Aqueous Extracts of Lippia turbinata andAloysia citriodora (Verbenaceae):Assessment of Antioxidant Capacity andDNA damage

Erika Portmann1, Marcela M. Lopez Nigro1, Claudia G. Reides2, Susana Llesuy2,Rafael A. Ricco3, Marcelo L. Wagner3, Alberto A. Gurni3, and Marta A. Carballo1

AbstractThe aim of the present work was to make a contribution to the knowledge of aqueous extracts of Lippia turbinata and Aloysiacitriodora (Verbenaceae; infusion and decoction) in relation with the establishment of its antioxidant activity and lack of DNAdamage, for its potential use in therapeutics. The cytogenotoxic profile was evaluated through genotoxic biomarkers such asmitotic index, cellular proliferation kinetics, sister chromatid exchanges, single-cell gel electrophoresis assay, and micronucleustest in human peripheral blood lymphocyte cultures. No statistical differences were found (P > .05) between control and exposedcultures, even between both aqueous extracts. The total antioxidant capacity was shown to be higher in the decoction than in theinfusion and both aqueous extracts protected against protein carbonylation and lipid peroxidation, the decoction being moreefficient than the infusion (P < .005). These results suggest the safe use of these medicinal plants as chemoecologic agents intherapeutics.

Keywordsbiomarkers, Lippia turbinata, Aloysia citriodora, Verbenaceae, antioxidant activity

Introduction

Medicinal herbs are some of our oldest medicines. Nowadays

increasing use is a clear evidence of public interest in having

alternatives to conventional medicine. However, they have not

been tested with required methods for conventional

pharmaceuticals.1

Currently the Verbenaceae family is composed of 100 kinds

and approximately 2000 species of wide geographical distribu-

tion, including tropical, subtropical, and moderate regions. In

Argentina, 26 kinds are described in 191 species of which 54

are endemic.2 The family is characterized for including aro-

matic species mostly used in the traditional and popular med-

icine. Among the most important, we found Lippia (poleos) and

Aloysia (cedrones) species. Thus, both Lippia and Aloysia spe-

cies are known to show insecticidal, antiplasmodial, antibac-

terial, and antifungal activities as well as hypotensive and

muscle relaxant effects.3–6

Aloysia citrodora Palau and Lippia turbinata Griseb,

vulgarly known as cedron and poleo, respectively, are herbal

species widely consumed in Argentina and commonly prepared

as aqueous extracts (decoction or infusion).7 Both species spon-

taneously grow in South America. Cedron (Aloysia triphylla

(L’Her) Britton, formerly classified as Aloysia citriodora (Cav)

Ort, Lippia citriodora (Ort) HBK, Verbena citriodora Cav, and

Verbena triphylla (L’Her)) has a long history of folk uses as

infusions for the treatment of asthma, cold, fever, colic, diar-

rhea and indigestion, insomnia, and anxiety.7,8 It is believed

that its essential oil and phenolic compounds (flavonoids) are

responsible for these properties.7 Likewise, L turbinata Griseb

is a very branchy, aromatic shrub 0.5 to 1.5 m high and its

leaves are used in folk medicine mainly as digestive, although

several other uses had been reported (diuretic, abortive, emme-

nagogue, stomachic, tonic, and stimulant).9

1 CIGETOX—Citogenetica Humana y Genetica Toxicologica—INFIBIOC,

Instituto de Fisiopatologıa y Bioquımica Clınica, FFyB, UBA, Buenos Aires,

Argentina2 Catedra de Qca, General e Inorganica, FFyB, UBA, Buenos Aires, Argentina3 Catedra de Farmacobotanica, FFyB, UBA, Buenos Aires, Argentina

Corresponding Author:

Marta A. Carballo, CIGETOX—Citogenetica y Genetica Toxicologica—

INFIBIOC, Instituto de Fisiopatologıa y Bioquımica Clınica, Facultad de

Farmacia y Bioquımica, UBA, Junın 956 (1113), Buenos Aires, Argentina

Email: macarballo@ffyb.uba.ar

International Journal of Toxicology31(2) 192-202ª The Author(s) 2012Reprints and permission:sagepub.com/journalsPermissions.navDOI: 10.1177/1091581812436726http://ijt.sagepub.com

The chemical composition of the essential oils from the

leaves of both plants have also been studied and reviewed.7,8,10

However, there is few literature data related to the chemical

composition of infusion and/or decoction extracts.9–11 Thus,

Carnat et al revealed large amount of polyphenolic compounds

in A citriodora’s infusion and Wernert et al, described the

presence of polyphenols (hydroxycinnamic acids and flavons)

in both species and higher complexity and concentration of

total phenols, tannins, and flavonoids in A citriodora leaves

than those obtained from L turbinata leaves.9,11

Natural antioxidants have generated considerable interest in

preventive medicine and in the food industry. Many of them

have been identified as a free radical or active oxygen scaven-

gers. Within these polyphenolic compounds, phytochemicals

with a large distribution in nature have this antioxidant activ-

ity.12 It is very interesting to study the role of reactive oxygen

species in several diseases and to evaluate the potential anti-

oxidant protective effect of natural compounds on affected

tissues. For considering a natural compound or a drug as an

antioxidant, it is necessary to investigate their antioxidant prop-

erties in vitro and then to evaluate their antioxidant functions in

biological systems.

