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Mara E. LETELIER, Araceli TERN, Marcela A. BARRA, Paula ARACENA-PARKS

Antioxidant properties of Rosmarinus officinalis and its effects on xenobiotic biotransformation
Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, vol. 8, nm. 6, noviembre, 2009,
pp. 487-497,
Universidad de Santiago de Chile
Chile
Available in: http://www.redalyc.org/articulo.oa?id=85617461005

Boletn Latinoamericano y del Caribe de Plantas

Medicinales y Aromticas,
ISSN (Printed Version): 0717-7917
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Universidad de Santiago de Chile
Chile

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2009 Boletn Latinoamericano y del Caribe de Plantas Medicinales y Aromticas, 8 (6), 487 - 497
BLACPMA ISSN 0717 7917
Artculo Original | Original Article

Antioxidant properties of Rosmarinus officinalis and its effects on

xenobiotic biotransformation
[Propiedades antioxidantes de Rosmarinus officinalis y sus efectos sobre la biotransformacin de xenobiticos]
Mara E. LETELIER, Araceli TERN, Marcela A. BARRA, Paula ARACENA-PARKS
Laboratory of Pharmacology, Department of Pharmacological and Toxicological Chemistry, Chemical and Pharmaceutical Sciences School,
Universidad de Chile, Santiago, Chile.

Abstract
Rosmarinus officinalis (rosemary) extracts display antioxidant properties mainly due to the presence of polyphenols. These properties have yet to be
systematically studied in biological systems. In this study, we used rat liver microsomes to evaluate the antioxidant capacity of a dry extract from rosemary
leaves. Noteworthy, compounds occurring in herbal extracts are xenobiotics to animals, thus mainly biotransformed in the hepatic endoplasmic reticulum.
Thus, we also studied the ability of this extract to inhibit different xenobiotics biotransformation pathways. The rosemary extract prevented all tested
oxidative processes that were evaluated in rat liver microsomes. It also inhibited the activities of: 1) UDP-glucuronyltransferase; 2) microsomal GSHtransferase; and 3) cytochrome P450 system (N-demethylating activity). Our results indicate that herbal extracts, in addition to act as antioxidants, display a
significant interaction with the metabolism of xenobiotics and/or endogenous compounds. Considerations about such interaction are discussed.
Keywords: Rosmarinus officinalis; Rosemary; Herbal antioxidant; Cu2+/ascorbate; Oxidative stress; Cytochrome P450 system; UDP-glucuronyltransferase;
GSH-transferase.

Resumen
Extractos de Rosmarinus officinalis (romero) presentan propiedades antioxidantes debido, principalmente, a la presencia de polifenoles. Dichas
propiedades no han sido sistemticamente estudiadas en sistemas biolgicos. En el presente estudio, utilizamos microsomas de hgado de rata para evaluar la
actividad antioxidante de un extracto seco de hojas de romero. Cabe destacar que los compuestos presentes en preparados herbales son xenobiticos para los
animales, por lo que son biotransformados principalmente en el retculo endoplsmico heptico. Por ello, tambin estudiamos la capacidad de este extracto
para inhibir diferentes vas de biotransformacin de xenobiticos. El extracto de romero protegi todos los procesos oxidativos que fueron evaluados en
microsomas hepticos de rata. ste tambin inhibi las actividades de: 1) UDP-glucuroniltransferasa; 2) GSH-transferasa microsmica; y 3) el sistema
citocromo P450 (actividad N-desmetilante). Nuestros resultados indican que los extractos herbales, adems de comportarse como antioxidantes, pueden
interaccionar significativamente con el metabolismo de xenobiticos y/o compuestos endgenos. Consideraciones acerca de dicha interaccin son sometidas a
discusin.
Palabras Clave: Rosmarinus officinalis; Romero; Antioxidantes; Cu2+/ascorbato; Estrs oxidativo; Sistema citocromo P450; UDP-glucuroniltransferasa;
GSH-transferasa.
ABBREVIATIONS: ABTS, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate); CYP450, cytochrome P450; DPPH, 1,1-diphenyl-2-picrylhydrazyl; G-6-P,
glucosephosphate; GST, glutathione S-transferase; ROS, reactive oxygen species; UDPGT, UDP-glucuronyltransferase.
Recibido | Received: March 2, 2009.
Aceptado en Versin Corregida | Accepted in Corrected Version: May 12, 2009.
Publicado en Lnea | Published Online: November 30, 2009.
Declaracin de intereses | Declaration of interests: Authors have no competing interests.
Financiacin | Funding: This work was not financially supported
This article must be cited as: Mara E. Letelier, Araceli Tern, Marcela A. Barra, Paula Aracena-Parks. 2009. Antioxidant properties of Rosmarinus officinalis and its effects on
xenobiotic biotransformation. Bol Latinoam Caribe Plant Med Aromat 8(6):487 497. {EPub November 30, 2009}.

