pediatría, Instituto Mexicano del Seguro Social, Centro Médico Nacional Siglo XXI, Av. Cuauhtémoc 330,
Col. Doctores, CP 06720, Delg. Cuauhtémoc, México DF, México 2División de Biologia Celular y Molecular, Centro de Investigacion
Biomedica del Noroeste, Instituto Mexicano del Seguro Social, Nuevo Leon, México 4Laboratorio de Inmunoquímica II, Departamento
de Inmunoquímica, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México DF, México
Tuberculosis (TB - Mycobacterium tuberculosis) is an ancient infectious disease that has appeared once again
as a serious worldwide health problem and now comprises the second leading cause of death resulting from a single
infection. The prevalence of multidrug resistance (MDR) TB is increasing and therapeutic options for treatment are
not always accessible; in fact, some patients do not respond to the available drugs. Therefore, there is an urgent
need to develop novel anti-TB agents. The aim of the present study was to screen extracts of Aristolochia taliscana,
a plant used in traditional Mexican medicine to treat cough and snake bites, for antimycobacterial activit y. The
hexanic extract of A. taliscana was tested by microdilution alamar blue assay against Mycobacterium strains and
bioguided fractionation led to the isolation of the neolignans licarin A, licarin B and eupomatenoid-7, all of which
had antimycobacterial activity. Licarin A was the most active compound, with minimum inhibitory concentrations
of 3.12-12.5 µg/mL against the following M. tuberculosis strains: H37Rv, four mono-resistant H37Rv variants and 12
clinical MDR isolates, as well as against five non-tuberculous mycobacteria (NTM) strains. In conclusion, licarin A
represents a potentially active anti-TB agent to treat MDR M. tuberculosis and NTM strains.
Key words: antimycobacterial neolignans - A. taliscana - M. tuberculosis H37Rv - MDR M. tuberculosis -
non-tuberculous mycobacteria
Financial suppor t: Instituto Mexicano del Seguro Social (FOFOI FP-
2003-0 09, 2005/1/I/102), CONACYT (to RLD)
+ Corresponding author: adelinaj@servidor.unam.mx
Received 4 June 2009
Accepted 4 August 2009
Medicinal plants are an important natural source of
novel leads in the field of antimycobacterial therapeu-
tics (Cantrell et al. 2001, Copp & Pearce 2007, Gutier-
rez-Lugo & Bewley 2008). According to ethnobotanical
data, some species of Aristolochia, such as Aristolochia
elegans and Aristolochia grandiflora have been widely
utilized in Mexican traditional medicine to treat cough
(Diaz 1976). A preliminary biological evaluation of the
hexanic extract from Aristolochia taliscana Hook roots
showed that it possessed an in vitro antimycobacterial ef-
fect against Mycobacterium tuberculosis H37Rv a nd My-
cobacterium avium [minimum inhibitory concentrations
(MIC's) = 50 µg/mL]. The plant is commonly known in
Mexico as guaco or raíz de guaco and neolignans with
antiprotozoal activity have already been identified in the
species (Enriquez et al. 1984, Abe et al. 2002).
In recent years, the number of patients with tuber-
culosis (TB) has increased rapidly due, in part, to the
appearance of multidrug-resistant (MDR) and exten-
sively drug-resistant (XDR) strains in both develop-
ing and developed countries. One-third of the world’s
population is currently infected with M. tuberculosis
and approximately 10% of these cases will develop
clinical manifestations, particularly those patients with
compromised immunological systems. The AIDS/HIV
pandemic has contributed to the worsening of the prob-
lem; in fact, about 30% of registered mortality has been
associated with TB, especially in developing countries
(Jain & Mondal 2008, Rivers & Mancera 2008). Patients
with AIDS are also susceptible to becoming infected
with non-tuberculous mycobacteria (NTM) such as M.
avium (Rodriguez et al. 2006). It is estimated that the
worldwide prevalence of MDR-TB is about 3.2% and
that 6.6% of these cases are XDR-TB (Rivers & Mancera
2008). The emergence of XDR-TB strains constitutes a
serious health problem because, at present, there is no
pharmaceutical alternative for treating patients infected
with such strains (Tomioka 2006, Zager & McNerney
2008). Consequently, novel drugs to treat or prevent the
disease are urgently needed (O´Brien & Spigelman 2005,
Gutierrez-Lugo & Bewley 2008).