The measurement of cytogenetic alterations in vitro is con-

sidered an initial step in the risk assessment procedures for

genotoxic agents. Short-term genetic toxicology tests are useful

tools for determining toxic effects in the genetic material of

cells. For this reason, they have been proposed for the identi-

fication of potential chemical carcinogens in order to protect

public health.13–16 Within the biological end points, mitotic

index (MI) and replication index (RI) were used as indicators

of adequate cell proliferation, micronuclei (MN) for genotoxic

evaluation,17 and sister chromatid exchange (SCE) as a sensi-

tive indicator of DNA damage and/or subsequent DNA repair

(chromosomal instability).18 Beside, single-cell gel electro-

phoresis (comet assay) was used to detect as much breakage

doubles like simple chain, although the nature and the mechan-

ism of the test are not clarified totally.19

Taking into account previous results from our group, where

the quantitative analysis of total phenols, tannins, and flavo-

noids were developed for both species in decoction and infu-

sion form, as well as preliminary studies of antioxidant activity

(2,20-azino-bis-(3-ethyllbenzo-tiazoline-6-sulfonic acid) dia-

mmonium salt [ABTS] method), the present study is aimed at

evaluating the antioxidant capacity through lipid peroxidation

and protein oxidation of the commonly employed aqueous

extracts (infusion and decoction) of A citrodora and L turbi-

nata, and assessing the development of a potential cytotoxic/

genotoxic in order to ensure a relatively safe use of these med-

icinal plants.

Materials and Methods

Plant Materials

Lippia turbinata and A citriodora were provided by the

Department of Calingasta, from Cuyo, collected at Barreal, San

Juan, Argentine (31� 400 73000 S, 69� 290 57700 W). The voucher

specimens of the plants were kept in the Herbarium Museum of

Pharmacobotany ‘‘Juan A. Dominguez,’’ School of Pharmacy

and Biochemistry of the University of Buenos Aires (BAF

9018, 9019).

Preparation of Aqueous Extracts

Decoction and infusion (extemporaneous aqueous prepara-

tions) were prepared according to the Argentine Pharmaco-

peia.20 For preparing decoction, air-dried aerial parts of the

plants (5 g) were boiled in water (100 mL) for 10 minutes

(100�C); while for preparing infusion, boiling water was added

to the herb material and left undisturbed for 10 minutes. Aqu-

eous extracts were prepared considering daily consumption in

our country (5 g), with total absorption and 5 L in volume

(1 mg/mL final concentration in culture). Besides, in order to

consider a potential genotoxic effect, we reduced the concen-

tration 10 times (0.1 mg/mL final concentration in culture).

Both preparations were made at a concentration of 5% (w/v)

and were sterilized through a 0.22-mm filter and stored at

�20�C. Replicates of the same batch were evaluated.

Chemicals

RPMI 1640 medium, fetal bovine serum (FBS), phytohemag-

glutinin (PHA), phosphate-buffered saline (PBS), low-melting

point agarose, and normal melting point agarose were pur-

chased from Gibco BRL (Argentine). Potassium chloride (pur-

ity >99%), methanol (purity >99%), glacial acetic acid (purity

>99%), Giemsa solution, H2O2, NaCl (purity >99%), dimethyl

sulfoxide (DMSO; purity >99%), and NaOH (purity >99%)

were from Merck (Argentine). Bromodeoxyuridine, colcemid

(purity >95%), cytochalasin B (purity >98%), ethidium bro-

mide (purity >95%), acridine orange (purity >95%), disodium

salt of ethylenediaminetetraacetic (Na2EDTA; purity >99%),

trizma (purity >99%), triton X-100, Hoechst 33258, and mito-

mycin C (purity >98%) were obtained from Sigma-Aldrich,

Argentine. Heparin was from ABBOT (Argentine) and

Ficoll-Paque was purchased from GE Healthcare (Argentine).

Trolox was purchased from Aldrich Chemicals, Milwaukee,

Wisconsin). 2,20-Azo-bis (2-amidinopropane) (ABAP) was

obtained from Acros Organics (New Jersey).

Study Participants

The School of Pharmacy and Biochemistry of the University of

Buenos Aires Ethical Committee established the regulations

for the development of the study and informed consent was

given by each individual prior to the beginning of the

evaluation.

Phytochemistry Analysis

Qualitative analysis of polyphenols. Qualitative analysis of

polyphenols was performed by 2-dimensional thin-layer chro-

matography on cellulose as described by Hagerman et al.21 The

Portmann et al 193

presence of flavonoids and proanthocyanidins was analyzed

according to the method of Markham.22

Determination of total polyphenols content. Total polyphenols

were determined by Folin–Ciocalteu procedure according to

Makkar et al.23 Aliquots (50 mL) of aqueous infusions and

decoctions were transferred into test tubes and their volumes

were made up to 0.5 mL with distilled water. Folin-

Ciocalteu reagent, 0.25 mL, and 20% aqueous sodium car-

bonate solution, 1.25 mL, were added and the test tubes

were vortexed. After 40 minutes, absorbance was measured

at 725 nm against blank.