*Contactos | Contacts: Mara Eugenia Letelier, M.Sc., Department of Pharmacological and Toxicological Chemistry, School of Chemical and Pharmaceutical
Sciences, Universidad de Chile. Olivos 1007, Independencia, Santiago, Chile. Phone: 56-2-9782885. Fax: 56-2-9782912. E-mail address: mel@ciq.uchile.cl

BLACPMA es una publicacin de la Cooperacin Latinoamericana y Caribea de Plantas Medicinales y Aromticas

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Letelier et al.

Antioxidant properties and xenobiotic biotransformation of Rosmarinus officinalis

INTRODUCTION
All aerobic organisms generate reactive oxygen
species (ROS) as part of highly regulated enzymes
and as a by-product of oxygen metabolism. ROS,
which include superoxide anion, hydroxyl radical,
and hydrogen peroxide, are highly reactive species
capable to oxidize biological molecules, such as
lipids, proteins, and nucleic acids (Droge, 2002).
Thus, several antioxidant mechanisms have evolved
to prevent oxidative damage to cells. This antioxidant
capacity includes non-enzymatic and enzymatic
mechanisms (Benzie, 2000). GSH and vitamin E are
classical examples of the non-enzymatic antioxidant
mechanism, as these molecules act as free radical
scavengers (Evstigneeva et al.,, 1998; Elias et al.,
2008). The enzymatic antioxidant system of cells
includes enzymes that catalyze the reduction of ROS,
and include superoxide dismutase and catalase (Elias
et al., 2008). GSH also plays a role in this system, as
a cofactor of GSH-peroxidase. When ROS generation
overwhelms the cells antioxidant capacity, oxidative
stress is ensued. Oxidative stress can lead to cell
damage and ultimately cell death (Halliwell and
Gutteridge, 1988). During oxidative stress, some
enzymes that are involved in drug metabolism, such
as GSH-transferases (GSTs), can also act as
antioxidants, through the metabolism of highly
electrophilic and lipophilic compounds (van der Aar
et al., 1996). In addition, some GSTs occurring in the
endoplasmic reticulum also display peroxidase
activity (Mosialou and Morgenstern, 1989). An
increase in the expression of such enzymes has been
also described during oxidative stress (Pinkus et al.,
1996; Mari and Cederbaum, 2001).
Increasing evidence has demonstrated that
compounds occurring in plants have antioxidant
capacity, mainly in virtue of their ability to scavenge
free radicals (Masella et al., 2005). Systematic
studies on the antioxidant capacity of total herbal
extracts are missing. This is mainly due to the
purification of specific compounds from extracts and
assessment of their free radical scavenging properties
using synthetic radicals such as 1,1-diphenyl-2picrylhydrazyl
(DPPH)
y
2,2'-azinobis-(3ethylbenzothiazoline-6-sulfonate)
(ABTS)
(Antolovich et al., 2002). Isolation of compounds
from herbal extracts allows the study and
understanding of the antioxidant properties of these
specific substances. Such studies, however, may
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overlook the potential synergy and/or additive

antioxidant effects that the various molecules
occurring in the whole extract exert through different
mechanisms. On the other hand, synthetic free
radicals are useful for the rapid screening of the
antioxidant capacities of herbal extracts or isolated
compounds. These free radicals display redox
mechanisms that are very different from those of
oxygen free radicals, which are the oxidants
occurring in biological systems. Therefore, the use of
such technique may not reflect a true biological
antioxidant capacity of herbal compounds (Letelier et
al., 2008).
By definition, all herbal antioxidant substances are
xenobiotics for mammals. Therefore, they are
metabolized
through
the
drug-metabolizing
pathways, occurring mainly in the endoplasmic
reticulum of liver cells. The enzymatic systems of
such pathways include the cytochrome P450
(CYP450) system (Kramer and Tracy, 2008), UDPglucuronyltransferase (UDPGT) (Tephly and
Burchell, 1990), and microsomal GST (Rinaldi et al.,
2002). Thus, all herbal antioxidant substances,
depending on their physicochemical properties, are
likely to behave as competitive inhibitors of these
enzymatic systems with respect to their activity
towards endogenous compounds and drugs.
Noteworthy, CYP450 is the main contributor of
oxidative stress in the endoplasmic reticulum,
because it catalyzes the generation of highly
electrophilic metabolites and also directly releases
ROS when uncoupled (James et al., 2003; Shimada,
2006). Thus, herbal compounds may display and
additional antioxidant mechanism by inhibiting
CYP450 (Johnson, 2008).
Rosmarinus officinalis (rosemary) belongs to the
Lamiaceae family, constituted by numerous
medicinal plant species. Rosemary can be found in
the Mediterranean basin of south Europe, north of
Africa and southwest Asia. This plant is produced
mainly in Spain, Tunisia, Morocco, and to a lesser
extent, Portugal, Turkey and India. Rosemary can
also be found in Chile, between the V and IX regions.
Although mainly used as a condiment, several
therapeutic actions are attributed to rosemary,
including antimicrobial, antispasmodic, hypertensive
and sedative activities (Simon et al., 1984).
Antioxidant properties of plants are thought to be
mainly due to polyphenols occurring in all herbal
extracts (i.e. flavonoids, flavones, flavonols,
anthocyanines, etc.) that are scavengers of ROS

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Letelier et al.