The aim of the present paper was to isolate and struc-
turally characterize A. taliscana hexane-extract com-
pounds that possessed activity against M. tuberculosis
H37Rv, mono-resistant variants of H37Rv, MDR M. tu-
berculosis clinical isolates and NTM.
MATERIALS AND METHODS
Plant materials - A. taliscana roots were pur-
chased at a medicinal plant ma rket i n city of Mexico,
Mexico. T he plant material was compa red with t he
botanical specimen deposited at the Herbarium of the
Instituto Mexicano del Seguro Social and a voucher
was deposited under code 1106.
Antimycobacterial neolignans from A. taliscana • Rosalba León-Díaz et al.
46
Extraction and isolation - Powdered air-dried roots
(1.5 kg) were macerated (3 × 48 h) with 12 L of n-hexane.
The extract was filtered and evaporated in vacuo to
yield 33 g of the crude extract. Open column chroma-
tography (CC) was performed employing the silica gel
60 GF254 (70-230 mesh, Merck) as the stationary phase
and silica gel 60 F254 pre-coated aluminium plates (0.2
mm, Merck) for analytical and preparative thin-layer
chromatography (TLC) analysis. Spots were visualized
by spraying with a 10% solution of H2SO4 followed by
heating the plates at 100°C.
For silica gel CC, the extract (15 g) was fractionated
by eluting with n-Hex: CHCl3(100→0) and CHCl3:MeOH
(100→0); 72 fractions (250 mL each) were obtained. The
primary fractions (F1-F15) were combined according to
the results from the TLC analysis. All primary fractions
were tested for antimycobacterial activity.
From the active fraction F5-F7, 975 mg of white
needles with a melting point (m.p.) of 82-86oC (lit. m.p.
89-90°C) crystallized. The compound was identif ied as
licarin B (1) and was also detected in fractions F8-F11.
Fractions F8-F11 (2.5 g) were re-chromatographed on
CC using silica gel (75 g) with a solvent gradient of n-
Hex:CHCl3 (100→0) and CHCl3:MeOH (100→0). This
process yielded 14 secondary fractions (FA-FN) of 150
mL each. Secondary fraction FD yielded 40 mg of 1 and
fraction FF (400 mg) was re-chromatographed in CC and
eluted with n-Hex: CHCl3(100→0) and CHCl3:MeOH
(100→0) to obtain eight tertiary fractions Fa-Fh. The
isolated maroon-coloured powder (80 mg) from Fc-Fe
was characterized as eupomatenoid-7 (2) with an m.p. of
100 -104 oC (lit. m.p. 105-106°C).
The secondary fraction FJ (300 mg) was further re-
c hr o ma to g ra p he d o n CC ut i li z i ng n- He x :C HC l3 a n d C HC l 3
as elution systems. Nine 50 mL tertiary fractions (Fa’-Fi’)
were obtained. From Fb’, 196 mg of a white product was
obtained by crystallization with an m.p. of 107-110°C (lit.
m.p. 133-134°C), which was identified as licarin A (3).
Chemical characterization - Chemical character-
ization of the isolated neolignans was determined by
1H-NMR (Eclipse 300 Jeol, 300 MHz) and 13C-NMR
(Variant Unity, 300 MHz) using tetramethylsilane as an
internal standard in CDCl3. Electron impact-mass spec-
tra were obtained on a Jeol AX-505 HA mass spectrom-
eter at 70 eV. Infrared (IR) spectra on film over NaCl in
a Bruker model Tensor 27 spectrometer, optical rotation
in a Perkin Elmer model 345 polarimeter at 25°C using
a sodium lamp (589 nm) and m.p. in a Fisher-Johns ap-
paratus. All the spectroscopic data (1H and 13C-NMR)
of each compound were compared with those previously
reported in the literature (Enriquez et al. 1984) and are
described in Tables I, II.