The amount of total polyphenols was expressed as mg tannic

acid/g dry plant material. Calibration curve of tannic acid was

developed. All measurements were done in triplicate.

Determination of total tannins. Total tannin content was deter-

mined by Folin–Ciocalteu procedure, after the removal of tan-

nin by precipitation with bovine serum albumin (BSA; 0.2 mol/

L acetate buffer pH 5.0, 0.17 mol/L sodium chloride, and

1.0 mg/mL BSA fraction V). One milliliter of BSA was added

to 1 mL of the extract and vortexed. After 15 minutes at room

temperature, the tubes were centrifuged at 5000g. Aliquots of

supernatant (50 mL) were transferred into the test tubes and

nonabsorbed phenolics were determined as described for total

phenols. Calculated values were subtracted from total polyphe-

nol contents and total tannin contents expressed as mg tannic

acid/g dry plant material. All measurements were done in

triplicate.

Determination of proanthocyanidins. Proanthocyanidins were

determined by butanol-HCl assay according to the method of

Porter et al.24 Aliquots (0.50 mL) of extracts were transferred

into the test tubes and 3.0 mL of butanol–HCl reagent (butanol:

HCl, 95:5 v/v) and 0.1 mL 2% ferric reagent (2% ferric ammo-

nium sulfate in 2 mol/L HCl) were added. Test tubes were

vortexed and put into a boiling water bath for 60 minutes. After

cooling, the absorbance was recorded at 550 nm against blank.

Proanthocyanidins were expressed as optical density at

550 nm. All measurements were done in triplicate.

Determination of flavonoids. 0.1 mL of each extract were

added to 1.4 mL of deionized water and 0.50 mL of flavonoids

reactive: 133 mg crystalline aluminum chloride and 400 mg of

crystalline sodium acetate dissolved in 100 mL of extracting

solvent (140 mL methanol, 50 mL water, and 10 mL acetic

acid). After 30 minutes at room temperature, the absorbance

was recorded at 430 nm against blank.25 Calibration curve of

rutin was developed. The amount of flavonoids was calculated

as mg rutin/g dry plant material.

Cytototoxic and Chromosome Instability Biomarkers (MI,RI, and SCE)

Lymphocyte Cultures and Cell Harvesting. Duplicate human per-

ipheral blood cultures were prepared according to the method

of Carballo et al with modifications.26 Briefly, 1 mL of each of

the blood samples were placed in a sterile flask containing

7.5 mL RPMI 1640 medium supplemented with 1.5 mL fetal

bovine serum, 120 mL PHA, and 32 mmol/L bromodeoxyuri-

dine. The prepared extracts were added in 2 different concen-

trations (0.1 and 1 mg/mL) at the beginning of the cultures,

which were then incubated for 72 hours at 37�C. Mitomycin C

(0.025 mmol/L) was used as a positive control. Fifty minutes

before harvesting, 100 mL of colcemid (10 mg/mL) was added

to each culture flask. For harvesting, cells were centrifuged at

approximately 800 to 1000 rpm for 10 minutes. The superna-

tant was removed and 5 mL of a prewarmed (37�C) 0.075 mol/

L KCl hypotonic solution were added. Cells were resuspended

and incubated at 37�C for 45 minutes. The supernatant was

removed by centrifugation, and 5 mL of methanol glacial acetic

acid (3:1) were added. The fixative was removed and the pro-

cedure was repeated twice. To prepare the slides, 5 drops of the

fixed cell suspension were dropped on clean glass slides and

air-dried. Cells were stained following a modified Fluores-

cence plus Giemsa technique 27 Slides were stained for 20 min-

utes in a 0.05% (w/v) Hoechst 33258 solution, rinsed with tap

water and placed under a near-ultraviolet (UV) lamp for 90

minutes, covered with Sorensen buffer, pH 6.8, and stained

with a 3% Giemsa solution in phosphate buffer (pH 6.8) for

8 minutes.

We chose 72 hours as the total duration of the experiment to

record the effects of the tested agent on all phases of the cell

cycle and to establish their impact on overcoming the G0 bar-

rier and entering the cell cycle by mitogen-induced

lymphocytes.

Microscopic EvaluationMitotic Index (MI). The MI was calculated as the proportion of

metaphase for 2000 cells, in each preparation, donor, and

concentration.

Cell Proliferation Kinetics (CPK). The proportion of first (M1),

second (M2), and third (M3) dividing cells was scored from 100

consecutive metaphases from each duplicate 72-hour culture

for each herb, preparation, donor, and concentration. The

RI was calculated according to the formula (RI ¼[M1 þ 2M2 þ3M3]/100).28

Sister Chromatid Exchanges. The frequency of SCE was

observed in 35 to 50 harlequin-stained metaphases, each with

46 centromeres for each herb, preparation, donor, and concen-

tration. The results were expressed as the frequency of SCE per

metaphase.