Antioxidant properties and xenobiotic biotransformation of Rosmarinus officinalis

(Middleton et al., 2000). Alcoholic extracts from

rosemary leaves, stems and flowers display
antioxidant properties (Pathak et al., 1991;
Caigueral et al., 1998). These extracts are enriched
in polyphenols, such as carnosol, rosmanol, carnosic
acid, and rosmarinic acid (del Bano et al, 2003;
Moreno et al, 2006).
In the present study, we first evaluated the
antioxidant properties of a dry extract of rosemary
leaves, in terms of protecting rat liver microsomes
lipids, thiol-containing proteins, and UDPGT activity
from damage induced by Cu2+/ascorbate, a ROS
generating system. We compared the biological
antioxidant activity of this extract with its free radical
scavenging activity using DPPH. In addition, we
evaluated the potential inhibitory effect of this extract
on the activities of the CYP450 system, UDPGT, and
microsomal GST. When evaluated separately, the
rosemary extract behaved as an antioxidant and as an
inhibitor of the tested enzymatic activities. When
evaluated simultaneously, however, we found that the
extract displayed a lower inhibitory effect, giving
precedence to its antioxidant activity. The
significance of these results on the potential toxicity
of herbal extracts through the inhibition they exert on
drug-metabolizing enzymes is discussed.
MATERIAL AND METHODS
Chemicals
5,5-dithiobis(2-nitrobenzoic acid) (DTNB), GSH,
p-Nitrophenol
(PNP),
UDP-glucuronic
acid
(UDPGA), p-Nitroanisole (PNA), Aminopyrine,
NADP, glucose-6-phosphate (G-6-P), glucose-6phosphate dehydrogenase, Tris-HCl, and bovine
albumin (Fraction IV) were purchased from Sigma
Chemical Co. (St. Louis, MO, USA). Trichloroacetic
acid (TCA), thiobarbituric acid (TBA), sodium
ascorbate, FeCl3, CuSO4, MgCl2, and FolinCiocalteus reagent were purchased from Merck Co.
Chile. 1-chloro-2,4-dinitrobenzene was purchased
from ACROS Organics (New Jersey, NJ, USA).
Other chemicals were of analytical grade. Dry extract
of Rosmarinus officinalis leaves (vegetable drug) was
graciously provided by Laboratorios Ximena Polanco
(Santiago, Chile), along with proprietary information
regarding yield. Stocks of this dry extract (0.2 to 1
mg/mL) were prepared in a mixture of water: ethanol
(1:1) the same day of the experiments.

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Animals
Adult male Sprague Dawley rats (200-250 g),
maintained at the vivarium of the School of Chemical
and Pharmaceutical Sciences (Universidad de Chile,
Santiago, Chile) were used. Rats were allowed free
access to pelleted food, maintained with controlled
temperature (22C) and photoperiod (lights on from
07:00 to 19:00 h). All procedures were performed
using protocols approved by the Institutional Ethical
Committee of the School of Chemical and
Pharmaceutical Sciences, Universidad de Chile, and
according to the guidelines of the Guide for the Care
and Use of Laboratory Animals (NRC, USA).
Isolation of rat liver microsomes
Microsomal fraction was prepared according to
Letelier et al. (2005). Rats were fasted for 15 h with
water ad libitum, and sacrificed by decapitation.
Livers were perfused in situ with 4 volumes of 25 ml
0.9 % W/V NaCl, excised, and placed on ice. All
homogenization and fractionation procedures were
performed at 4C and all centrifugations were
performed using either a Suprafuge 22 Heraeus
centrifuge or an XL-90 Beckmann ultracentrifuge.
Liver tissue (9 -11 g wet weight), devoid of
connective and vascular tissue, was homogenized
with five volumes of 0.154 M KCl, with eight strokes
in a Dounce Wheaton B homogenizer. Homogenates
were centrifuged at 9,000xg for 15 min and
sediments were discarded. Supernatants were then
centrifuged at 105,000xg for 60 min. Pellets
(microsomes) were stored at -80C until use. Protein
determinations were performed according to Lowry
et al. (1951).
Determination of polyphenols
Polyphenols were determined by the method
described by Letelier et al. (2008). Fifty L herbal
extract were mixed with 250 L Folin Ciocalteau
reagent, 750 L 20% w/v sodium carbonate and
distilled water to a final volume of 5 mL. Blanks
contained all reagents other than herbal extract.
Reaction mixtures were then incubated for 2 h
protected from light. Afterwards, the absorbance of
the samples was determined at 760 nm in a UV3
Unicam UVVIS spectrophotometer, using their
respective blanks as reference. Catechin, a
polyphenol compound, was used as a reference
standard. Results were expressed as nmol catechin/g
rosemary extract.

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Letelier et al.

