Effect of boldo (Peumus boldus Molina) infusion on lipoperoxidation induced by cisplatin in mice liver

Abstract

Peumus boldus Molina (Monimiaceae), commonly referred to as 'boldo', is used in traditional Chilean medicine to treat hepatic and gastrointestinal diseases. Its leaves are rich in antioxidant compounds, principally alkaloids and flavonoids. This study evaluates the protective effect of a complete boldo leaf infusion on lipoperoxidation (MDA determination at 532 nm) induced by cisplatin in mice liver. To determine if the observed effect can be explained by the action of boldine or catechin, each compound was studied separately. The mice were divided into 8 groups (n = 6): (I) not treated; (II) treated with cisplatin 6 mg/Kg b.w.; (III) treated with boldo leaf infusion 5%; (IV) pretreated with boldo leaf infusion 5% and treated with cisplatin 6 mg/Kg b.w.; (V) treated with boldine 50 mg/Kg b.w.; (VI) pretreated with boldine 50 mg/Kg b.w. and treated with cisplatin 6 mg/kg.b.w.; (VII) treated with catechin; and (VIII) pretreated with catechin 50 mg/Kg b.w. and treated with cisplatin 6 mg/Kg b.w. As expected, the treatment with cisplatin significantly increased (p < 0.01) lipoperoxidation in comparison with the non-treated group. Pretreatment with boldo leaf infusion significantly diminished (p < 0.05) the lipoperoxidation induced by cisplatin with respect to the animals not pretreated with the infusion. The pretreatments with boldine and catechin significantly diminished (p < 0.05) the lipoperoxidation induced by cisplatin with respect to the group treated only with cisplatin. The results suggest that the boldo infusion is acting as a protector with respect to the oxidative hepatic damage caused by cisplatin, and that this protective ability would be due to the presence in the infusion of the natural antioxidants boldine and principally catechin. These findings suggest the potential use of the infusion as a chemoprotector.

Full text

Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 23, 1024–1027 (2009)
DOI: 10.1002/ptr
1024 J. FERNÁNDEZ ET AL.
Copyright © 2009 John Wiley & Sons, Ltd.
PHYTOTHERAPY RESEARCH
Phytother. Res. 23, 1024–1027 (2009)
Published online 14 January 2009 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/ptr.2746
Effect of boldo (Peumus boldus Molina)
infusion on lipoperoxidation induced by
cisplatin in mice liver
J. Fernández, P. Lagos, P. Rivera and E. Zamorano-Ponce*
Universidad del Bío-Bío, Casilla 447, Chillán, Chile
Peumus boldus Molina (Monimiaceae), commonly referred to as ‘boldo’, is used in traditional Chilean medi-
cine to treat hepatic and gastrointestinal diseases. Its leaves are rich in antioxidant compounds, principally
alkaloids and flavonoids. This study evaluates the protective effect of a complete boldo leaf infusion on
lipoperoxidation (MDA determination at 532 nm) induced by cisplatin in mice liver. To determine if the
observed effect can be explained by the action of boldine or catechin, each compound was studied separately.
The mice were divided into 8 groups (n ==
==
= 6): (I) not treated; (II) treated with cisplatin 6 mg/Kg b.w.; (III)
treated with boldo leaf infusion 5%; (IV) pretreated with boldo leaf infusion 5% and treated with cisplatin
6mg/Kg b.w.; (V) treated with boldine 50 mg/Kg b.w.; (VI) pretreated with boldine 50 mg/Kg b.w. and treated
with cisplatin 6 mg/kg.b.w.; (VII) treated with catechin; and (VIII) pretreated with catechin 50 mg/Kg b.w. and
treated with cisplatin 6 mg/Kg b.w. As expected, the treatment with cisplatin significantly increased (p <<
<<
< 0.01)
lipoperoxidation in comparison with the non-treated group. Pretreatment with boldo leaf infusion significantly
diminished (p <<
<<
< 0.05) the lipoperoxidation induced by cisplatin with respect to the animals not pretreated with
the infusion. The pretreatments with boldine and catechin significantly diminished (p <<
<<
< 0.05) the lipoperoxidation
induced by cisplatin with respect to the group treated only with cisplatin. The results suggest that the boldo
infusion is acting as a protector with respect to the oxidative hepatic damage caused by cisplatin, and that
this protective ability would be due to the presence in the infusion of the natural antioxidants boldine
and principally catechin. These findings suggest the potential use of the infusion as a chemoprotector.