Single-Cell Gel ElectrophoresisCell viability. Cell viability was determined using the ethidium

bromide/acridine orange assay described by Mc Gahon et al.29

Briefly, 4 mL of dye-mix solution (100 mg/mL ethidium bro-

mide and 100 mg/mL acridine orange) was added to 100 mL of

cell suspension. Cells were observed with a 40� objective

using a fluorescent microscope. At least 200 cells were

counted, and the results expressed as percentage of viable and

nonviable cells.30

194 International Journal of Toxicology 31(2)

Alkaline comet assay. The procedure described by Singh et al

was used with modifications.31 Each sample was processed

with a duplicate including negative and positive (H2O2

50 mmol/L) controls. Aqueous extracts (infusion and decoc-

tion) were assayed in 2 different concentrations (0.05 and

0.5 mg/mL) and were added at beginning of the experiment.

Fifty microliters of freshly prepared cell suspension was added

to 950 mL RPMI 1640, incubated for 2 hours at 37�C, and then

centrifuged at 1000 rpm for 5 minutes. Pellets were mixed with

200 mL of 1% low-melting point agarose solution at 37�C and

were spread onto slides precoated with 1% normal melting

point agarose. The lysis solution (2.5 mol/L NaCl,

100 mmol/L Na2EDTA, 10 mmol/L Trizma, 1% Triton X-

100 and DMSO 10%, and pH 10) allows the rupture of cellular

and nuclear membranes of embedded cells. The slides were

submerged in cold, freshly prepared solution and left overnight

at 4�C. Afterward, they were placed in a cold electrophoresis

alkaline buffer (10 N NaOH, 200 mmol/L Na2EDTA, and pH

>13) and the embedded cells were exposed for 20 minutes to

allow DNA unwinding. Electrophoresis was performed in the

same buffer at 25 V and 300 mA (0.75 V/cm) for 20 minutes at

4�C. The slides were washed with neutralization buffer (Tris

0.4 mol/L, pH 7.5) and DNA was stained with 50 mL of ethi-

dium bromide (0.02 mg/mL) and observed using a fluorescent

microscope at 40�. All the procedures were done in darkness

to avoid additional DNA damage. Totally 100 randomly

selected cells were analyzed visually on a scale 0 to 4 (cate-

gories depending on DNA damage level). Damage index comet

assay (DICA) was calculated according to the formula

DICA ¼ 1n1 þ 2n2 þ 3n3 þ 4n4, where n1 to n4 represent

the number of cells with 0 to 4 damage level.

Micronucleus Test (Cytokinesis-Blocked Micronucleus)

Human lymphocytes were isolated using Ficoll-Paque density

gradients. Briefly, blood was diluted 1:1 with PBS and layered

onto Ficoll-Paque (4:3). The blood was centrifuged and the

lymphocyte layer was removed and washed twice in PBS, and

then washed with RPMI 1640 media. Cell density was counted

with a hemocytometer. Typically, each culture consisted of an

initial density of 1 � 106 cells in 1 mL of culture medium. The

culture medium consisted of RPMI 1640 supplemented with

10% FBS and PHA (10 mg/mL final concentration). Negative

and positive controls (mitomycin C 0.025 mmol/L) were devel-

oped. The lymphocyte cultures were grown in a humidified

incubator with 5% CO2 at 37�C in polystyrene plates. Aqueous

extracts were added to the cultures at 24 hours after PHA

stimulation, followed by the addition of cytochalasin B

(4.5 mg/mL) at 44 hours and harvested at 72 hours after the

beginning of the culture.30 At 72 hours, lymphocytes cultures

were spun directly (600 rpm, 5 minutes) onto glass slides using

a cytocentrifuge (Shandon, Cytospin 3, Microlat). Slides were

allowed to air-dry before methanol fixation at room tempera-

ture for 10 minutes. Slides were stored at �20�C in a sealed

box. Before scoring, the slides were stained with Giemsa 10%.

1000 binucleated lymphocytes (those that have undergone

one mitotic division) were scored for the number of MN in

accordance with recently published validation of the MN

assay.32 Scoring was performed by 2 scorers, with 10% of the

slides being rescored.

Antioxidant Capacity

On Biological SystemsSpontaneous chemiluminescence (CL) of brain homogenates. Rat

brains were obtained from female Wistar rats weighing

150 + 20 g. Rats were maintained in cages in a standar-

dized environment and fed a laboratory diet and water ad

libitum. Rats were killed by decapitation and the essentially

blood-free brains were excised and placed in an ice-cold

glass. The tissues were homogenized (1:5) in 30 mmol/L

potassium phosphate buffer (pH 7.4) containing

120 mmol/L KCl and centrifuged at 3000 rpm for 10 min-

utes at 4�C. The supernatants were immediately diluted 3

times their volume in the same buffer. Portions (3 mL) of

the dilute brain homogenate were transferred to 15-mL glass

vials for luminescence studies.

The CL of brain homogenates was measured in a Packard

liquid scintillation counter, at room temperature, in the out-of-

coincidence model.

