Antioxidant Properties of Rosmarinus Officinalis and Its Effects on Xenobiotic Biotransformation

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Red de Revistas Científicas de América Latina, el Caribe, España y Portugal

Sistema de Información Científica

María E. LETELIER, Araceli TERÁN, Marcela A. BARRA, Paula ARACENA-PARKSAntioxidant properties of Rosmarinus officinalis and its effects on xenobiotic biotransformation

Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, vol. 8, núm. 6, noviembre, 2009,

pp. 487-497,

Universidad de Santiago de Chile

Chile

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Boletín Latinoamericano y del Caribe de Plantas

Medicinales y Aromáticas,

ISSN (Printed Version): 0717-7917

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Universidad de Santiago de Chile

Chile

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Letelier et al. Antioxidant properties and xenobiotic biotransformation of Rosmarinus offi

www.blacpma.org Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol. 8 (6) 200

INTRODUCTION

All aerobic organisms generate reactive oxygenspecies (ROS) as part of highly regulated enzymesand as a by-product of oxygen metabolism. ROS,which include superoxide anion, hydroxyl radical,and hydrogen peroxide, are highly reactive speciescapable to oxidize biological molecules, such aslipids, proteins, and nucleic acids (Droge, 2002).Thus, several antioxidant mechanisms have evolvedto prevent oxidative damage to cells. This antioxidantcapacity includes non-enzymatic and enzymaticmechanisms (Benzie, 2000). GSH and vitamin E are

classical examples of the non-enzymatic antioxidantmechanism, as these molecules act as free radicalscavengers (Evstigneeva et al.,, 1998; Elias et al.,2008). The enzymatic antioxidant system of cellsincludes enzymes that catalyze the reduction of ROS,and include superoxide dismutase and catalase (Eliaset al., 2008). GSH also plays a role in this system, asa cofactor of GSH-peroxidase. When ROS generationoverwhelms the cell’s antioxidant capacity, oxidativestress is ensued. Oxidative stress can lead to celldamage and ultimately cell death (Halliwell andGutteridge, 1988). During oxidative stress, someenzymes that are involved in drug metabolism, such

as GSH-transferases (GSTs), can also act asantioxidants, through the metabolism of highlyelectrophilic and lipophilic compounds (van der Aaret al., 1996). In addition, some GSTs occurring in theendoplasmic reticulum also display peroxidaseactivity (Mosialou and Morgenstern, 1989). Anincrease in the expression of such enzymes has beenalso described during oxidative stress (Pinkus et al.,1996; Mari and Cederbaum, 2001).

Increasing evidence has demonstrated thatcompounds occurring in plants have antioxidantcapacity, mainly in virtue of their ability to scavengefree radicals (Masella et al., 2005). Systematicstudies on the antioxidant capacity of total herbalextracts are missing. This is mainly due to the purification of specific compounds from extracts andassessment of their free radical scavenging propertiesusing synthetic radicals such as 1,1-diphenyl-2- picrylhydrazyl (DPPH) y 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS)(Antolovich et al., 2002). Isolation of compoundsfrom herbal extracts allows the study andunderstanding of the antioxidant properties of thesespecific substances. Such studies, however, may

overlook the potential synergy and/or ad

antioxidant effects that the various moloccurring in the whole extract exert through difmechanisms. On the other hand, syntheticradicals are useful for the rapid screening oantioxidant capacities of herbal extracts or iscompounds. These free radicals display mechanisms that are very different from thooxygen free radicals, which are the oxoccurring in biological systems. Therefore, the such technique may not reflect a true biolantioxidant capacity of herbal compounds (Leteal., 2008).

By definition, all herbal antioxidant substanc

xenobiotics for mammals. Therefore, theymetabolized through the drug-metabo pathways, occurring mainly in the endoplreticulum of liver cells. The enzymatic systesuch pathways include the cytochrome (CYP450) system (Kramer and Tracy, 2008), glucuronyltransferase (UDPGT) (Tephly Burchell, 1990), and microsomal GST (Rinaldi2002). Thus, all herbal antioxidant substdepending on their physicochemical propertielikely to behave as competitive inhibitors of enzymatic systems with respect to their atowards endogenous compounds and Noteworthy, CYP450 is the main contributoxidative stress in the endoplasmic retic because it catalyzes the generation of electrophilic metabolites and also directly reROS when uncoupled (James et al., 2003; Shi2006). Thus, herbal compounds may displayadditional antioxidant mechanism by inhiCYP450 (Johnson, 2008).