Licarin B (1) - White needles soluble in CHCl3, m.p.
82-86°C, [α]D
25°C = -0.262 (MeOH), IR: νma x 2900, 1600
and 1050-1200. IE-MS: m/z (rel. int.) 324 [M+ (100)], 309
(12), 293 (8), 278 (28), 202 (6), 135 (20), 121 (8), 91 (7),
77 (14) and 46(5).
TABLE I
1H-NMR spectral data (δ scale) and coupling constant (J, Hz) for compounds 1-3 isolated from Aristolochia taliscana
Proton Compound 1 Compound 2 Compound 3
25.09
(J2-3 = 8.97)
-5.09
(J2-3 = 9.5)
33.37-3.45 -3.39-3.47
46.72- 6.79
(J4-6 = 1.5)
7.0 3
(J4-6 = 1.5)
6.77
(J4-6 = 1.5)
66.79 6.82
(J6-4 = 1.5 )
6.77
(J4-6 = 1.5)
2´ 6.92
(J2´-5´ = 1.7)
7.01
(J2´-5´ = 2.0)
6.97
(J2´-5´ = 1.9)
5´ 6.87
(J5´-6´ = 8.3)
7.2 5 -7. 32
(J5´-6´ = 8.2)
6.89
(J5´-6´ = 8.3)
6´ 6.87
(J6´-5´ = 8.3, J2´- 6´ = 0- 44)
6.98
(J5´-6´ = 8.2, J2´-6´ = 0.6)
6.89
(J5´-6´ = 8.3, J2´- 6´ = 0.44)
α6.35 6.49 6.36
β6.04-6.15 6.15-6.27 6.04-6.16
γ1.86 1.90 1.86
CH31.37 2.40 1.37
OCH33.88 3.97 3.87
OCH3-4.03 3.89
OCH2O5-95 - -
OH -5.75 5.62
all compounds had Jα-β = 15.87 Hz, Jα-γ = 1.7 Hz and Jβ-γ = 6.5 Hz. Compounds 1 and 3 had J3-Me = 6.8 Hz. Record in CDCl3 at 300 MHz.
47
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 105(1), February 2010
Eupomatenoid-7 (2) - Maroon-coloured powder, solu-
ble in CHCl3, m.p. 100-104°C, [α]D
25°C = -0.280 (MeOH),
IR: νmax 3429, 2937, 2849, 1725, 1604, 1513, 1452, 1371,
1267, 1221, 1147 and 1056. IE-MS: m/z (rel. int.) 324 (100),
309 (20), 293 (15), 123 (6), 91 (9), 77 (5) and 31 (15).
Licarin A (3) - White powder, soluble in CHCl3, m.p.
107-110°C, [α]D
25ºC = -0.15 (MeOH), IR: νma x 3541, 2938,
1673, 1608, 1496, 1269 and 1143. IE-MS: m/z (rel. int.)
326 (100), 311 (20), 308(7), 295 (5), 202 (10), 123 (8), 91
(10), 77 (8) and 31 (25).
Mycobacterium strains - The following mycobacte-
ria from the American Type Culture Collection (ATCC)
were used: M. tuberculosis H37Rv (27294); mono-resis-
tant strains: H37Rv isoniazid-resistant (35822), H37Rv
streptomycin-resistant (35820), H37Rv rifampicin-resis-
tant (35838) and H37Rv ethambutol-resistant (35837); M.
avium (35717) and Mycobacterium smegmatis (35798).
In addition, drug-resistant M. tuberculosis clinical iso-
lates (12 strains) obtained from Mexican patients with
pulmonary disease were also tested. Drug-resistant M.
tuberculosis clinical isolates were selected based on their
drug susceptibility patterns against antimycobacterial
drugs employing the microdilution alamar blue assay
(MABA) test. In addition, the following clinical or envi-
ronmental non-TB mycobacteria isolates were included:
Mycobacterium chelonae, Mycobacterium fortuitum
and Mycobacterium non-chromogenicum. The strains
were cultured in Middlebrook 7H9 broth supplemented
with 10% OADC enrichment (Becton Dickenson, USA)
at 37°C until a logarithmic growth phase was achieved.