Spontaneous brain CL was measured in the presence or

absence of infusion or decoction of different aliquots of

poleo and cedron in the reaction medium, this allows the

calculation of 50% CL inhibitory concentration (IC50). The

emission was expressed in counts per minute (cpm)/mg of

protein.33

Sample preparation. Rat brain homogenates were subjected

to oxidation by incubating in the presence of ABAP hydro-

chloride, which starts generating radicals in aqueous phase.

Incubation was performed by mixing 1 mL of homogenate,

500 mL of ABAP 200 mmol/L at 37�C, and 200 mL of a dilution

of infusion or decoction 1/100 of cedron or 1/25 of poleo. A

final volume of 2 mL was assessed with phosphate buffer (pH

7.4) containing 120 mmol/L of KCl. After 2 hours, the oxida-

tion products of lipids or proteins were analyzed using the

thiobarbituric acid reactive substance or protein carbonylation

(PC) techniques, respectively.

Thiobarbituric acid reactive products of lipid peroxidation. One

microliter of samples prepared as described above or the buffer

(blank) was diluted with 100 mL butylated hydroxytoluene and

1 mL of trichloroacetic acid to precipitate proteins. The pre-

cipitate was removed by centrifugation and the supernatant was

incubated with 0.67% thiobarbituric acid for 10 minutes at

100�C and centrifuged for 10 minutes. A volume of 1 mL of

thiobarbituric acid is added to the supernatant. The mixture was

heated for 1 hour at 100�C. The absorbance was measured at

535 nm (e ¼ 156 mmol L�1 cm�1).34

Protein carbonylation. One milliliter of sample obtained as

described above was used to assess the oxidation of proteins.

Portmann et al 195

The results are expressed as the percentage inhibition of protein

oxidation.

Protein carbonyl groups were detected with 2,4-

dinitrophenylhydrazine (DNPH), which leads to the forma-

tion of a stable 2,4-dinitrophenylhydrazone (DNP). The

DNP absorbs UV light so that the total carbonyl content

of a protein can be quantified by a spectrophotometric

assay at 370 nm. The measurement of protein carbonyls

following their covalent reaction with DNPH was pioneered

by Levine and has become the most widely utilized mea-

sure of protein oxidation in several diseases.35

On ExtractsTotal reactive antioxidant potential (TRAP). Total reactive

antioxidant potential was measured by CL in a Luminoskan

V 1.2-0 liquid scintillation counter. The reaction medium

consisted of 20 mmol/L ABAP, 40 mmol/L luminol,

0.05 mmol/L and 0.50 mL of infusion or decoction of cedron

and poleo, respectively. The system is calibrated with differ-

ent concentrations of Trolox (0.25-0.50 mmol/L). A compar-

ison of the induction time after the addition of Trolox or

different aliquots of infusion and decoction allows the calcu-

lation of the total antioxidant capacity (TRAP) as the equiv-

alent of Trolox concentration necessary to produce the same

induction time.36,37

Scavenging of 2,20-azino-bis-(3-ethyllbenzo-tiazoline-6-sulfonicacid) diammonium salt radical cation. The reaction mixture con-

sisted of ABTS 0.36 mmol/L and ABAP 18 mmol/L in

50 mmol/L phosphate buffer. After 45 minutes of incubation

at 45�C, different aliquots of the decoction and infusion were

added to 3 mL of the solution. The absorbance was read at

734 nm at fixed time (3 or 4 minutes), stirring constantly.38

Scavenging of 2,2-diphenyl-2-picryl hydrazyl (DPPH) radical. The

method consists of measuring the consumption of DPPH

(stable radical) spectrophotometrically through decreased

absorbance at 515 nm. Different aliquots of the decoction or

infusion were added to 3 mL of the solution prepared by dis-

solving 2.5 mg of DPPH in 100 mL of methanol. The

absorbance was read at 515 nm at fixed time (10 minutes),

stirring constantly.39

Statistical Analysis

Results were expressed as mean + standard deviation.

Statistical comparisons between groups were performed with

Student t test for independent observations. Statistical analysis

of genotoxic biomarkers was performed using 1-way analysis

of variance (ANOVA). In the cases of paired samples, the

differences between treatments were evaluated by paired t test.

A value of P < .05 was considered as statistically significant for

all the end points evaluated.

Results

Phytochemistry Analysis

Qualitative analysis of polyphenols—Chromatography. Both

infusion and decoction show the polyphenol profiles character-

ized by caffeic acid derivatives (hydroxycinnamic acids) and

flavonoids (flavones). The origin of flavone as an orange-

colored complex with natural product (NP) reagent suggests

the presence of O-dihydroxy groups on the B-ring of the fla-

vonoid molecule. Besides, the compounds that have only 1 OH

group originating as yellow complexes with the same reagent

(NP) are detected.

Quantitative analysis. Table 1 showed that both extracts have

high-value total phenols and total flavonoids. The low values

found for total tannins are compatible with the absence of

proanthocyanidins (condensed tannins).