Antioxidant properties and xenobiotic biotransformation of Rosmarinus officinalis

Determination of DPPH bleaching activity

This assay was determined according to Letelier et
al. (2008). In a final volume of 1 mL, increasing
concentrations of rosemary extract were mixed with
20 g/mL of DPPH. Fresh DPPH stock solutions
were prepared in ethanol. Blanks contained herbal
extract and vehicle. DPPH bleaching activity of all
mixtures was recorded continuously at 37 C for 20
min at 517 nm in a UV3 Unicam UV-VIS
Spectrophotometer. Reaction rates were determined
in conditions where product formation was linearly
dependent with time.
Microsomal lipid peroxidation assay
The extent of lipid peroxidation following pretreatment of microsomes with Cu2+/ascorbate was
estimated assaying thiobarbituric acid reactive
species (TBARS), according to Letelier et al. (2005).
Mixtures (1 mL final volume) contained 1 mg/mL
microsomal protein, 25 nM CuSO4, 1 mM sodium
ascorbate in 50 mM phosphate buffer, pH 7.4. Blanks
contained all the reagents but microsomal protein.
Blanks and samples were incubated for 20 min at
37C with constant agitation. TBARS were
calculated using the 532=156/mM/cm and expressed
as Data are presented as nmol TBARS/min/mg
protein.
Determination of microsomal thiol content
Thiol groups were titrated with DTNB as
described by Letelier et al. (2005). Microsomes (1
mg protein/mL) were incubated with 25 M CuSO4,
1 mM sodium ascorbate in 50 mM phosphate buffer,
pH 7.4. Blanks contained all reagents but microsomal
protein. Blanks and samples were incubated for 30
min at 37 C with constant agitation. Afterwards,
microsomal thiol content was titrated with 0.6 mM
DTNB. To evaluate the antioxidant effect of
rosemary extract, microsomes (1 mg protein/mL)
were incubated for 5 min with this extract and then
20 min with Cu2+/ascorbate prior determination of the
microsomal thiol content. Thiol concentration was
estimated on the basis of the equimolar apparition of
5-thio-2-nitrobenzoic acid (410=13,600/M/cm) and is
presented as nmol thiols/min/mg protein.
Assay of UDPGT activity
p-nitrophenol conjugation with UDPGA, reaction
catalyzed by UDPGT was assayed according to
Letelier et al. (2007). Activity was assayed by
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determining the remaining p-nitrophenol after 15 min

incubation of microsomes (2 mg protein/mL) in the
following conditions: 0.5 mM p-nitrophenol, 2 mM
UDPGA, 4 mM MgCl2, in 100 mM Tris-HCl buffer,
pH 8.5. Blanks were assayed in the absence of
UDPGA. Reactions were stopped by addition of TCA
to 5% (W/V) final concentration; samples were then
centrifuged at 10,000g for 10 min in a Suprafuge 22
Heraeus centrifuge and supernatants were collected.
NaOH was added to the supernatants to final 0.5 M.
Reaction rates were determined in conditions where
product formation was linearly-dependent to time and
protein concentration. To calculate UDPGT activity,
a standard curve of p-nitrophenol was generated. To
evaluate the antioxidant effect of herbal extract,
microsomes (1 mg protein/mL) were incubated 5 min
with this extract and then 20 min with Cu2+/ascorbate
before determining UDPGT activity. Data are
presented as nmol conjugate/min/mg protein.
Assay of GST activity
Conjugation
of
1-chloro-2,4-dinitrobenzene,
reaction catalyzed by GST, was assayed according
Letelier et al.(2006). The reaction mixture contained
0.1 mg/mL microsomal protein, 1 mM 1-chloro-2,4dinitrobenzene, and 4 mM GSH in 100 mM
phosphate buffer, pH 6.5. Blanks were assayed in the
absence of GSH. Apparition of the conjugated
product was continuously recorded for 3 min at 25C,
at 340 nm in a UV3 Unicam UV-VIS
spectrophotometer. To calculate GST activity, the
340=9.6 mM/cm of the conjugate was used. To
evaluate the antioxidant effect of rosemary extract,
microsomes (1 mg protein/mL) were incubated for 5
min with this extract and then 20 min with
Cu2+/ascorbate before determining GST activity. Data
are presented as mol conjugate/min/mg protein.
Assay of the CYP450 system activity
Activity of the CYP450 system was assayed as the
N-demethylating activity on Aminopyrine, according
to Letelier et al. (1985). The reaction mixture
contained microsomes (1 mg protein/mL), 5 mM
aminopyrine, 3.5 mM MgCl2, 0.1 M G-6-P, 10 mM
NADP, 5 Units/mL G-6-P dehydrogenase, in 35 mM
Tris-HCl buffer, pH 8.0. Blank contained all reagents
but G-6-P dehydrogenase. All mixtures were
incubated for 15 min at 37 C. Reactions were
stopped by addition of TCA to 5% (W/V) final
concentration; samples were then centrifuged at
10,000g for 10 min in a Suprafuge 22 Heraeus

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Letelier et al.