M. tuberculosis and non-TB mycobacteria were diluted
in 7H9 at the ratios of 1:20 and 1:50, respectively. Bacte-
rial suspensions were fresh when utilized in the assays.
Antimycobacterial assay - Extracts, fractions and
the pure compounds were evaluated by the previously
described MABA assay (Jimenez-Arellanes et al. 2003,
2007). Briefly, samples were dissolved in dimethyl sulfox-
ide (DMSO) (20 mg/mL) under sterile conditions. Serial
two-fold dilutions of each sample (range, 100-3.12 µg/mL)
were prepared to a final volume of 100 µL with 7H9 broth
and 100 µL of each mycobacterium suspension was added
to 96-well sterile microplates (Nunc). For M. tuberculosis,
plates were incubated at 35°C during five days, whereas
non-TB mycobacteria were incubated for two days. MIC
is expressed as the lowest concentration of the compound
that causes 99% inhibition of mycobacterium growth. All
assays were run in duplicate and streptomycin (0.5 µg/mL,
Sigma), isoniazid (0.06 µg/mL, Sigma) and rifampicin (0.1
µg/mL, Sigma) were utilized as positive controls.
Cytotoxicity assay - The assay was carried out in a
J774A.1 murine macrophage cell line (ATCC HB-197)
using the trypan blue exclusion test. Briefly, purified
neolignans were dissolved in DMSO at a concentra-
tion of 20 µg/mL. Cells were grown in 24-well plates
using DMEM supplemented with 10% foetal bovine se-
rum (FBS) and antibiotics. Immediately prior to testing,
monolayers were washed with warm Hanks’ balanced
salt solution. Serial two-fold dilutions of each compound
were prepared in DMEM supplemented with 10% FBS
(1-1/16 of MIC against M. tuberculosis H37Rv) and 1
mL/well of each dilution was added. To evaluate cell
viability, controls were included in the microplate by
adding DMEM media with DMSO; cell viability was
determined after a 24-h incubation period. Trypan blue
solution was added and the percentage of viable cells
was calculated to determine the cytotoxic index (IC50).
The assay was run in triplicate.
Acute toxicity in mice - Male Balb/c mice (22 ± 2.2 g)
were used to determine the acute toxicity parameter fol-
lowing the methodology previously described by Lorke
(1983) and according to the guidelines of the local Ethi-
cal Committee for Experimentation in Animals. Animals
were maintained under standard environmental conditions
at 12-h light/dark photoperiods with free access to food
and water. Mice were randomly divided into five groups
of three animals each. Group 1 received the control ve-
hicle (Tween 20:H2O 2:8), while Groups 2-5 were treated
orally with the crude extract at doses of 0.6, 1.0, 1.6 and
2.9 g/kg. The same design was employed to test the most
active primary fraction (F8-F11) and the pure compound
(l ic ari n A). A ll sam ples were solubiliz ed in Tween 20:H2O
(2:8) and were intragastrically administered in a volume
that was less than 10 mL/kg of body weight. Treatment
response was monitored at 1, 2, 4, 6 and 24 h and daily for
14 days, registering any signal of toxicity. At the end of
the experimental period, the animals were sacrificed in a
CO2 chamber to obtain the internal organs (lung, kidney,
heart, spleen and liver) for pathological analysis.
TABLE II
13C-NMR spectral data for compounds 1-3 in CDCl3 isolated
from Aristolochia taliscana
Carbon Compound 1 Compound 2 Compound 3
293.38 151. 48 9 3.77
345.75 110 .19 45.61
3ª 133.08 133.05 133.28
4113.3 4 10 9.16 113. 32
5132.21 133.61 132.20
6109. 29 104.42 10 9.29
7144 .10 17 7.82 144.43
7ª 146 .50 142.09 146. 58
1’ 134.32 123.67 132.11
2’ 106 .77 10 9.43 108.94
3’ 147. 87 146 .58 14 6. 66
4’ 147. 58 109.43 145.58
5’ 108.04 114.44 114 .07
6’ 120.18 120.62 119. 95
α130.91 131.4 6 130.93
β123.44 124.36 123.45
γ17.8 7 18.41 18 .33
CH3 (3) 18. 33 9.57 17. 56
OCH3 (7) 55.94 56.05 55.93
OCH3 (3’) - 56.09 55.9 7
OCH2101.06 - -
record in CDCl3 at 300 MHz.