Cytogenotoxic profile. Table 2 shows the biomarkers evaluated

in lymphocytes cell cultures were exposed to infusion and

decoction of cedron and poleo. No significant differences in

the frequency of mitosis and cell proliferation kinetics repre-

sented against the RI were detected (P > .05) when compared

with negative control.

The analysis of chromosomal instability through SCE did

not show modifications when compared with control cultures

and between treatments.

Table 1. Phytochemistry Analysis of A citriodora and L turbinata

Phytochemistry Analysis

A citriodora L turbinata

Infusion, X + SD Decoction, X + SD Infusion, X + SD Infusion, X + SD

Total phenols quantification (mg tannicacid/g of dry material)

51.85 + 3.17 51.35 + 3.49 10.62 + 0.62 13.36 + 0.26

Total tannins quantification (mg tannicacid/g of dry material)

5.95 + 0.65 8.90 + 0.90 1.60 + 0.17 1.98 + 0.20

Condensed tannins quantification(proanthocyanidins; mg tannic acid/gof dry material)

Not detected Not detected Not detected Not detected

Total flavonoids quantification (mgtannic acid/g of dry material)

20.82 + 1.80 20.67 + 1.70 5.95 + 0.60 8.68 + 0.90

196 International Journal of Toxicology 31(2)

The evaluation of genotoxic damage biomarker (MN)

showed the same behavior for both decoctions and concen-

trations without statistically significant differences with

negative control although both donors exhibit different sus-

ceptibility. In both donors, positive control show significant

difference (P < .0001; Table 3).

When we analyze cell viability in order to develop comet

assay, we observe 80% viable cells in control and treated sam-

ples. The results of the comet analysis in peripheral blood

lymphocytes are shown in Figures 1 and 2 for poleo and

cedron, respectively. All the groups exhibited a high proportion

of type I comets, whereas types II, III, and IV comets were not

found in any experimental unit except in positive control,

which was characterized by types II and III comets. No signif-

icant difference was found between negative control and

treated cells.

Antioxidant capacity. In all the assays used for the determina-

tion of the antioxidant capacity, aqueous extracts infusion and

decoction (5% w/v) were diluted to final concentration of 3 mg

of dry material/mL for poleo and 1 mg/mL for cedron.

Brain homogenates and plasma were used to determine the

effect on lipid peroxidation and to evaluate the protection over

PC, respectively. Lipid peroxidation was quantified by sponta-

neous brain CL and TBARS.

Autooxidation of brain was determined by the CL method,

in the presence or absence of infusion or decoction of aqueous

extracts. Table 4 shows the percentage of CL at 90 minutes in

the presence of infusion or decoction in the reaction medium.

The same procedure was applied using different volumes of

infusion or decoction. This allows calculating the volume that

inhibited 50% of CL (IC50). Among the tested preparations,

decoction showed the lowest value of IC50.

Table 5 shows 2 model antioxidants as positive control

catechin and quercetin. A relative value of the scavenging

capacity is provided by the concentration of antioxidant or

aqueous extract required to decrease PC or TBARS to 50%of that observed in the absence of additives.33

The protection over PC was performed on normal plasma

oxidized with ABAP and the results show an inhibition of

96% and 94% for decoction and infusion of cedron, respec-

tively, whereas the poleo produced an inhibition of 72% for

decoction and 29% for infusion (control value:

0.78 + 0.08 nmol/mg protein). TBARS determination in

brain homogenates oxidized with ABAP was decreased

74% for decoction and 54% for infusion of cedron. Decoc-

tion of poleo produced an inhibition of 65% and infusion of

poleo 21% (control value: 0.92 + 0.10 nmoles/mg protein;

Table 6).

Table 7 shows the total in vitro antioxidant activity evalu-

ated as TRAP, ABTS, and DPPH. Values are higher in decoc-

tion of both aqueous extracts.

Discussion

The use of plants for healing purposes is becoming increasingly

popular, as they are believed to be beneficial and free from side

Table 3. Micronuclei Frequency in Peripheral Blood LymphocytesTreated With Aqueous Plant Extracts (Infusion and Decoction)a

Poleo (Inf) Cedron (Inf) Poleo (Dec) Cedron (Dec)

Treatment Ind 1, X + SD Ind 2, X + SD

Negative C 3.74 + 1.7 5.06 + 0.98Positive C 61.45 + 5,8 85.82 + 11.5Cc1 4.00 + 1,1 3.80 + 1.4 7.70 + 3.8 6.97 + 2.4Cc2 3.60 + 1.5 3.20 + 1.9 5.37 + 1.8 4.40 + 3.5

Abbreviations: Ind., Individual; Cc1, 0.1 mg/mL; Cc2, 1 mg/mL; Dec, decoction;Inf, infusion; Negative C, negative control; Positive C, positive control: mito-mycin C, 0.025 mmol/L.a Analysis of variance (ANOVA) P > .05.