Antioxidant properties and xenobiotic biotransformation of Rosmarinus officinalis

centrifuge and supernatants were collected. To

develop the colorimetric reaction, 0.5 mL of a
mixture of 0.1 mL of 2,4-pentanodione and 25 mL of
4 M ammonium acetate was added to 1 mL of
supernatant and mixtures were incubated for 2 h
protected from light. Absorbance of samples was
then determined at 400 nm in a UV3 Unicam UV
VIS spectrophotometer, using their respective blanks
as reference. To calculate the N-demethylating
activity of the CYP450 system, a standard curve of
formaldehyde was generated. Reaction rates were
determined in conditions where product formation
was linearly-dependent to time and protein
concentration. To evaluate the antioxidant effect of
rosemary extract, microsomes (1 mg protein/mL)
were incubated for 5 min with this extract and then
20 min with Cu2+/ascorbate before determining this
activity.
Generation of the CYP450 monooxygenase
spectrum
CYP450 monooxygenase spectrum was obtained
according to Omura and Sato (Omura and Sato,
1964). This method uses the ability of the carbon
monoxide to coordinate with the heme group of the
monooxygenase; the resulting conjugate has a peak
of maximum absorbance at 450 nm (=91/mM/cm.
The reaction mixture (blank and sample) contained 1
mg/mL microsomal protein, 5 mM sodium dithionite
and 50 mM phosphate buffer, pH 7.4. The spectrum
was generated following addition of carbon
monoxide to the sample in a UV3 Unicam UVVIS
spectrophotometer. To assess the possible interaction
of compounds occurring in the rosemary extract with
the CYP450 monooxygenase, 28 g of extract were
added to microsomes prior or after the addition of
sodium dithionite. A decrease in the absorbance at
450 nm of the CYP450 monooxygenase when
incubated with the rosemary extract was interpreted
as an interaction.
Statistical analysis
Data presented correspond to the mean of at least
4 independent experiments SEM. Statistical
significance (ANOVA) and regression analyses were
performed using Graph Pad Prism 5.0. Differences
were considered as significant when p<0.05.

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RESULTS
Antioxidant capacity of Rosmarinus officinalis
We first studied the antioxidant properties of
an aqueous solution of a dry extract from rosemary
leaves. As an initial test, we determined the content
of polyphenols of this aqueous preparation, as
detailed in Materials and Methods. We obtained
detectable values of polyphenols content, equivalent
to 0.53 0.009 (n=4) mol of catechin/mg extract or
87.9 1.53 mol of catechin/g vegetable drug. Thus,
most likely the rosemary extract used will display
antioxidant properties.
DPPH bleaching is a common measure of
antioxidant capacity of herbal extracts. As depicted in
Fig. 1, rosemary extract induced DPPH bleaching, in
a concentration-dependent manner. The dependence
with concentration followed a semi-logarithmic curve
(r=0.983). From the semi-logarithmic regression, we
calculated that 19.02 0.87 g extract/mL (n=4)
elicited the half-maximum bleaching effect on DPPH
in the conditions tested (see Materials and Methods).
Figure 1. Effect of the rosemary extract on DPPH bleaching.

The dry extract from rosemary leaves was dissolved in H2O:

ethanol (1:1) to 0.2 mg/mL and DPPH was dissolved in ethanol.
The DPPH bleaching assay was performed as detailed in Material
and Methods. Data are plotted as the mean SEM (n>4) of
DPPH in Abs517/20min in a semi-logarithmic scale against the
extract concentration. The IC50 value was calculated from the
dose-response inhibition regression using the log [extract]
(r=0.983).

As expected, the rosemary extract used in our

study displayed antioxidant capacity as measured by
the DPPH method. In our experience, this antioxidant
capacity must be validated with biological systems to
allow conclusion regarding the overall antioxidant
properties of any herbal extract (Letelier et al., 2008).

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Letelier et al.

Antioxidant properties and xenobiotic biotransformation of Rosmarinus officinalis

For this purpose, we used rat liver microsomes as a

biological system and Cu2+/ascorbate as a ROS
generating system. We tested the antioxidant capacity
of the rosemary extract, as its ability to prevent
several oxidative consequences of ROS generation by
the Cu2+/ascorbate system: 1) lipid and thiol
oxidation, 2) oxidative activation of UDPGT activity,
and 3) oxidative changes on the CYP450
monooxygenase.
Effect of rosemary extract on microsomal lipid
peroxidation
Incubation of rat liver microsomes with
Cu2+/ascorbate led to the generation of 2.36 0.018
nmol TBARS/min/mg protein (n=4). As depicted in
Fig. 2, pre-incubation of microsomes with rosemary
extract prevented this lipoperoxidative effect in a
linear-dependent manner (r=0.999), with an IC50
value of 5.63 g extract/mg protein.

reflected in functional changes. Thus, titration of

thiol groups in biological samples can be used as a
marker for oxidative damage in proteins. Incubation
of microsomes with Cu2+/ascorbate decreased the
microsomal thiol content from 28.4 0.12 to 19.4
0.23 nmol thiol/mg protein (about 30%, n=4). As
shown in Fig. 3, pre-incubation with the rosemary
extract prevented the thiol loss in a linear-dependent
manner (r=0.994), with an IC50 value of 13.05 g
extract/mg protein.
Figure 3. Effect of the rosemary extract on the microsomal thiol
content loss elicited by Cu2+/ascorbate.