Antimycobacterial neolignans from A. taliscana • Rosalba León-Díaz et al.
48
RE SULTS
Chemical characterization of the isolated neolig-
nans - The three neolignans were characterized by com-
paring spectral data (Tables I, II) with those previously
reported in the literature (Enriquez et al. 1984) and the
respective molecular structures of the compounds are
illustrated in Fig 1.
Biological evaluation - As shown in Table III, a
MIC of 50 µg/mL was determined for the hexanic crude
extract against M. tuberculosis H37Rv and M. avium.
Primary fractionation yielded F8-F11 as the most ac-
tive fractions, with MIC’s of 12.5-50 µg/mL against
M. tuberculosis H37Rv strains and 12.5-100 µg/mL
against M. avium. These fractions, as well as F5-F7,
were active against all tested mono-resistant strains
of H37Rv and MDR M. tuberculosis clinical isolates
(SIN3, SI N4, MMDO and HG8) and the MIC values
obtained ranged from 12.5-50 µg/mL (Table IV). In ad-
dition, fractions F8-F11 inhibited the growth of NTM
as follows: M. non-chromogenicum (MIC = 25 µg/mL)
and M. smegmatis, M. chelonae and M. fortuitum (MIC
= 50 µg/mL); the fractions were less active against M.
avium (MIC = 100 µg/mL). By contrast, fractions F5-
F7 were highly active against M. non-chromogenicum
(MIC = 12.5 µg/mL) (Table V).
Antimycobacterial activity of the pure isolated com-
pounds is shown in Table VI. Licarin B (1) was moderately
active against H37Rv and against mono-resistant variants
(MICs, 25-50 µg/mL), but was highly active against the
majority of MDR M. tuberculosis clinical isolates tested
(with MIC values ranging from 12.5-50 µg/mL). Eupo-
matenoid-7 (2) was active against H37Rv strains (MIC
= 25 µg/mL), the four mono-resistant variants of H37Rv
and three of the MDR clinical isolates tested (MIC values
ranging from 12.5-25 µg/mL). The most clinically rele-
vant activity of this compound (MIC = 6.25 µg/mL) was
against an M. tuberculosis clinical isolate (SIN4) that is
resistant to first- and second-line drugs (Table VI).
Finally, while licarin A (3) exhibited moderate activ-
ity against M. tuberculosis H37Rv (MIC = 25 µg/mL), this
compound was highly active against all mono-resistant and
MDR M. tuberculosis strains tested, with MIC's ranging
from 3.12-12.5 µg/mL (Table VI). Clinical isolates with
highest sensitivity to this compound included MMDO,
HG8 and SIN4. I n addition , lica rin A (3) inhibited the NT M
M. a vium , M. smegm atis, M. fortuitum (all with MIC = 6.25
µg/mL) and M. chelonae (MIC = 3.12 µg/mL) (Table V).
Chemical str uctu res of the active neolignans (1-3) from Aristolo-
chia taliscana.
TABLE III
Antimycobacterial activity of the hexanic extract and pri-
mary f ractions from Aristolochia taliscana
Sample
Minimum inhibitory concentrations
(µg/m L)
Mycobacterium
tuberculosis H37Rv
Mycobacterium
avium
Hexanic extract 50 50
F1 ND ND
F2 100 200
F3 ND ND
F4 200 200
F5-F 7 100 200
F8 50 100
F9 25 100
F10 12.5 12.5
F11 50 50
F12 200 100
F13-F15 200 200
ND: non deter mined.