Copyright © 2009 John Wiley & Sons, Ltd.
Keywords: Peumus boldus Molina, Cisplatin; lipoperoxidation; antioxidants; boldine; catechin.
INTRODUCTION
Even though cisplatin (cis-diaminodicloroplatin) is one
of the most active cytotoxic agents used for decades to
treat diverse solid cancerous tumors, evidence indicates
that treatment with this drug produces several collateral
effects, derived from oxidative stress that is induced at
both the renal and hepatic level (Pratibha et al., 2006).
A large number of natural products and dietary com-
ponents have been evaluated for their capacity to act
as antioxidants (Dragland et al., 2003), including plant
extracts (Cai et al., 2004; Zamorano-Ponce et al., 2004,
2006). Since many of these plant extracts have long
been used in traditional medicine, there is an emerging
scientific interest to better understand the mechanisms
that are involved with the different biological effects
(Valerio and Gonzales, 2005).
Boldo (Peumus boldus Molina), a tree of the Moni-
miaceae family that is endemic to Chile, has been known
for its medicinal properties since it was used by diverse
indigenous groups, including the Mapuche ethnia who
lived in Chile prior to the arrival of the Spanish in
the fifteenth century (Ferrer, 1904). Actually, boldo is
widely used in Chilean folk medicine and is recognized
as a medicinal herb in pharmacopoeia. Its use is
recommended as a regulator of the hepatic function,
colagogue, antispasmodic, digestive stimulant, and
nervous sedative. It is most commonly taken as a leaf
infusion that is drunk after eating (Muñoz et al., 2001).
Boldo leaves are rich in alkaloids, flavonoids, and
essential oils (O’Brien et al., 2006).
The present study evaluates the oxidative damage
induced by cis-DDP in Mus musculus, Balb-C mouse
hepatic tissue and investigates the possible protective
effect provided by the complete infusion of boldo leaves
as well as each of the natural antioxidants present in
boldo leaves, boldine and catechin, separately, deter-
mining the lipoperoxidation (MDA measurement at
532 nm) as an oxidative stress cellular marker (Hwang
and Kim, 2007).
MATERIALS AND METHODS
Animals. Males Mus musculus, Balb-C strain, 6–8 weeks
old, weighing approximately 25 g were supplied by the
Animal House of the Faculty of Science of University
of Bío-Bío. Animal use and care in all experiments
comply with Chilean ethical laws on animal manipula-
tion. Mice were housed in plastic cages at 22 ± 1°C,
60 ± 10% humidity and 12 h light/12 h dark cycle during
Received 15 May 2008
Revised 21 October 2008
Accepted 21 October 2008
* Correspondence to: E. Zamorano-Ponce, Laboratorio de Genética
Toxicológica GENETOX, Departamento de Ciencias Básicas, Facultad
de Ciencias, Universidad del Bío-Bío, Casilla 447, Chillán, Chile.