Figure 2. Effect of the rosemary extract on microsomal lipid

peroxidation elicited by Cu2+/ascorbate.

Microsomes (1 mg protein/mL) were incubated with increasing

concentrations of the rosemary extract prior to incubation with
Cu2+/ascorbate; thiol groups were titrated with DTNB according
to Material and Methods. Data are presented as % residual thiols
using untreated microsomes as control (28.4 0.12 nmol
thiol/mg protein, n=4) and represent the mean SEM of at least 4
independent experiments. The IC50 value was obtained from the
linear regression of the data (r=0.994).

Rat liver microsomes (1 mg protein/mL) were incubated with

increasing concentrations of the rosemary extract prior to
incubation with Cu2+/ascorbate; lipid peroxidation was assayed as
TBARS generation, as described in Materials and Methods. Data
are presented as % lipid peroxidation using untreated microsomes
as control (2.36 0.018 nmol TBARS/min/mg protein, n=4) and
represent the mean SEM of at least 4 independent experiments.
The IC50 value was obtained from the linear regression of the
data (r=0.999).

Effect of rosemary extract on microsomal thiol

oxidation
Thiol groups occurring in the lateral chain of
proteins are reactive to oxidation by ROS, likely
leading to structural changes in proteins that are
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Effect of rosemary extract on the oxidative

damage of CYP450 monooxygenase
The Cu2+/ascorbate system causes conformational
changes on the CYP450 monooxygenase, which are
reflected as a loss in the characteristic absorbance at
450 nm of the protein conjugate with carbon
monoxide (see Materials and Methods). As shown in
Fig. 4, Cu2+/ascorbate led to a progressive loss of this
absorbance in time, effect that was completely
abolished when pre-incubating microsomes with the
rosemary extract.
Effect of rosemary extract on the oxidative
activation of the UDPGT activity
UDPGT is a microsomal enzyme that becomes
activated by the Cu2+/ascorbate system (Letelier et
al., 2007). Indeed, incubation of microsomes with

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Letelier et al.

Antioxidant properties and xenobiotic biotransformation of Rosmarinus officinalis

this system elicited the increase in the UDPGT

activity, in terms of p-Nitrophenol conjugation, from
12.57 0.30 to 26.6 0.16 nmol conjugate/min/mg
protein (~2-fold, n=4). As depicted in Fig. 5, preincubation of microsomes with low concentrations of
rosemary extract (up to 28 g extract/mg protein)
prevented the UDPGT activation in a concentrationdependent
manner.
Remarkably,
higher
concentrations of rosemary extract (56 and 112 g
extract/mg protein) elicited inhibition of the UDPGT
activity compared with the untreated control without
Cu2+/ascorbate. This is suggestive of additional nonoxidative mechanisms taking place under the
conditions tested. It is possible that the polyphenols
occurring in the rosemary extract may be inhibiting
this enzymatic activity by competing with its
substrate p-nitrophenol. In agreement to this
postulate, pre-incubation of microsomes with
increasing concentrations of rosemary extract, in the
absence of Cu2+/ascorbate, inhibited the conjugation
of p-nitrophenol in a linear-dependent manner
(r=0.999), as shown in Fig. 6.
Figure 4. Effect of the rosemary extract on the changes of
UDPGT activity elicited by Cu2+/ascorbate.

Rat liver microsomes (1 mg protein/mL) were incubated with

increasing concentrations of the rosemary extract prior to
incubation with Cu2+/ascorbate; UDPGT activity was assayed
using p-Nitrophenol as substrate and UDPGA, as detailed in
Materials and Methods. Data are presented as fold UDPGT
activity change in a semi-logarithmic plot, using untreated
microsomes as control (12.57 0.30 nmol conjugate/min/mg
protein, n=4, dotted line) and represent the mean SEM of at
least 4 independent experiments. The solid line depicts the doseresponse inhibition regression using the log [extract] (r=0.998).

Figure 5. Effect of the rosemary extract on the CYP450

monooxygenase content loss elicited by Cu2+/ascorbate.

Rat liver microsomes (1 mg protein/mL) were incubated with

increasing concentrations of the rosemary extract prior to
incubation with Cu2+/ascorbate; CYP450 monooxygenase content
was assayed as described in Materials and Methods. Data are
presented as a time plot of the % residual CYP450 monooxygenase content using the initial Abs450 as 100%, and represent
the mean SEM of at least 4 independent experiments. Closed
circles: control (without incubation with rosemary extract) and
closed squares: microsomes pre-incubated with rosemary extract.

Figure 6. Effect of the rosemary extract on UDPGT activity.

Rat liver microsomes (1 mg protein/mL) were incubated with

increasing concentrations of the rosemary extract and UDPGT
activity was assayed using p-Nitrophenol as substrate and
UDPGA, according to Materials and Methods. Data are presented
as % residual UDPGT activity using untreated microsomes as
control (12.57 0.30 nmol conjugate/min/mg protein, n=4) and
represent the mean SEM of at least 4 independent experiments.
The solid line depicts the linear regression of the data (r=0.999).