Rosmarinus officinalis (rosemary) belongs Lamiaceae family, constituted by nummedicinal plant species. Rosemary can be fouthe Mediterranean basin of south Europe, noAfrica and southwest Asia. This plant is promainly in Spain, Tunisia, Morocco, and to a extent, Portugal, Turkey and India. Rosemaralso be found in Chile, between the V and IX reAlthough mainly used as a condiment, stherapeutic actions are attributed to roseincluding antimicrobial, antispasmodic, hypertand sedative activities (Simon et al., Antioxidant properties of plants are thought mainly due to polyphenols occurring in all extracts (i.e. flavonoids, flavones, flavanthocyanines, etc.) that are scavengers of


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Determination of DPPH bleaching activity

This assay was determined according to Letelier etal. (2008). In a final volume of 1 mL, increasingconcentrations of rosemary extract were mixed with20 μg/mL of DPPH. Fresh DPPH stock solutionswere prepared in ethanol. Blanks contained herbalextract and vehicle. DPPH bleaching activity of allmixtures was recorded continuously at 37 ºC for 20min at 517 nm in a UV3 Unicam UV-VISSpectrophotometer. Reaction rates were determinedin conditions where product formation was linearlydependent with time.

Microsomal lipid peroxidation assay

The extent of lipid peroxidation following pre-treatment of microsomes with Cu2+/ascorbate wasestimated assaying thiobarbituric acid reactivespecies (TBARS), according to Letelier et al. (2005).Mixtures (1 mL final volume) contained 1 mg/mLmicrosomal protein, 25 nM CuSO4, 1 mM sodiumascorbate in 50 mM phosphate buffer, pH 7.4. Blankscontained all the reagents but microsomal protein.Blanks and samples were incubated for 20 min at37°C with constant agitation. TBARS werecalculated using the ε532=156/mM/cm and expressedas Data are presented as nmol TBARS/min/mg

protein.

Determination of microsomal thiol content

Thiol groups were titrated with DTNB asdescribed by Letelier et al. (2005). Microsomes (1mg protein/mL) were incubated with 25 ηM 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 30min at 37 °C with constant agitation. Afterwards,microsomal thiol content was titrated with 0.6 mMDTNB. To evaluate the antioxidant effect ofrosemary extract, microsomes (1 mg protein/mL)

were incubated for 5 min with this extract and then20 min with Cu2+/ascorbate prior determination of themicrosomal thiol content. Thiol concentration wasestimated on the basis of the equimolar apparition of5-thio-2-nitrobenzoic acid (ε410=13,600/M/cm) and is presented as nmol thiols/min/mg protein.

Assay of UDPGT activity

p-nitrophenol conjugation with UDPGA, reactioncatalyzed by UDPGT was assayed according toLetelier et al. (2007). Activity was assayed by

determining the remaining p-nitrophenol after 1

incubation of microsomes (2 mg protein/mL) following conditions: 0.5 mM p-nitrophenol, 2UDPGA, 4 mM MgCl2, in 100 mM Tris-HCl b pH 8.5. Blanks were assayed in the absenUDPGA. Reactions were stopped by addition ofto 5% (W/V) final concentration; samples wercentrifuged at 10,000 g for 10 min in a SuprafuHeraeus centrifuge and supernatants were coll NaOH was added to the supernatants to final 0Reaction rates were determined in conditions product formation was linearly-dependent to tim protein concentration. To calculate UDPGT aca standard curve of p-nitrophenol was generate

evaluate the antioxidant effect of herbal emicrosomes (1 mg protein/mL) were incubated with this extract and then 20 min with Cu2+/asc before determining UDPGT activity. Dat presented as nmol conjugate/min/mg protein.

Assay of GST activity

Conjugation of 1-chloro-2,4-dinitrobenreaction catalyzed by GST, was assayed accoLetelier et al.(2006). The reaction mixture con0.1 mg/mL microsomal protein, 1 mM 1-chlordinitrobenzene, and 4 mM GSH in 100 phosphate buffer, pH 6.5. Blanks were assayed absence of GSH. Apparition of the conju product was continuously recorded for 3 min atat 340 nm in a UV3 Unicam UVspectrophotometer. To calculate GST activityε340=9.6 mM/cm of the conjugate was useevaluate the antioxidant effect of rosemary emicrosomes (1 mg protein/mL) were incubatedmin with this extract and then 20 min Cu2+/ascorbate before determining GST activityare presented as µmol conjugate/min/mg protein