E-mail: ezamoran@ubiobio.cl

EFFECT OF BOLDO INFUSION ON LIPOPEROXIDATION INDUCED BY CISPLATIN IN MICE LIVER 1025
Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 23, 1024–1027 (2009)
DOI: 10.1002/ptr
Table 1. Protocols and treatment
Treatment Groups (n = 6) Dose
Untreated 1 –
Cis-DDP 2 6 mg/Kg b.w.a
Boldo infusion 3 5%/30 daysb
Infusion/cis-DDP 4 5%, 30 daysb/6 mg/Kg b.w.a
Boldine 5 50 mg/Kg b.w. /30 daysb
Boldine/cis-DDP 6 50 mg/Kg b.w. 30 daysb/6 mg/kg b.w.a
Catechin 7 50 mg/Kg b.w. /30 daysb
Catechin/cis-DDP 8 50 mg/Kg b.w. 30 daysb/6 mg/Kg b.w.a
a Intraperitoneal, 72 hours before sacrifice.
b Oral administration.
the acclimatization period to laboratory conditions
and throughout the entire experimental period. Water
was available ad libitum and the animals were fed a
conventional laboratory diet (pellet Kimber®). Mice
were separated in eight experimental groups with six
animals each, as shown in the schedule presented in
Table 1.
Once each treatment was complete, the animals were
sacrificed by cervical dislocation. The liver (0.5 g) was
removed, washed with cold sodium phosphate buffer
(50 mM, pH = 7.0), and homogenized in sodium phos-
phate buffer to give a 10% w/v homogenate. The homo-
genate was centrifuged at 750 × g for 10 min and the
supernatant (1 ml) was used for malondialdehyde
(MDA) determination by thiobarbituric acid reaction
(Ohkawa et al., 1979). The absorbance was measured
at 532 nm (Spectronic® Genesys™ .5 spectrophoto-
meters) and compared with those obtained from MDA
standards (
ε
= 2, 23 × 105M−1cm−1).
Plant material. Fresh Peumus Boldus Molina leaves
were sampled in March 2006 in the Campus Fernando
May, Universidad del Bío-Bío, Chillán, Chile (S 36°
36′ 05,1″, W 72° 04′ 38,1″) at 120 m above sea level.
The plant material was identified by Dr Patricio
Peñailillo Brito at the Institute of Plant Biology and
Biotechnology, Universidad de Talca, Chile. Voucher
specimen (No. 3332) has been kept at the Herbarium
of Universidad de Talca. Lead samples were dried at
room temperature.
Preparation of infusion. The infusion was made by
pouring 100 ml of boiling water on 5 g of plant mate-
rial. The mixture was left to stand for 30 min, filtered,
and then used. The infusion was freshly prepared for
each experiment.
Chemical and dosing. All chemicals were purchased
from Sigma Chemical Company (St Louis, MO, USA).
Boldine was used at a concentration of 50 mg/Kg b.w.
(Jimenez and Speisky, 2000). To prepare the solution
of boldine, it was first dissolved in ethanol and once
the ethanol was evaporated, the aqueous solution of
Boldine was prepared. Catechin was used at a concen-
tration of 50 mg/Kg b.w. (Isbrucker et al., 2006). The
dose of cis-DDP was 6 mg/Kg b.w.i.p. (Sadzuca, 1991).
The chemical solution dosing volume was determined
based on each animal’s body weight, considering a
volume of 0.5 ml. for a mouse of 20 g.
Statistical analysis. Experimental data were expressed
as a mean ± SD and statistically analyzed using U – t
test of Graph Pad Software. p values ≤ 0.05 were con-
sidered significant.
RESULTS
As shown in Table 2, lipoperoxidation significantly
increased (p < 0.01) in the cisplatin-treated animals in
comparison with the non-treated group. No significant
difference (p > 0.05) was found between lipoperoxida-
tion in untreated animals and the boldo infusion-treated
animals. Pretreatment with boldo (P. Boldus) infusion
was found to significantly reduce (p < 0.05) the lipo-
peroxidation induced by a single cis-DDP injection.
The data (Table 3) indicate that lipoperoxidation in
the group pretreated with boldine prior to the cis-
DDP injection significantly (p < 0.05) decreased in
comparison with animals treated only with cis-DDP.