TABLE IV
Activity of primary fractions against Mycobacterium tuberculo-
sis H37Rv (reference strain), its four monoresistant variants and
against multidrug-resistant clinical isolates of M. tuberculosis
M. tuberculosis MIC (µg/mL)
Monoresistant F5-F7 F 8 -F11
INH-R 25 25
RIF-R 50 25
STR-R 12.5 25
EMB-R 25 25
Clinical isolates
SIN3 25 25
SIN4 50 25
MMDO 12.5 25
HG8 25 12.5
EMB-R: ethambutol-resistant of M. tuberculosis H37Rv; INH-
R: isoniazid-resistant; MIC: minimum inhibitory concentrations;
RIF-R: rifampicin-resistant; STR-R: streptomycin-resistant.
49
Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 105(1), February 2010
Cytotoxicity assay of the pure neolignans on murine
macrophage J774A.1 cell line yielded values of IC50 =
6.25 µg/mL for licarin A and B and IC50 = 3.12 µg/mL
for eupomatenoid-7. The acute toxicity of the crude ex-
tract, F8-F11 and its most active component, licarin A,
determined in mice was > 1.706 mg/kg.
TABLE V
Antimycobacterial effect of the primary fractions and the pure compounds against non-tuberculous Mycobacteria strains
Strain
MIC (µg/mL)
Primary fractions Pure compounds
F5-7 F8 -11 1 2 3
Mycobacterium non- chromogenicum 12.5 25 12.5 25 ND
Mycobacterium smegmatis > 200 50 > 200 25 6.25
Mycobacterium fortuitum > 200 50 > 200 50 6.25
Mycobacterium chelonae > 200 50 > 200 25 3.12
Mycobacterium avium > 200 100 > 200 50 6.25
MIC: minimum inhibitory concentrations; ND: non determined.
TABLE VI
Antimycobacterial activity of the pure compounds from Aristolochia taliscana against Mycobacterium tuberculosis H37Rv
and of the clinical isolates of multidrug-resistant M. tuberculosis
Strain Drug resistance patterna , b
MIC of pure compounds
(µg/mL )
M. tuberculosis 1 2 3
H37Rv INH, RIF, STR, EMB susceptible 50 25 25
Monoresistant
INH-R 25 25 3.12
RIF-R 50 12.5 6.25
STR-R 25 25 3.12
EMB-R 25 25 6.25
Clinical isolates
MMDO INH, EMB 12.5 12.5 3.12
MTY650 STR, INH 12.5 50 6.25
MTY663 STR, INH, RIF, EMB, PZA 12.5 50 12.5
MTY675 STR, INH, EMB 12.5 50 12.5
MTY282 STR, INH, EMB, PZA 12.5 50 12.5
HG8 EMB, CLR, ETH 25 25 3.12
SIN3 STR, INH, R IF, EMB, RFB, CLR, ETH 25 25 6.25
MTY234 STR, INH, RIF, PZA 25 50 12.5
MTY112 STR, INH, RIF, EMB 25 50 12.5
MTY559 STR, EMB 25 50 12.5
SIN4 STR, INH, RIF, EMB, RFB, ETH, OFX 50 6.25 3.12
MTY172 INH, PZA 50 50 12.5
a: clinical isolates resistant to: streptomycin (STR), isoniazid (INH), rifampicin (RIF), ethambutol (EMB), rifabutin (RFB),
ethionamide (ETH), clarithromycin (CLR), ofloxacin (OFX), pyrazinamide (PZA); b: resistant pattern was determined by the
microdilution alamar blue assay. MIC: minimum inhibitory concentrations.
DISCUSSION
TB is a severe global health problem and the search
for novel therapeutic molecules is a necessity due to
the appearance of resistance to the anti-mycobacterial
drugs currently in use (Cantrell et al. 2001, O’Brien
& Spigelman 2005, Tomioka 2006, Gutiérrez-Lugo &
Antimycobacterial neolignans from A. taliscana • Rosalba León-Díaz et al.
50
Bewley 2008). Medicinal plants comprise a promising
natural source for the discovery of anti-TB dr ugs and
the in vitro activity of several secondary metabolites
has already been recognized. At present, 12-demeth-
ylmulticauline isolated from Salvia multicaulis (MIC=
0.46 µg/mL), micromolide from Micromelum hirsutum
(MIC= 1.5 µg/mL) and (E)-phytol from Leucas volkensii
(MIC= 2 µg/mL) are the most highly active compounds
reported against M. tuberculosis H37Rv (Cantrell et al.