Assay of the CYP450 system activity

Activity of the CYP450 system was assayed N -demethylating activity on Aminopyrine, accoto Letelier et al. (1985). The reaction mcontained microsomes (1 mg protein/mL), 5aminopyrine, 3.5 mM MgCl2, 0.1 M G-6-P, 1 NADP, 5 Units/mL G-6-P dehydrogenase, in 3Tris-HCl buffer, pH 8.0. Blank contained all re but G-6-P dehydrogenase. All mixtures incubated for 15 min at 37 ºC. Reactions stopped by addition of TCA to 5% (W/V)concentration; samples were then centrifug10,000 g for 10 min in a Suprafuge 22 He


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For this purpose, we used rat liver microsomes as a

biological system and Cu2+/ascorbate as a ROSgenerating system. We tested the antioxidant capacityof the rosemary extract, as its ability to preventseveral oxidative consequences of ROS generation bythe Cu2+/ascorbate system: 1) lipid and thioloxidation, 2) oxidative activation of UDPGT activity,and 3) oxidative changes on the CYP450monooxygenase.

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 inFig. 2, pre-incubation of microsomes with rosemaryextract prevented this lipoperoxidative effect in alinear-dependent manner (r=0.999), with an IC50 value of 5.63 μg extract/mg protein.

Figure 2. Effect of the rosemary extract on microsomal lipid peroxidation elicited by Cu2+/ascorbate.

Rat liver microsomes (1 mg protein/mL) were incubated withincreasing concentrations of the rosemary extract prior toincubation with Cu2+/ascorbate; lipid peroxidation was assayed as

TBARS generation, as described in Materials and Methods. Dataare presented as % lipid peroxidation using untreated microsomesas control (2.36 ± 0.018 nmol TBARS/min/mg protein, n=4) andrepresent the mean ± SEM of at least 4 independent experiments.The IC50 value was obtained from the linear regression of thedata (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, likelyleading to structural changes in proteins that are

reflected in functional changes. Thus, titrati

thiol groups in biological samples can be usedmarker for oxidative damage in proteins. Incuof microsomes with Cu2+/ascorbate decreasemicrosomal thiol content from 28.4 ± 0.12 to 10.23 nmol thiol/mg protein (about 30%, n=4shown in Fig. 3, pre-incubation with the rosextract prevented the thiol loss in a linear-depemanner (r=0.994), with an IC50 value of 13.extract/mg protein.

Figure 3. Effect of the rosemary extract on the microsomacontent loss elicited by Cu2+/ascorbate.

Microsomes (1 mg protein/mL) were incubated with increaconcentrations of the rosemary extract prior to incubation wCu2+/ascorbate; thiol groups were titrated with DTNB accoto Material and Methods. Data are presented as % residualusing untreated microsomes as control (28.4 ± 0.12 nmolthiol/mg protein, n=4) and represent the mean ± SEM of atindependent experiments. The IC50 value was obtained frolinear regression of the data (r=0.994).

Effect of rosemary extract on the oxi

damage of CYP450 monooxygenase The Cu2+/ascorbate system causes conforma

changes on the CYP450 monooxygenase, whireflected as a loss in the characteristic absorba

450 nm of the protein conjugate with cmonoxide (see Materials and Methods). As shoFig. 4, Cu2+/ascorbate led to a progressive loss oabsorbance in time, effect that was compabolished when pre-incubating microsomes wirosemary extract.

Effect of rosemary extract on the oxi

activation of the UDPGT activity UDPGT is a microsomal enzyme that bec

activated by the Cu2+/ascorbate system (Leteal., 2007). Indeed, incubation of microsomes



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this system elicited the increase in the UDPGT

activity, in terms of p-Nitrophenol conjugation, from12.57 ± 0.30 to 26.6 ± 0.16 nmol conjugate/min/mg protein (~2-fold, n=4). As depicted in Fig. 5, pre-incubation of microsomes with low concentrations ofrosemary extract (up to 28 µg extract/mg protein) prevented the UDPGT activation in a concentration-dependent manner. Remarkably, higherconcentrations of rosemary extract (56 and 112 µgextract/mg protein) elicited inhibition of the UDPGTactivity compared with the untreated control withoutCu2+/ascorbate. This is suggestive of additional non-oxidative mechanisms taking place under theconditions tested. It is possible that the polyphenols

occurring in the rosemary extract may be inhibitingthis enzymatic activity by competing with itssubstrate p-nitrophenol. In agreement to this postulate, pre-incubation of microsomes withincreasing concentrations of rosemary extract, in theabsence of Cu2+/ascorbate, inhibited the conjugationof 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 ofUDPGT activity elicited by Cu2+/ascorbate.