No statistical difference (p > 0.05) was found between
the lipoperoxidation found in untreated animals and
boldine-treated animals. The data (Table 4) indicate
that lipoperoxidation in the group pretreated with
Table 3. Effect of boldine on the lipoperoxidation induced by
cis-DDP in mice liver
Lipoperoxidation
Pretreated Treatment (nmol MDA/g tissue)
None None 381.8 ± 75.58
None Cis-DDP 661.8 ± 145.34
None Boldine 448.4 ± 168.99
Boldine Cis-DDP 432.0 ± 168.18
Table 2. Effect of boldo infusion on the lipoperoxidation induced
by cis-DDP in the liver of mice
Lipoperoxidation
Pretreated Treatment (nmol MDA/g tissue)
None None 381.8 ± 75.58
None Cis-DDP 661.8 ± 145.34
None Boldo infusion 353.2 ± 125.11
Boldo infusion Cis-DDP 496.8 ± 58.83

Copyright © 2009 John Wiley & Sons, Ltd. Phytother. Res. 23, 1024–1027 (2009)
DOI: 10.1002/ptr
1026 J. FERNÁNDEZ ET AL.
by cis-DDP since under our experimental conditions
mice pretreated with a boldo infusion and then treated
with cis-DDP presented significantly lower lipoper-
oxidation in the hepatic tissue than found in the mouse
group treated only with cis-DDP (p < 0.05).
The action of the bold infusion could be explained
by the presence of boldine and catechin, substances
with recognized antioxidant activity due to their ability
to act as free radical scavengers (Schmeda-Hirschmann
et al., 2003). Our results indicate that lipoperoxidation
is significantly lower in mice pretreated with boldine
and catechin followed by cis-DDP treatment than in
mice only treated with cis-DDP (P < 0.05). Boldine
and catechin activity is attributed to the formation
of phenoxy radical and other free radical species due
to the oxidation of boldine’s aporfinic structure and
catechin’s polyphenolic structure (Ubeda et al., 1993),
which act decomposing superoxide anions, hydrogen
peroxides and hydroxyl radicals. Additionally, boldine
has been shown to restore the activity of some enzymes
that participate in antioxidant systems such as gluta-
thione S – transferase in Hepa – 1 cells (Kubínová
et al., 2001), glutathione peroxidase in rats’ liver mito-
chondria (Jang et al., 2000), enzymes whose activity
diminishes in the liver and kidney after cis-DDP admini-
stration (Sadzuka et al., 1992).
Based on our study’s results and considering that
boldine’s solubility in water is minimal, we propose that
the observed effect for the total boldo leaf infusion
is principally due to the presence of catechin, which is
consistent with the highest free radical scavenging
capacity of the infusion attributed to catechin by other
authors (Schmeda-Hirschmann et al., 2003).
In conclusion, the data obtained in our study allow
us to recommend the use of a complete boldo leaf
infusion as an effective chemoprotector agent due to
its activity that prevents the oxidative damage caused
by cis-DDP at the hepatic level.
Future studies should precisely determine if boldo
infusion’s activity lies only in its capacity to act on free
radicals or if it also includes a restoring activity at the
level of some oxidative enzymes without interfering in
cis-DDP’s antitumoral activity. In this regard we must
inform that we are conducting experiments to know if
the boldo infusion has any preventive effect on genetic
damage induced by cis-DDP. Our preliminary results
indicate that the infusion can prevents genetic damage
induced by this antitumoral drug.
Acknowledgments
This work was supported by the grant No. 055109 3/R from the
Universidad del Bío-Bío, Chile. The authors are very grateful to
Dr Patricio Peñailillo Brito from the Universidad de Talca for the
identification of plant material and to Mr Gerardo Quezada Silva for
his technical assistance and collection of plant material.
Table 4. Effect of catechin on the lipoperoxidation induced by
cis-DDP in mice liver
Lipoperoxidation
Pretreated Treatment (nmol MDA/g tissue)
None None 381.8 ± 75.58
None Cis-DDP 661.8 ± 145.34
None Catechin 495.6 ± 31.67
Catechin Cis-DDP 405.4 ± 129.71
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