2001). Unfortunately, little information is available con-
cerning the activity of natural compounds against MDR
M. tuberculosis strains (Newton et al. 2002, Gibbons et
al. 2003, Luna-Herrera et al. 2007).
While the use of Aristolochia species has been dis-
cussed extensively because of its content of aristolochic
acid (Chen et al. 2007), this toxic compound was not
detected in A. taliscana-root hexane extract. Moreover,
the LD50 for the hexanic extract determined in mice was
> 1706 mg/kg. When evaluating this extract against M.
tuberculosis H37Rv and M. avium, moderate in vitro
activity (MIC = 50 µg/mL) was determined. Activity
against M. avium is of interest because currently, there
is a high frequency of TB cases associated with this spe-
cies in HIV/AIDS cases.
Bioguided fractionation of the extract led to the iso-
lation of the previously identified neolignans licarin B,
eupomatenoid-7 and licarin A (Enriquez et al. 1984, Abe
et al. 2002). While several biological effects (antibacte-
rial, antioxidant, anticancer, trypanocidal, neuroprotec-
tive, insecticidal and anti-inflammatory) of these com-
pounds have been reported, to our knowledge, this is the
first report on their anti-TB activity (Tsai et al. 2001,
Abe et al. 2002, Lee et al. 2004, Ma et al. 2004, 2005,
Park et al. 2004, Saleem et al. 2005).
Licarin A (LD50 > 1706 mg/kg) displayed the most
potent effect against all tested mono-resistant strains of
H37Rv and MDR clinical isolates of M. tuberculosis
(MIC's ranging from 3.12-12.5 µg/mL). Likewise, licarin
A was active against the non-TB mycobacteria M. avi-
um, M. chelonae, M. fortuitum and M. smegmatis (MICs
ranging from 3.12-6.25 µg/mL). A drug that is able to in-
hibit MDR M. tuberculosis and M. avium growth, such as
licarin A, would be of extremely high value in the clinic,
particularly in cases of HIV/AIDS and MDR/XDR.
Lignans are well-known secondary metabolites be-
cause of the cytotoxic effect they produce in several
cell lines (Tsai et al. 2001, Park et al. 2004, Kong et al.
2005). The cytotoxic activity of licarin A has also been
reported against P-388, KB16 and HT-29 cell lines (Tsai
et al. 2001) and this activity for licarin B (100 µM) has
been described against the human promyelocytic leu-
kaemia HL-60 cells, as it is the compound inactive for
caspase-3 activation. Meanwhile licarin A induces an
apoptotic effect by means of caspase-3 activation (Park
et al. 2004). On the other hand, Lee et al. (2004) reported
that licarin A is a potent inhibitor of phospholipase Cγ1
(PLCγ1) with an IC50 = 15.8 ± 1.3 µM and that it exerts
antiproliferative effects against three human cancer cell
lines [A-549 (lung), MCF7 (breast) and HCT-15 (colon)],
suggesting the use of licarin A as a cancer chemothera-
peutic and chemopreventive agent (Lee et al. 2004, Park
et al. 2004). The cytotoxicity of A. taliscana-isolated
neolignans on murine macrophages was IC50 = 3.25-
6.25 µg/mL; these values were similar to those deter-
mined for the MIC parameter.
The results obtained here permit us to suggest further
biological evaluation of the effect of licarin A against
macrophages infected with MDR M. tuberculosis, to de-
termine the compound’s intracellular activity.
In conclusion, a low-toxicity neolignan was isolated
from the hexane extract of A. taliscana roots, structur-
ally identified as licarin A and shown to be the most ac-
tive compound against all mycobacteria tested. Licarin
A is a new prototype molecule that exerts a relevant bio-
logical effect against the mycobacteria responsible for
MDR-TB, a pandemic that is increasing at present and
represents a serious health problem worldwide. In vivo
experimental studies are in progress to establish the anti-
TB potential of this compound.
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