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

increasing concentrations of the rosemary extract prior toincubation with Cu2+/ascorbate; UDPGT activity was assayedusing p-Nitrophenol as substrate and UDPGA, as detailed inMaterials and Methods. Data are presented as fold UDPGTactivity change in a semi-logarithmic plot, using untreatedmicrosomes as control (12.57 ± 0.30 nmol conjugate/min/mg

protein, n=4, dotted line) and represent the mean ± SEM of atleast 4 independent experiments. The solid line depicts the dose-response inhibition regression using the log [extract] (r=0.998).

Figure 5. Effect of the rosemary extract on the CYP450

monooxygenase content loss elicited by Cu

2+

/ascorbate.

Rat liver microsomes (1 mg protein/mL) were incubated wincreasing concentrations of the rosemary extract prior toincubation with Cu2+/ascorbate; CYP450 monooxygenase was assayed as described in Materials and Methods. Data

presented as a time plot of the % residual CYP450 mono-oxygenase content using the initial Abs450 as 100%, and rethe mean ± SEM of at least 4 independent experiments. Clcircles: control (without incubation with rosemary extract)closed squares: microsomes pre-incubated with rosemary

Figure 6. Effect of the rosemary extract on UDPGT activi

Rat liver microsomes (1 mg protein/mL) were incubated wincreasing concentrations of the rosemary extract and UDPactivity was assayed using p-Nitrophenol as substrate andUDPGA, according to Materials and Methods. Data are pras % residual UDPGT activity using untreated microsomescontrol (12.57 ± 0.30 nmol conjugate/min/mg protein, n=4represent the mean ± SEM of at least 4 independent experiThe solid line depicts the linear regression of the data (r=0

Effects of Rosmarinus officinalis on the metabolism pathway

The inhibition of UDPGT activity microsomes are pre-incubated with rosemary e



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

agreement with the idea that polyphenols from thisextract may behave as xenobiotic compounds. Assuch, they would be a substrate of drug-metabolizingenzymes occurring in rat liver microsomes, includingUDPGT, explaining the inhibitory effect on theconjugation of p-nitrophenol. If this postulate iscorrect, then rosemary extract should also behave asan inhibitor of other drug-metabolizing enzymes,decreasing the activities of GST and/or the CYP450system. Therefore, we studied the effect of therosemary extract on: 1) the conjugation of 1-chloro-2,4-dinitrobenzene with GSH, reaction catalyzed byGST, 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 andhighly electrophilic compounds with GSH. Weassayed the microsomal GST activity using 1-chloro-2,4-dinitrobenzene as a substrate, with a control valueof 1.12 ± 0.003 µmol conjugate/min/mg protein(n=4). As shown in Fig. 7, re-incubation ofmicrosomes with increasing concentrations ofrosemary extract led to inhibition of microsomal GSTactivity, in a concentration-dependent manner. Thesedata are in agreement with the rosemary extract potentially behaving as a competitor of theconjugation of 1-chloro-2,4-dinitrobenzene with GSH by microsomal GST.

Effect of the rosemary extract on the N- demethylation of Aminopyrine by the CYP450

system

The CYP450 system catalyzes the oxidation oflipophilic compounds (Phase I). We assayed the N-demethylation activity of the CYP450 onAminopyrine, and obtained a control value of 39.02 ±0.53 nmol product/min/mg protein (n=8). As depictedin Fig. 8, pre-incubation of microsomes withrosemary extract slightly inhibited this activity. Asobserved with GST, these data are in agreement withthe idea that the rosemary extract may act as acompetitor of the CYP450 system metabolism.

Figure 7. Effect of the rosemary extract on microsomal GS

activity.

Rat liver microsomes (0.1 mg protein/mL) were incubated increasing concentrations of the rosemary extract and GSTactivity was assayed using 1-chloro-2,4-dinitrobenzene assubstrate and GSH, according to Materials and Methods. D

presented as residual GST activity (in µmol conjugate/min protein) and represent the mean ± SEM of at least 4 indepeexperiments. * p


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Figure 8. Effect of the rosemary extract on the N-demethylatingactivity of the CYP450 system.

Figure 9. Effect of the rosemary extract on the CYP450monooxygenase content.

Rat liver microsomes (1 mg protein/mL) were incubated withincreasing concentrations of the rosemary extract and the N-demethylating activity of the CYP450 system was assayed usingAminopyrine as substrate and a NADPH-generating system,according to Materials and Methods. Data are presented asresidual CYP450 activity (in nmol product/min/mg protein) andrepresent the mean ± SEM of at least 4 independent experiments.

* p


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Antioxidant Properties of Rosmarinus Officinalis and Its Effects on Xenobiotic Biotransformation

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