Table 2. Mitotic Index, Replication Index, and Sister Chromatid Exchanges in Peripheral Blood Lymphocytes Treated With Aqueous PlantExtracts (Infusion and Decoction)a

Extracts Treatment Cc

Cytogenotoxic Biomarkers

MI, X + SD RI, X + SD SCE, X + SD

1 2 1 2 1 2

Negative control 3.35 + 1.20 2.33 + 0.28 1.47 + 0.05 1.43 + 0.18 3.28 + 1.75 4.83 + 3.00Cedron Inf Cc1 2.39 + 0.00 3.08 + 0.31 1.54 + 0.08 1.38 + 0.02 3.83 + 1.45 6.55 + 3.09

Cc2 2.35 + 0.21 2.18 + 0.16 1.51 + 0.01 1.29 + 0.06 3.88 + 1.40 5.92 + 1.88Poleo Inf Cc1 2.95 + 0.22 3.23 + 0.52 1.39 + 0.14 1.70 + 0.02 3.83 + 2.06 7.15 + 3.72

Cc2 3.20 + 0.99 2.20 + 0.48 1.34 + 0.05 1.60 + 0.04 3.72 + 1.73 6.88 + 3.36Negative control 4.30 + 0.28 3.75 + 0.21 1.72 + 0.04 1.43 + 0.08 3.14 + 1.72 3.41 + 1.46Cedron Dec Cc1 4.00 + 0.28 4.90 + 0.28 1.60 + 0.14 1.35 + 0.08 3.72 + 1.88 3.88 + 2.12

Cc2 3.80 + 0.28 4.55 + 0.07 1.72 + 0.11 1.30 + 0.04 4.14 + 1.86 4.62 + 2.63Poleo Dec Cc1 5.20 + 0.40 4.25 + 1.48 1.86 + 0.11 1.54 + 0.11 3.88 + 1.80 4.78 + 2.18

Cc2 4.10 + 1.56 5.00 + 0.14 1.76 + 0.05 1.31 + 0.20 3.24 + 1.73 3.57 + 1.86

Abbreviations: Cc1, 0.1 mg/mL; Cc2, 1 mg/mL; Dec, decoction; Inf, infusion; MI, mitotic index; RI, replication index; SCE, sister chromatid exchange.a SCE-positive control: 12.8 + 3.64 (individual 1); 13.98 + 5.24 (individual 2). Analysis of variance (ANOVA) P > .05.

Portmann et al 197

Figure 1. Damage index in peripheral blood lymphocytes treated with infusion and decoction of poleo. *Paired t test P < .05.

Figure 2. Damage index in peripheral blood lymphocytes treated with infusions and decoctions of cedron. *Paired t test P < .05.

198 International Journal of Toxicology 31(2)

effects. However, most of the information available on many

medicinal herbs has no supporting scientific data and their use

as medicaments is based solely on traditional folk usage that

has been perpetuated down through the generations.40 Never-

theless, some of them can cause adverse effects or have the

potential to interact with other medications.41

Our results in relation to phytochemical studies showed that

infusions and decoctions of both medicinal plants are charac-

terized by the presence of hydroxicinamic acids and flavonoids

(flavones). Chromatograms from infusion and decoction of A

citriodora leaves have polyphenol profiles characterized by the

presence of cinnamic acids and flavones (luteolin and apigenin

derivatives), and these compounds are responsible for their

antioxidant activity. When a comparative analysis was per-

formed between both medicinal plant extracts, the total phenol

and total flavonoids showed to be higher in the extract of A

citriodora than that of L turbinata.

Mitotic index and RI are used as indicators of adequate cell

proliferation biomarkers. Mitotic index measures the

proportion of cells in the M-phase of the cell cycle and its

inhibition could be considered as cellular death or a delay in

the cell proliferation kinetics.28 We did not observe modifica-

tions in relation to MI, consequently a relation with a cytotoxic

profile is absent, in our experimental conditions. On the other

hand, RI did not change with any of the aqueous extract con-

centrations assayed, showing that the extracts do not induce

any modifications in the cellular proliferation kinetics.

Sister chromatid exchanges represent a symmetrical

exchange of complementary DNA strands between chromatid

within a single chromosome.42 When SCE/cell was analyzed,

we could not find a significant increase in its frequency, so it

could be concluded that aqueous extracts do not induce chro-

mosomal instability.

In this experimental design, micronuclei assay is the only

biomarker that allows the simultaneous evaluation of aneugenic

and clastogenic effects easily detected in interphase cells. The

speed and easy usability of MN analysis and the non-

requirement of metaphase cells made this assay very popular.43

Micronuclei frequency assay in human lymphocytes from periph-

eral blood cultures has been shown to be a sensitive and useful

index method to evaluate genotoxicity.44 In our experimental

design, this biomarker showed the same performance than others,

without significant differences from control cultures for both

extracts (P > .05).