Effects of Rosmarinus officinalis on the drugmetabolism pathway

The inhibition of UDPGT activity when
microsomes are pre-incubated with rosemary extract
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Letelier et al.

Antioxidant properties and xenobiotic biotransformation of Rosmarinus officinalis

in the absence of Cu2+/ascorbate (Fig. 6) is in

agreement with the idea that polyphenols from this
extract may behave as xenobiotic compounds. As
such, they would be a substrate of drug-metabolizing
enzymes occurring in rat liver microsomes, including
UDPGT, explaining the inhibitory effect on the
conjugation of p-nitrophenol. If this postulate is
correct, then rosemary extract should also behave as
an inhibitor of other drug-metabolizing enzymes,
decreasing the activities of GST and/or the CYP450
system. Therefore, we studied the effect of the
rosemary extract on: 1) the conjugation of 1-chloro2,4-dinitrobenzene with GSH, reaction catalyzed by
GST, and 2) N-demethylation of aminopyrine
reaction catalyzed by the CYP450 system.
Effect of rosemary extract on GST activity
GST isoenzymes, along with UDPGT, catalyze
reactions of conjugation of xenobiotics (Phase II).
GSTs catalyze the conjugation of lipophilic and
highly electrophilic compounds with GSH. We
assayed the microsomal GST activity using 1-chloro2,4-dinitrobenzene as a substrate, with a control value
of 1.12 0.003 mol conjugate/min/mg protein
(n=4). As shown in Fig. 7, re-incubation of
microsomes with increasing concentrations of
rosemary extract led to inhibition of microsomal GST
activity, in a concentration-dependent manner. These
data are in agreement with the rosemary extract
potentially behaving as a competitor of the
conjugation of 1-chloro-2,4-dinitrobenzene with GSH
by microsomal GST.
Effect of the rosemary extract on the Ndemethylation of Aminopyrine by the CYP450
system
The CYP450 system catalyzes the oxidation of
lipophilic compounds (Phase I). We assayed the Ndemethylation activity of the CYP450 on
Aminopyrine, and obtained a control value of 39.02
0.53 nmol product/min/mg protein (n=8). As depicted
in Fig. 8, pre-incubation of microsomes with
rosemary extract slightly inhibited this activity. As
observed with GST, these data are in agreement with
the idea that the rosemary extract may act as a
competitor of the CYP450 system metabolism.

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Figure 7. Effect of the rosemary extract on microsomal GST

activity.

Rat liver microsomes (0.1 mg protein/mL) were incubated with

increasing concentrations of the rosemary extract and GST
activity was assayed using 1-chloro-2,4-dinitrobenzene as
substrate and GSH, according to Materials and Methods. Data are
presented as residual GST activity (in mol conjugate/min/mg
protein) and represent the mean SEM of at least 4 independent
experiments. * p<0.05 compared to control (one-way ANOVA).

As indicated above (Materials and Methods), to

measure the structural changes of the CYP450
monooxygenase, microsomes are treated with sodium
dithionite to reduce the heme group to a Fe2+ form. In
this condition, the Fe2+ is able to react with carbon
monoxide, and the product displays a characteristic
and classical peak of absorbance at 450 nm (Omura
and Sato, 1964). Substrates of the CYP450 system
bind, as a first step, to the heme group of the CYP450
monooxygenase in the Fe3+ form. If the rosemary
extract is competing CYP450 system substrates, preincubation of microsomes with this extract before
reduction and formation of the CYP450 monooxygenase-CO complex should lead to a loss in the
absorbance at 450 nm. The non-specific binding of
rosemary extract compounds to the membrane was
assessed by performing this assay incubating microsomes with this extract after reduction and formation
of the CYP450-CO complex. Thus, the difference in
the absorbance loss (Fig. 9, inset) will correspond to
the specific binding. As shown in Fig. 9, both
conditions led to a decrease in the absorbance at 450
nm, but this decrease was partially prevented by
reduction and formation of the CYP450 monooxygenase-CO complex prior to incubation with the
extract.

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Antioxidant properties and xenobiotic biotransformation of Rosmarinus officinalis

Figure 8. Effect of the rosemary extract on the N-demethylating

activity of the CYP450 system.

Figure 9. Effect of the rosemary extract on the CYP450

monooxygenase content.

Rat liver microsomes (1 mg protein/mL) were incubated with

increasing concentrations of the rosemary extract and the Ndemethylating activity of the CYP450 system was assayed using
Aminopyrine as substrate and a NADPH-generating system,
according to Materials and Methods. Data are presented as
residual CYP450 activity (in nmol product/min/mg protein) and
represent the mean SEM of at least 4 independent experiments.
* p<0.05 compared to control (one-way ANOVA).

Rat liver microsomes (1 mg protein/mL) were incubated with

the rosemary extract prior to (closed circles, total loss) or
following (closed squares, nonspecific loss) the reduction of the
Fe3+-form of the CYP450 monooxygenase, formation of its
complex with carbon monoxide, and measurement of the
absorbance at 450 nm, according to Material and Methods. Data
are presented as residual CYP450 monooxygenase (in nmol/mg
protein) and represent the mean SEM of at least 4
independent experiments. The inset depicts the difference
calculated between the total and non-specific loss.