The alkaline comet assay is increasingly used in industrial

genotoxicity testing in vitro45–47 and is also becoming an impor-

tant tool for evaluating the genotoxic potential of compounds in

vivo.48,49 In the literature, this test is being considered a fast

method to predict the genotoxic damage of an agent in experi-

mental designs, as much in alive as in vitro.50 The results obtained

with the administration of extract per se did not produce any

Table 5. Concentration of Antioxidant or Aqueous Extract Required to Decrease PC or TBARS to 50% of That Observed in the Absence ofAdditives (IC50)a

IC50

CedronDecoction(mg/mL)

CedronInfusion (mg/mL)

PoleoDecoction (mg/mL)

PoleoInfusion (mg/mL)

Catechin(mg/mL)

Quercetin(mg/mL)

PC 16.8 + 0.9 17.1 + 1.0 92.3 + 5.0 220.5 + 11.4 6.1 + 0.3 13.5 + 0.7TBARS 21.9 + 2.1 29.7 + 2.7 98.6 + 4.5 306.9 +11.7 2.0 + 0.1 1.5 + 0.1

Abbreviations: IC50, 50% chemiluminescence inhibitory concentration; PC, protein carbonylation (control value: 0.78 + 0.08 nmol/mg protein); TBARS,thiobarbituric acid test (control value: 0.92 + 0.10 nmol/mg protein).a Values are expressed as mean + standard error.

Table 4. Spontaneous Brain Chemiluminescence and 50% Chemiluminescence Inhibitory Concentration in the Presence of Poleo and Cedron(Infusion–Decoction) in the Reaction Mediuma

Control Cedron Decoction Cedron Infusion Poleo Decoction Poleo Infusion

CL (cpm/mg protein) 723 + 41 318 + 25b 463 + 30b 591 + 41 634 + 39IC50 (mg/mL) – 1.1 1.8 4.0 6.0

Abbreviations: CL, spontaneous chemiluminescence; IC50, 50% chemiluminescence inhibitory concentration.a Values are expressed as mean + standard error.b P < .001.

Table 6. Percentage of Inhibition of TBARS and Protein Carbonyla-tion With Infusion or Decoction of Poleo and Cedrona

% Inhibition

CedronDecoction

CedronInfusion

PoleoDecoction

PoleoInfusion

PC 96 + 4 94 + 5 72 + 3 29 + 2TBARS 74 + 4 54 + 3 65 + 4 21 + 3

Abbreviations: PC, protein carbonylation (control value: 0.78 + 0.08 nmol/mgprotein); TBARS, thiobarbituric acid test (control value: 0.92 + 0.10 nmol/mgprotein).a Values are expressed as mean + standard error.

Portmann et al 199

statistically significant change in the parameter when compared

to the negative control. Further studies are needed in order to show

that our results may possibly represent the first evidence that both

extracts (infusion and decoction) could have an effective antige-

notoxic activity in an in vitro system using comet assay.48,51,52 In

vivo previous studies in A citriodora agree with our findings.53,54

Much attention of preventive medicine research is focused on

natural antioxidants. This interest refers not only to isolation and

identification of new biologically active molecules by the phar-

maceutical industry but also to the emergent public interest in

using crude plant extracts, such as infusion for self-medication.

In a normal diet, the intake of herbs may contribute significantly

to the total intake of plant antioxidants and even be a better source

of dietary antioxidants than many other food groups.55

Scavenging of different types of reactive oxygen and nitro-

gen species, mostly free radicals, is thought to be one of the

main mechanisms of the antioxidant action exhibited by phe-

nolic phytochemicals. In this study, 3 different radical scaven-

ging models were used, showing that decoction and infusion

had varying degrees of scavenging actions against the radicals

used. All tests used showed that infusion and decoction of both

extracts have antioxidant properties, decoction being more effi-

cient than infusion.

Several studies are focused in the relationship between the

antioxidant activity of the phenolic compounds as hydrogen

donating free radical scavengers and their chemical structure.

It has been shown that the presence of the –CH¼CH–COOH

group in the hydroxylated cinnamates ensures greater H-

donating ability and subsequent radical stabilization than the

carboxylate group in the hydroxy benzoates.56

The physiological relevance of antioxidant capacity of both

extracts was evaluated by the possible protection against protein

oxidation in plasma and lipid peroxidation of brain homogenates

measured as TBARS or spontaneous CL. Both extracts inhibited

lipid peroxidation evaluated as spontaneous brain CL and

TBARS, and protein oxidation evaluated as PC. In both cases,

it appears that decoction produces a higher percentage of inhibi-

tion than infusion (P < .005). Furthermore, cedron has proven to

be 4 times more effective than poleo. An important remark to be

made is that decoction seemed to be more efficient in protecting

lipid peroxidation and protein oxidation than the infusion.

Furthermore, A citriodora has proven to be 4 times more effec-

tive than L turbinata, suggesting their interest as ingredients for

antioxidants formulations since cedron presents higher values

than poleo, which are correlated with the greatest content of

polyphenols in the leaves of cedron.9

Conclusion

Recently, interest has increased considerably in finding natu-

rally occurring antioxidant for use in food or medicinal mate-

rials to replace synthetic antioxidants, which are being

restricted due to their side effect such as carcinogenicity.57 In

this article, we assess the antioxidant properties as well as the

absence of genotoxic effect of infusion and decoction on both

aqueous extracts. These results would suggest the benefits of

using both kinds of preparations as natural source of antioxi-

dants and possible protective action against DNA damage.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to

the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the

research, authorship, and/or publication of this article: UBACYT-

20020100100123 2011-2014

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