These data confirm that there are some molecules

occurring in the rosemary extract that are likely
competitive inhibitors of the CYP450 system activity.
DISCUSSION
When employing widely accepted methods to
study the antioxidant capacity of the rosemary extract
used in this study, we detected polyphenols occurring
in the hydro-alcoholic solution of the dry extract. In
addition, this extract displayed DPPH bleaching
activity (Fig. 1). In our experience, the use of DPPH
is only useful when screening the relative antioxidant
capacity of several herbal extracts or isolated compounds. This determination, however, may underestimate the true antioxidant capacity of compounds
in biological systems, since DPPH and oxygen free
radicals display different redox mechanisms (Letelier
et al., 2008). In agreement with this, the IC50 for the
antioxidant activity of the rosemary extract in terms
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of microsomal lipid peroxidation and thiol oxidation

by the Cu2+/ascorbate system were 4- and 2-fold
lower, respectively, than that obtained for the DPPH
bleaching assay (Figs. 2 and 3). Moreover, this
extract was able to completely prevent the oxidative
structural damage on the CYP450 monooxygenase
elicited by the Cu2+/ascorbate system (Fig. 4). In
summary, the antioxidant capacity of a substance is a
property comprising much more than just a free
radical scavenging activity. Therefore, we strongly
advice a systematic analysis of the antioxidant
capacity of herbal extracts/compounds using
biological systems, in addition to the screening with
synthetic free radicals, such as DPPH or ABTS.
Remarkably, when studying the antioxidant
capacity of the rosemary extract in terms of
protecting UDPGT from oxidative activation by the
Cu2+/ascorbate system (Letelier et al., 2007), we
found that the extract inhibited this activity at the
highest concentrations tested (Fig. 5). The inhibitory

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Letelier et al.

Antioxidant properties and xenobiotic biotransformation of Rosmarinus officinalis

property of the rosemary extract on the UDPGT

activity, however, gave precedence to its antioxidant
capacity. On the other hand, this result suggested that
compounds found in the rosemary extract could
behave as inhibitors of the drug-metabolizing
enzymes occurring in microsomes: UDPGT,
microsomal GST and the CYP450 system. A
systematic analysis of the concentration-dependent
effect of the rosemary extract on reactions catalyzed
by each of these enzymes (Figs. 6, 7, and 8) showed
the inhibitory properties of the rosemary extract on
all reactions tested. This strongly suggested that
molecules occurring in this extract, being xenobiotic
compounds, are likely to be substrates of these
enzymes and, consequently, competitive inhibitors of
the reactions these enzymes catalyze. This was
further confirmed for the case of the CYP450 system,
in which we could detect specific binding of
compounds occurring in the herbal extract to the
Fe3+-form of the CYP450 monooxygenase (Fig. 9).
Taken together, these findings indicate that the
rosemary extract must contain lipophilic substances
that are substrates of the CYP450 system. Because
this extract is able to inhibit the N-demethylation
activity of this system, we postulate that is likely to
contain lipophilic substituted amines. Noteworthy,
most of the drugs used for the treatment of
psychotropic disorders, such as antidepressants and
anxiolytic medications, contain substituted amine
groups. Therefore, it can be postulated that this
rosemary extract may display psychotropic activity.
Recently, it has been reported (conference abstract)
that the same extract indeed displays antidepressant
and anxiolytic activity in rats, mimicking the
activities of fluoxetine and diazepam, respectively
(Diaz-Veliz et al.,, 2008). Additional research is
needed to test our postulates.
In addition to a potential competitive effect of
rosemary compounds on N-demethylation by the
CYP450 system, there are other possible mechanisms
for rosemary extract to have a psychotropic activity,
such as the presence of sedative compounds
(Gonzalez-Trujano et al, 2007; Machado et al, 2009).
In this regard, it has been shown that rosemary
extract contain certain polyphenols with sedative
activity (Machado et al, 2009). Because polyphenols
are biotransformed mainly by UDPGT, we believe it
is unlikely these type of compounds can be substrates
(and therefore, competitive inhibitors) of the CYP450
system. More research will be required to address if
these are exclusive mechanisms or not.
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Rosemary is mainly used as a condiment. Our data

demonstrate that a dry extract from rosemary leaves
displays antioxidant activity in biological systems, as
evaluated in rat liver microsomes. Furthermore,
compounds occurring in this extract are able to
inhibit the drug-metabolizing pathways in a manner
that gives precedence to their antioxidant capacity.
CONCLUSION
Our results suggest that the presence of
compounds with substituted amine groups in herbal
extracts, assessed as their capacity to competitively
inhibit the N-demethylating activity of the CYP450
system, is related to a potential psychotropic activity.
To the best of our knowledge, this is the first
indication of potential psychotropic activity in an
herbal extract. We are currently researching this
possibility for rosemary and other herbal extracts.
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